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Effect of Interleukin-2 Pretreatment on Paclitaxel Absorption and Tissue Disposition after Oral and Intravenous Administration in Mice

Unité Propre de Recherche et de l'Enseignement Supérieur, Equipe d'Accueil 2706, Faculty of Pharmaceutical Sciences, Université Paris Sud XI, Châtenay Malabry, France (B.H., F.B., R.F., L.B.-F.); Laboratory of Pharmacology, Paul Brousse Hospital AP-HP, Villejuif, France (C.A., L.B.-F.); and Zoology Department, Institut-Gustave-Roussy, Villejuif, France (B.P., A.D., P.G.)

(Received October 4, 2007; Accepted May 27, 2008)

	Abstract

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The aim of the present study was to investigate the effects of recombinant interleukin (rIL)-2 treatment on paclitaxel (PLX) pharmacokinetics in the plasma and tissue of Lewis lung carcinoma-bearing mice (lung tissues and s.c. tumors). PLX pharmacokinetics studies were conducted after oral and i.v. administration of 15 and 4 mg/kg, respectively, either alone or after 3 days of rIL-2 pretreatment. The noncompartmental approach was used to determine the mean pharmacokinetic parameters using WinNonlin software (Pharsight, Mountain View, CA). The influence of rIL-2 pretreatment on physiological P-glycoprotein (P-gp) expression in lung and intestine was investigated by Western blot analysis. After oral administration of PLX, areas under the curve (AUC) in plasma, lung, and s.c. tumors were significantly higher (2.98, 2.66, and 3.41-fold, respectively) in the rIL-2 + PLX group as compared with the PLX group. However, no significant effect of rIL-2 pretreatment was observed in plasma or lung following i.v. administration of PLX. PLX AUC in s.c. tumors was significantly higher (1.37-fold) with rIL-2 pretreatment as compared with the PLX-alone group after i.v. injection. Pretreatment with rIL-2 appeared to have no effect on PLX plasma terminal half-life when PLX was administered orally or i.v. However, prolongation of PLX terminal half-life estimated from lung and s.c. tumors data had been observed. Increased PLX tissue absorption in the rIL-2-pretreated group may be explained by a decrease of P-gp expression in the intestines and lung or decreased functionality due to rIL-2. Oral administration allowed the targeted tissues a much higher PLX exposure as compared with i.v. administration.

PLX is a P-glycoprotein (P-gp) substrate (Gottesman and Pastan, 1993). Furthermore, it has been shown that PLX activates the steroid and xenobiotic receptor and regulates expression of CYP3A4 and P-gp in human cells (Synold et al., 2001).

P-gp, a well known modulator of anticancer agent pharmacokinetics, leads to an increase of drug efflux and reduces drug accumulation in tumor cells. Enhanced expression of P-gp is considered to be a major mechanism of PLX resistance (Schinkel et al., 1997). In addition, intestinal P-gp reduces oral bioavailability of drugs that are P-gp substrates such as vincristine and PLX (Kimura et al., 2002). P-gp inhibitors such as cyclosporine, PSC833, or GF120918 have been used in combination with anticancer drugs (P-gp substrates) to overcome the multidrug resistance (MDR) phenotype. The coadministration of cyclosporine and PLX results in enhanced oral bioavailability of PLX due to increased intestinal absorption (Kimura et al., 2002). However, P-gp inhibitors appear to be too toxic for routine use (Gallo et al., 2003). On the other hand, it has been shown that P-gp is expressed at the apical side of alveolar epithelial cells and lung vascular endothelium, suggesting that P-gp could play a role in drug transport from interstitium to lumen (van der Deen et al., 2005). Although P-gp has been characterized in several cell types in lung tissue, its effect on drug pharmacokinetics is not clear (Scheffer et al., 2002).

Patients diagnosed with cancer who are in a state of a primary immunosuppression, specifically, a lymphopenia or a deficit in endogenous production of interleukin (IL)-2, respond poorly to chemotherapy (Cerea et al., 2001). Also, long-term survival of patients with small cell lung cancer is correlated with IL-2 secretion at diagnosis, with long-term survival and complete response to chemotherapy only observed in patients with high IL-2 secretion at diagnosis (Fischer et al., 2000). In patients diagnosed with gastric cancer, perisurgery immunotherapy with IL-2 corrects the postsurgical immune deficit and increases survival (Romano et al., 2004). Indeed, the IL-2 group showed lower post surgical complications (p < 0.05) and higher lymphocyte infiltration into the tumor (p < 0.001). Moreover, disease free survival was longer in the IL-2 group than in the control group, although these findings were not statistically significant (p < 0.06).

