Héloïse Ragelle

Sylvie Crauste-Manciet

Johanne Seguin

Denis Brossard

Daniel Scherman

Philippe Arnaud

Guy G Chabot
email: guy.chabot@parisdescartes.fr

Nanoemulsion formulation of fisetin improves bioavailability and antitumour activity in mice

Keywords: Fisetin, 3,3',4',7-tetrahydroxyflavone, EAhy 926, immortalized human umbilical vein endothelial cells, HLB, hydrophilic-lipophilic balance, HPLC, high performance liquid chromatography, PDI, polydispersity index Nanoemulsion, flavonoids, fisetin, pharmacokinetics, bioavailability, antitumor activity

The natural flavonoid fisetin (3,3',4',7-tetrahydroxyflavone) has shown antitumour activity but its administration is complicated by its low water solubility. Our aim was to incorporate fisetin into a nanoemulsion to improve its pharmacokinetics and therapeutic efficacy.

Solubility and emulsification tests allowed to develop an optimal nanoemulsion composed of Miglyol ® 812 N / Labrasol ® / Tween ® 80 / Lipoid E80 ® / water (10%/10%/2.5%/1.2%/76.3%). The nanoemulsion had an oil droplet diameter of 153  2 nm, a negative zeta potential (-28.4  0.6 mV) and a polydispersity index of 0.129. The nanoemulsion was stable at 4°C for 30 days, but phase separation occurred at 20°C. Pharmacokinetic studies in mice revealed that the fisetin nanoemulsion injected intravenously (13 mg/kg) showed no significant difference in systemic exposure compared to free fisetin. However, when the fisetin nanoemulsion was administered intraperitoneally, a 24-fold increase in fisetin relative bioavailability was noted, compared to free fisetin. Additionally, the antitumour activity of the fisetin nanoemulsion in Lewis lung carcinoma bearing mice occurred at lower doses (36.6 mg/kg) compared to free fisetin (223 mg/kg). In conclusion, we have developed a stable nanoemulsion of fisetin and have shown that it could improve its relative bioavailability and antitumour activity.

Introduction

Among the plant-derived compounds that have been linked to the chemoprevention and treatment of cancer, the flavonoids occupy a special place due to their abundance in human food and their relative non toxicity [START_REF] Havsteen | The biochemistry and medical significance of the flavonoids[END_REF][START_REF] Lopez-Lazaro | Flavonoids as anticancer agents: structure-activity relationship study[END_REF][START_REF] Middleton | The effects of plant flavonoids on mammalian cells: implications for inflammation, heart disease, and cancer[END_REF]Surh, 2003).

In a program aimed at finding new antiangiogenic agents in the flavonoid family, we have recently identified the natural flavonoid fisetin (3,3',4',7-tetrahydroxyflavone) as an interesting lead that can stabilize endothelial cells in vitro at non cytotoxic concentrations (Touil et al., 2009). Fisetin is found in several fruits, vegetables, nuts and wine [START_REF] Arai | Dietary intakes of flavonols, flavones and isoflavones by Japanese women and the inverse correlation between quercetin intake and plasma LDL cholesterol concentration[END_REF][START_REF] Kimira | Japanese intake of flavonoids and isoflavonoids from foods[END_REF] and displays a variety of biological effects including antioxidant, anti-inflammatory [START_REF] Park | Anti-inflammatory activity of fisetin in human mast cells (HMC-1)[END_REF]Woodman and Chan, 2004), anti-carcinogenic and in vitro anti-angiogenesis [START_REF] Fotsis | Flavonoids, dietary-derived inhibitors of cell proliferation and in vitro angiogenesis[END_REF]. Fisetin has been shown to inhibit several molecular targets, including cyclin-dependent kinases (Lu et al., 2005a;Lu et al., 2005b;Sung et al., 2007), DNA topoisomerases I and II [START_REF] Constantinou | Flavonoids as DNA topoisomerase antagonists and poisons: structure-activity relationships[END_REF][START_REF] Olaharski | Chromosomal malsegregation and micronucleus induction in vitro by the DNA topoisomerase II inhibitor fisetin[END_REF], urokinase [START_REF] Jankun | Nutraceutical inhibitors of urokinase: potential applications in prostate cancer prevention and treatment[END_REF], actin polymerization [START_REF] Böhl | Flavonoids affect actin functions in cytoplasm and nucleus[END_REF], and androgen receptor signalling [START_REF] Khan | A novel dietary flavonoid fisetin inhibits androgen receptor signaling and tumor growth in athymic nude mice[END_REF].