Recombinant interleukin (rIL)-2 is already used in the treatment of metastatic renal adenocarcinoma. It has been shown that rIL-2 decreases MDR1 mRNA and P-gp expression in human colon carcinoma cultured cells (Stein et al., 1996) and in intestine and brain of Swiss mice (Bonhomme-Faivre et al., 2002). Furthermore, we have shown previously that rIL-2 decreases rhodamine transport through the reversed gut sac of Swiss mice (Veau et al., 2002). In addition, rIL-2 pretreatment increased PLX efficacy measured by the significant decrease of s.c. tumor growth and lung metastasis number in the Lewis lung carcinoma model (Hosten et al., 2006).

The aim of the present study was to document the pharmacokinetics of PLX in plasma and tissues after rIL-2 pretreatment in the Lewis lung carcinoma model after oral and i.v. administration and to investigate the effect of rIL-2 pretreatment on the intestinal and lung P-gp expression in mice by Western blot analysis.

	Materials and Methods

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Chemicals. Cell culture media and reagents were purchased from Invitrogen (Carlsbad, CA). PLX (Taxol) (paclitaxel powder from Taxus yannanesis; Sigma-Aldrich, Saint-Quentin Fallavier, France) was diluted in methanol to obtain a stock solution (1 mg/ml). Docetaxel (Taxotere) (Sigma-Aldrich) was diluted in methanol to obtain a stock solution (1 mg/ml). Throughout the study, acetonitrile, triethylamine, hexane, ammonium acetate, ethyl acetate, and methanol (HPLC grade) were purchased from VWR (Fontenay sous Bois, France), and water for injections was purchased from C.D.M. Lavoisier (Paris, France).

Drugs and Treatment. Proleukin, rIL-2, was purchased from Chiron (Suresnes, France). Recombinant IL-2 was reconstituted, and 1 ml was diluted with 5% dextrose solution to obtain a final concentration of 1.8 MIU/ml. Taxol, PLX formulated with Cremophor EL and ethanol [1:1 (v/v)], was diluted with isotonic sodium chloride solution to obtain a final concentration of 2 mg/ml.

Animals. Animal experiments were carried out according to French and European laws and regulations. The protocol ethics were institutionally approved. Anesthesia was induced with 5% isoflurane and maintained with 2.5% isoflurane in air. Female C57BL/6 mice (Charles River Laboratories, Les Oncins, France), 6 to 8 weeks old, weighing 20 to 25 g, were used. The mice were given water and food ad libitum, in accordance with the European Community guidelines. The animals were housed under these conditions for at least 1 week before use. Lewis lung carcinoma (LLC) tumor cells were s.c. injected into the right back of the mice to obtain an s.c. tumor and lung metastasis.

Tumor Cells. LLC cells were kept frozen in fetal bovine serum/dimethyl sulfoxide [90:10 (v/v)]. The tumor cells were supplied by the research laboratory Unité Mixte de Recherche 8121, Gustave-Roussy Institute (Villejuif, France). They were cultured for 10 days in RPMI 1640 medium containing glutamate supplemented with 10% heat-inactivated fetal bovine serum and a mixture of antibiotics (10,000 IU/ml sodium penicillin G and 10,000 µg/ml streptomycin sulfate). LLC cells (10⁶) were injected on day 0 in a volume of 0.2 ml of isotonic sodium chloride into the backs of mice.

PLX Pharmacokinetic Studies. Oral and i.v. administration. On day 5, when tumors reached a volume of 100 mm³ on average, mice were randomized into four groups: group 1, PLX alone (15 mg/kg p.o. on day 8); group 2, rIL-2 (0.27 MIU i.p.) twice daily from days 5 to 7, then PLX (15 mg/kg p.o.) on day 8; group 3, PLX alone (4 mg/kg i.v.) on day 8; and group 4, rIL-2 twice daily from days 5 to 7, then PLX (4 mg/kg i.v.) on day 8.

For the PLX assay, blood samples (0.5 ml on average) were collected in heparinized tubes before PLX administration and at 0.5, 1, 2, 3, 6, 16, 24, and 48 h after its oral administration or 0.25, 0.5, 1, 2, 3, 6, 16, 24, and 48 h after i.v. administration, with three to four mice per time point. Blood samples were centrifuged for 10 min at 3500 rpm. Plasma was harvested into clean tubes and stored at -20°C until analysis. After blood sampling, mice were sacrificed. Lung tissues and s.c. tumors were collected and stored at -80°C until analysis.

PLX quantification in plasma. PLX plasma concentrations were measured using a revised RP-HPLC/UV method (Willey et al., 1993). Quantification followed a solid-phase extraction. For analysis, 100 µl of plasma was mixed with 100 µl of 0.2 M ammonium acetate and 50 µl of 5 µg/ml docetaxel [internal standard (IS)]. The mixture was vortexed for 20 s. Sample extraction was accomplished using SPE cyano 1-ml Bond Elut cartridges (Interchim, Fontenay sous Bois, France). Cartridges were washed using 1 ml of water and 1 ml of 20% methanol in 0.01 M ammonium acetate solution, followed by 1 ml of n-hexane. The cartridges were then dried under vacuum for 2 min. PLX and the IS were then eluted using 2 ml of triethylamine (0.1%) in acetonitrile. The eluent was evaporated under a gentle stream of nitrogen at 30°C. The residue was reconstituted in 100 µl of a water/acetonitrile mixture [1:1 (v/v)] and vortexed for 20 s. An aliquot of 50 µl was then injected into the chromatographic system.

Chromatographic analysis was achieved on a Nucleosil C₁₈ column (4.6 x 250 mm, 5-µm i.d.) (Interchim) with a mobile phase composed of acetonitrile and water [1:1 (v/v)] delivered at a flow rate of 1.2 ml/min. The eluent was monitored at 227 nm. The quantification method was validated according to the Food and Drug Administration recommendations (Shah et al., 2000).

The PLX standard curve was correctly described by unweighted least-square linear regression. Over the PLX plasma concentration range of 50 to 5000 ng/ml, the determination coefficient (R²) of the calibration curves remained >0.99. Based on quality control samples, the overall relative S.D. (an index of precision) was less than 12%. The overall relative error (an index of accuracy) was less than 10%. The lower limit of quantification (LLOQ) was 50 ng/ml. The stability of plasma samples at -20°C was confirmed by analyzing six quality control samples at the end of the study.

PLX quantification in lung tissues and s.c. tumors. PLX tissue concentrations were measured using a validated RP-HPLC/UV method using modifications of the HPLC procedure described by Mohamed et al. (2003). Calibration standards of PLX were prepared in drug-free lung tissues from C57BL/6 mice by spiking with concentrated standards to obtain a concentration range between 100 to 10,000 ng/g. Quality control samples for PLX were prepared in drug-free lung tissues from C57BL/6 mice by spiking with concentrated standards. Three quality controls were prepared, low (600 ng/g), medium (4000 ng/g), and high (9000 ng/g). PLX tissue concentrations were measured using an RP-HPLC/UV method. This quantification followed a liquid/liquid extraction. PLX was extracted from 100 mg of weighed lung tissue or s.c. tumor. For analysis, the tissue samples were mixed with 100 µl of docetaxel (2 µg/ml) (IS) and 400 µl of acetonitrile. The mixture was vortexed for 20 s and centrifuged (10,000 rpm/min). The supernatant was evaporated under nitrogen at 30°C. The residue was reconstituted in 200 µl of water and mixed with 3 ml of ethyl acetate.

The mixture was agitated for 20 min and evaporated under nitrogen at 30°C. Mobile phase was then added, 100 µl of acetonitrile/water [48:52 (v/v)], and the sample was vortexed for 20 s. An aliquot of 50 µl was injected into the chromatographic system.

Chromatographic analysis was performed on a Nucleosil C₁₈ column (4.6 x 125 mm, 3-µm i.d.) (Interchim) with a mobile phase composed of acetonitrile and water [48:52 (v/v)], delivered at a flow rate of 1.2 ml/min with monitoring at 227 nm. Food and Drug Administration recommendations were followed to validate this method (Shah et al., 2000).

The standard curve of PLX was well described by 1/X²-weighted least-square linear regression (X = concentration). Over the PLX tissue concentration range of 100 to 10,000 ng/g, the determination coefficient (R²)ofthe calibration curves remained >0.99. The LLOQ was 100 ng/g. The precision and accuracy concerning the within- and between-days reproducibility were always less than 12%.

View larger version (15K): [in this window] [in a new window]   FIG. 1. Mean PLX concentration versus time profiles in plasma. Diamonds, PLX treatment alone i.v. at 4 mg/kg. Squares, PLX (i.v.) at 4 mg/kg after 3 days of pretreatment with rIL-2 administered i.p. at 0.27 MIU twice a day. Triangles, PLX alone p.o. at 15 mg/kg. Crosses, PLX (p.o.) at 15 mg/kg after 3 days of pretreatment with rIL-2 administered i.p. at 0.27 MIU twice a day. Data represent the mean PLX concentration ± S.E.M. in plasma (n = 3–4 per time point).

0

0

0 last

Semiquantitative Determination of P-gp Expression by Western Blot Analysis. P-gp expression was measured in mouse lung and intestine (ileum segment) by Western blot analysis. The lungs and intestines were removed 1 hour after the last rIL-2 injection from four female C57BL/6 mice that had been treated with rIL-2 (16.5 µg) twice a day for 3 days and four female C57BL/6 mice without treatment. Lung and intestine samples were homogenized in a glass tube in buffer (10 mM triethanolamine + 8.5% saccharose) containing protease inhibitors. The crude membranes obtained were solubilized with lysis buffer (1 M Tris, 0.5 M EDTA, 3 M NaCl, 10% Triton, 20% SDS, and protease inhibitor). Protein concentrations were determined using the colorimetric bicinchoninic assay kit (Sigma-Aldrich), with bovine serum albumin as a standard. Proteins (15 µg) were separated by SDS-polyacrylamide gel electrophoresis on an 8% polyacrylamide gel and transferred onto a nitrocellulose membrane (GE Healthcare, Chalfont St. Giles, UK). The nitrocellulose membranes were then incubated with a primary antibody (C219, diluted to 1:100; DakoCytomation Denmark A/S, Glostrup, Denmark), washed, and finally incubated with a peroxidase-conjugated anti-mouse IgG secondary antibody. The immunoreactive bands were visualized by the enhanced Chemi-luminescent system (Perkin Elmer Life Science, Boston, MA). The nitrocellulose membranes were also incubated with anti-β actin (diluted to 1:5000; Sigma-Aldrich, St. Louis, MO) to normalize P-gp expression. The autoradiographs of P-gp and β actin protein were scanned and analyzed by densitometry using the Scion Image program (Scion Corporation, Frederick, MD) to obtain a quantitative evaluation of the levels in the lung and intestine.

Statistical Analysis. Pharmacokinetic studies. PLX maximal concentrations were compared using a Student's t test. AUC_{0 Tlast} of the two groups (rIL-2 + PLX versus PLX alone) were compared using Bailer's method (Bailer, 1988). The mean AUC and their S.E. values were calculated using the equations:

(1)

(2)

Where y_i and SE_i are the mean paclitaxel concentration and its S.E. at time i (i = 0, 1, 2, n). The test for equality of the mean AUC between treatments A and B is performed using the standard Wald statistic:

(3)

Under the null hypothesis, which indicates that AUC are equal, this statistic follows a normal distribution. The null hypothesis is rejected if Z_obs is greater than 1.96.

Western blot studies. In the Western blot analysis of the P-gp expression, the mean P-gp/β actin ratio was compared using the Student's t test. The P-gp expression was expressed as the mean P-gp/β actin ratio ± S.E.M. (n = 4). The Student's t test was used to compare the results obtained with or without rIL-2 pretreatment.

	Results

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Noncompartmental Analysis in Plasma after Oral and i.v. Administration. The plasma PLX concentration versus time profiles observed after oral and i.v. administration are shown in Fig. 1. The main pharmacokinetic parameters observed in plasma after oral and i.v. administration were obtained after a noncompartmental analysis, as summarized in Table 1. The plasma PLX maximal concentration (C_max) in the rIL-2 + PLX group was 2.6-fold higher than in the PLX alone group after oral administration (p < 0.01). The plasma PLX total exposure, measured by AUC, was 3-fold higher in the rIL-2 + PLX group as compared with the PLX-alone group after oral administration (p < 0.001). After i.v. administration, plasma PLX C_max, AUC, and the terminal half-lives in the two groups were not significantly different.

View larger version (15K): [in this window] [in a new window]   FIG. 2. Mean PLX concentration versus time profiles in lungs. Diamonds, PLX alone i.v. at 4 mg/kg. Squares, PLX (i.v.) at 4 mg/kg after 3 days of pretreatment with rIL-2 administered i.p. at 0.27 MIU twice per day. Triangles, PLX alone p.o. at 15 mg/kg. Crosses, PLX (p.o.) at 15 mg/kg after 3 days of pretreatment of rIL-2 administered i.p. at 0.27 MIU twice a day. Data represent the mean PLX concentration ± S.E.M. in lung tissue (n = 3–4 per time point).

0 last

0 last

lung 0

plasma 0

Noncompartmental Analysis in s.c. Tumors. The s.c. PLX concentration versus time profiles observed after oral and i.v. administration are shown in Fig. 3. The main pharmacokinetic parameters observed in s.c. tissue after oral and i.v. administration were obtained with a noncompartmental analysis as summarized in Table 3. After oral administration, PLX C_max and AUC_{0 last} in the rIL-2 + PLX group were increased by 2.1- and 3.41-fold, respectively, as compared with the PLX-alone group (p < 0.01). A prolongation of output half-life was also observed in the rIL-2 + PLX group as compared with the PLX-alone group (6.8 versus 2.69 h, respectively).

View larger version (16K): [in this window] [in a new window]   FIG. 3. Mean PLX concentration versus time profiles in s.c. Diamonds, PLX alone i.v. at 4 mg/kg. Squares, PLX (i.v.) at 4 mg/kg after 3 days of pretreatment of rIL-2 administered i.p. at 0.27 MIU twice a day. Triangles, PLX alone p.o. at 15 mg/kg. Crosses, PLX (p.o.) at 15 mg/kg after 3 days of pretreatment of rIL-2 administered i.p. at 0.27 MIU twice a day. Data represent the mean PLX concentration ± S.E.M. in s.c. tumor (n = 3–4 per time point).

After i.v. administration, the PLX C_max in the two groups was comparable with a prolonged output half-life (11.6 versus 4.9 h in the rIL-2 + PLX and PLX-alone groups, respectively). A significant 1.4-fold increase in AUC_{0 last} was observed in the rIL-2 + PLX group as compared with the PLX-alone group (p = 0.015).

After oral administration, the AUC_{s.c. tumor 0}/AUC_{plasma 0} ratios were 6.7 and 8.3 in the PLX-alone and rIL-2 + PLX groups, respectively. After i.v. administration, the same ratios were 2.6 and 4.8 in the PLX-alone and rIL-2 + PLX groups, respectively.

Semiquantitative Determination of P-gp Expression by Western Blot Analysis in Intestine and Lung. Western blot analysis of P-gp expression was performed on lung and intestine from four control mice and four mice treated with rIL-2 (16.5 µg twice a day) for 3 days. P-gp expression was significantly decreased in the lungs and intestine of the rIL-2-treated group as compared with the control group (p < 0.05) (Figs. 4 and 5).

View larger version (31K): [in this window] [in a new window]   FIG. 4. P-gp immunodetection from the intestine of mice treated with 3 days of rIL-2 or control. The mean P-gp/β actin ratio ± S.E.M. values from mouse intestines (n = 4 per group) are represented in the histogram.

View larger version (29K): [in this window] [in a new window]   FIG. 5. P-gp immunodetection from lung of mice treated with 3 days of rIL-2 or control. The mean P-gp/β actin ratio ± S.E.M. values from mouse lung (n = 4 per group) are represented in the histogram.

	Discussion

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Pretreatment with rIL-2 increased the plasma PLX AUC 3-fold and C_max 2.6-fold after oral administration. In contrast, AUC and C_max were unaffected by rIL-2 pretreatment when PLX was administered i.v. This result suggests an enhanced PLX intestinal absorption, resulting in an increased total exposure without altering its terminal half-life. The pharmacokinetic changes could be explained by either an inhibition of cytochrome P450-mediated intestinal and hepatic PLX metabolism or by an inhibition of PLX transport via intestinal P-gp (Sonnichsen et al., 1995). PLX total exposure appeared to be unaffected by rIL-2 pretreatment after i.v. administration, suggesting that rIL-2 had no significant effect on hepatic PLX metabolism by cytochrome P450. Previous studies showed that rIL-2 administered i.p. at 0.27 MIU inhibited intestinal P-gp activity by decreasing intestinal P-gp functionality in mice (Veau et al., 2002). In addition, it has been demonstrated that the coadministration of PLX with a P-gp inhibitor (MS-209) (Naito et al., 2002) caused an increase of AUC and C_max without modifying the other pharmacokinetic parameters (Kimura et al., 2002). Taken together, these findings suggest that rIL-2 inhibited intestinal P-gp functionality or expression without excluding an effect on cytochrome P-450. This hypothesis was strengthened by the intestinal Western blot analysis, which showed a significant decrease in intestinal P-gp expression in the rIL-2-pretreated group as compared with the control group.

The noncompartmental analysis of PLX lung data showed that rIL-2 pretreatment increased PLX AUC and C_max after oral administration of PLX. No significant effect on total exposure or inlet of PLX administered i.v. was observed with rIL-2 treatment.

The PLX terminal half-life appeared to be prolonged after rIL-2 pretreatment, independently of the route of PLX administration. These results suggest that elevations in PLX AUC and C_max after oral administration are probably related to higher PLX systemic exposure (plasma AUC) after rIL-2 pretreatment. The prolongation of PLX terminal half-life in lung observed in mice pretreated with rIL-2 could be the result of an inhibition of the expression or functionality of P-gp in lung. Indeed, the Western blot analysis of lung showed a significant decrease in lung P-gp expression in rIL-2-pretreated mice as compared with the control group. Two previous studies demonstrated the inhibition of P-gp function in lung. Campbell et al. (2003) showed that the coincubation of rat alveolar epithelial cell population with verapamil leads to inhibition of P-gp activity and to greater intracellular accumulation of P-gp substrate Rhodamine 123 during the efflux phase. In addition, Roerig et al. (2004) showed that the uptake of the P-gp substrate Rhodamine is increased in lung tissue in the presence of verapamil and GF120918 (P-gp inhibitors). In our study, rIL-2 pretreatment resulted in prolongation of PLX terminal half-life from lung after oral and i.v. administration of PLX, suggesting that rIL-2 inhibited P-gp expression or function in lung.

The noncompartmental analysis of PLX in s.c. tumors showed that rIL-2 pretreatment increased PLX AUC and C_max after PLX oral administration. After i.v. administration of PLX, rIL-2 pretreatment led to a significant increase in PLX total exposure. PLX terminal half-life also appeared to be prolonged after rIL-2 pretreatment with either route of PLX administration. These results suggest that the increases in PLX AUC and C_max after oral administration are probably related to higher PLX systemic exposure (plasma AUC) after rIL-2 pretreatment. Nevertheless, the increase of PLX total exposure and terminal half-life (regardless of the route of PLX administration) in s.c. tumors in mice pretreated with rIL-2 could be explained by other properties of rIL-2. Indeed, rIL-2 is known to induce capillary leak syndrome (Assier et al., 2005), meaning that rIL-2 may have increased vascular permeability and facilitated PLX penetration, especially in peripheral tissues. Moreover, it is well known that rIL-2 enhances the antitumor immune response, which decreases tumor growth and metastasis development in mouse tumor models (Taieb et al., 2006). PLX total exposure in the s.c. tumors may also be increased by rIL-2 by modifying the tumor cell density and the PLX distribution in the tumor, even when the systemic exposure to i.v. PLX was the same in the PLX and rIL-2 + PLX groups. However, if rIL-2 induced the capillary leak syndrome or anticancer effects, these hypotheses do not explain the PLX terminal half-life prolongation in s.c. tumors after rIL-2 pretreatment. Finally, rIL-2 could have decreased the P-gp expression or function in s.c. tumors, which may explain the prolonged PLX terminal half-life. However, the P-gp expression levels of the s.c. tumors were too low and variable as visualized by Western blot analysis to provide evidence for a significant decrease in P-gp expression induced by rIL-2 (data not shown).

In any event, the mechanism by which rIL-2 decreases P-gp expression may be explained by in vitro studies. Indeed, NF-B and NF-AT (nuclear factor of activated T cells) are two nuclear transcription factors for IL-2 (Park et al., 2007). When IL-2 binds its receptor, it activates several proteins including Janus tyrosine kinase (JAK) and signal transducer and activator of transcription 1, 3, 5, and 6 (O'Sullivan et al., 2007). Signal transducer and activator of transcription 3, however, inhibits the antiapoptosis factor NF-B (Hoentjen et al., 2005). A study has shown that NF-B activates the orphan receptors pregnane X receptor/steroid and xenobiotic receptor, which induce the MDR1 gene transcription and P-gp expression. This study also reported that inhibition of NF-B significantly reduces P-gp expression in HCT 15 cells (Bentires-Alj et al., 2003). Hence, by binding its receptor, rIL-2 could inhibit NF-B (via STAT3 recruitment) and thus decrease the P-gp expression in vivo.

Oral administration of PLX has recently become a topic of great interest (Veltkamp et al., 2007). Indeed, oral treatment with cytotoxic agents is preferred because it reduces administration costs and facilitates the use of long-term treatment (Malingré et al., 2001). Treatment with anticancer drugs as PLX is limited by nonhematological and hematological toxicity, which is related to its systemic exposure (Gelderblom et al., 2002; Mielke et al., 2005). The present comparison between the two routes of administration provided evidence for two principle findings. The first, oral administration allowed the targeted tissues a much higher PLX exposure as compared with i.v. administration (AUC_{tissues 0} /AUC_{plasma 0} ratios were higher after oral administration as compared with the i.v. treatment). This result could be explained in part by the presence of Cremophor El in the formulated PLX, which is not orally absorbed and may be responsible for the nonlinear pharmacokinetics of PLX after i.v. administration (van Tellingen et al., 1999). Indeed, after oral administration, when Cremophor is not present, plasma levels of PLX represent a higher fraction of free drug, which will result in enhancement of the availability of PLX to lung tissue and s.c. tumors. In contrast, after i.v. administration, entrapment of PLX in Cremophor EL micelles in the plasma compartment causes an increase of PLX affinity for this compartment (Veltkamp et al., 2007), which may decrease the PLX availability for lung tissue and s.c. tumors. Second, rIL-2 pretreatment allowed better PLX tissue penetration as compared with the PLX-alone group. The significant influence of rIL-2 on the PLX pharmacokinetics, combined with its intrinsic anticancer properties on tumor tissue in the Lewis lung carcinoma model, could represent an additional argument for therapeutic association of rIL-2 and orally administered anticancer drugs that act as efflux pump substrates.

	Acknowledgments

 
We thank Simone Orbach-Arbouys and Karine Ser-Leroux for kind help with the project. We thank Pharsight Corporation for allowing the University to benefit from the Academic Licensing Program.

	Footnotes

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

doi:10.1124/dmd.107.019091.

ABBREVIATIONS: PLX, paclitaxel; P-gp, P-glycoprotein; PSC833, 6-[(2S,4R,6E)-4-methyl-2-(methylamino)-3-oxo-6-octenoic acid]cyclosporin D; GF120918, N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide; MDR, multidrug resistance; IL, interleukin; rIL, recombinant interleukin; HPLC, high-performance liquid chromatography; MIU, Million International Unit; LLC, Lewis lung carcinoma; RP, reverse phase; IS, internal standard; LLOQ, lower limit of quantification; C_max, maximal concentration; T_max, time of the maximal concentration; AUC, area(s) under the curve; NF, nuclear factor; JAK, Janus tyrosine kinase.

Address correspondence to: Benoît Hosten, Laboratory of Pharmacology, Paul Brousse Hospital AP-HP, 14 Avenue Paul-Vaillant Couturier, 94800 Villejuif, France. E-mail: hostenb{at}yahoo.fr

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