In vivo, fisetin has recently been shown to possess interesting anticancer activity in mice bearing lung carcinoma (Touil et al., 2011), prostate tumours [START_REF] Khan | A novel dietary flavonoid fisetin inhibits androgen receptor signaling and tumor growth in athymic nude mice[END_REF], and human embryonal carcinoma (Tripathi et al., 2011). Its in vivo mechanism of action appears rather complex and includes antiangiogenic, antiandrogenic and anti-metastatic activities [START_REF] Chien | Antimetastatic potential of fisetin involves inactivation of the PI3K/Akt and JNK signaling pathways with downregulation of MMP-2/9 expressions in prostate cancer PC-3 cells[END_REF][START_REF] Khan | A novel dietary flavonoid fisetin inhibits androgen receptor signaling and tumor growth in athymic nude mice[END_REF]Touil et al., 2011;Tripathi et al., 2011).

Despite its highly interesting properties for cancer therapy, fisetin administration in vivo remains problematic partly due to its poor water solubility (Guzzo et al., 2006;[START_REF] Mignet | Development of a liposomal formulation of the natural flavonoid fisetin[END_REF]. Fisetin bioavailability must therefore be significantly improved in order to optimize its delivery to tumours after in vivo administration. Although the design of suitable being developed for therapy, and suitable molecular carriers for flavonoids have yet to be designed and tested [START_REF] Leonarduzzi | Design and development of nanovehicle-based delivery systems for preventive or therapeutic supplementation with flavonoids[END_REF]. To do so, we therefore chose to formulate fisetin into nanoemulsion in order to hopefully achieve a better bioavailability.

Nanoemulsions represent good vehicles to formulate hydrophobic active molecules [START_REF] Sarker | Engineering of nanoemulsions for drug delivery[END_REF]. For example, nanoemulsions are widely used for parenteral administration of lipids, and have also been employed for intravenous administration of anticancer drugs like paclitaxel [START_REF] Kan | Development of nonionic surfactant/phospholipid o/w emulsion as a paclitaxel delivery system[END_REF] and chlorambucil [START_REF] Ganta | Pharmacokinetics and pharmacodynamics of chlorambucil delivered in parenteral emulsion[END_REF]. Also noteworthy, nanoemulsion has also been recently reported to contribute to the in vivo increase in efficacy of anticancer drugs, e.g., dacarbazine (Tagne et al., 2008) and camptothecin [START_REF] Han | A novel camptothecin derivative incorporated in nanocarrier induced distinguished improvement in solubility, stability and anti-tumor activity both in vitro and in vivo[END_REF].

The aim of the present study was therefore to design and characterize a nanoemulsion of fisetin that could be suitable for parenteral administration. We also evaluated the fisetin nanoemulsion pharmacokinetics after intravenous (i.v.) or intraperitoneal (i.p.) administration in mice, and determined its relative i.p. bioavailability compared to the i.p. administration of the free fisetin. Finally, the antitumour activity of the fisetin nanoemulsion was compared to the administration of free fisetin in Lewis lung carcinoma bearing mice.

A c c e p t e d M a n u s c r i p t 5

Materials and methods

Materials

Fisetin (98% purity) was purchased from Shanghai FWD Chemicals Limited (Shanghai, China). or of the highest analytical purity available.

Fisetin solubility studies

Fisetin solubility was assessed according to the approached solubility method [START_REF] Mulak | Solubilité et constante diélectrique[END_REF]. Fisetin solubility was first separately assessed in different oil phases and surfactants, and thereafter in various mixtures of oil/surfactant (Tables 123). Increasing fisetin concentrations were introduced in the various phases under agitation and heating at 60  2°C until a precipitate was observed. The solubility was determined after cooling at room temperature.

Nanoemulsion preparation

Fisetin was dissolved in several fractions in a mixture of Labrasol ® /Tween ® 80 heated at 60  2°C under sonication (Sonifier ® 450, Branson, Danbury, CT, U.S.A.). This mixture was added to the oil phase (Miglyol ® 812) in which lecithin (Lipoid E80 ® ) has previously been dispersed by heating. Aqueous and oil phases have been heated at 70  2°C thereafter.

Emulsification was accomplished by phase inversion [START_REF] Becher | Technique of emulsification[END_REF], i.e., the aqueous phase was added to the oil phase. The mixture was then submitted to a high shear mixer (Ultraturrax ® T25 , Ika, Staufen, Germany) for 10 min at 21,500 rpm (set at 5) that allowed the formation of a crude emulsion. An additional 15 min sonication in the cold of the previous emulsion using a Sonifier ® 450 set at 90% and output 3 allowed to obtain a submicron emulsion. For intravenous (i.v.) and intraperitoneal (i.p.) administration, sodium hydroxide 0.1 N was used to adjust pH to 7.0 and glycerol 2.25% was added to adjust tonicity. Two

Nanoemulsion characterization and stability studies

Nanoemulsions were visually inspected for eventual creaming, coalescence, phase separation and/or precipitation. After dilution (1/1000) in water, the mean droplet size, size distribution, the zeta potential (), and the polydispersity index (PDI) were determined using a Zetasizer Nano ZS (Malvern Instruments, Orsay, France). The PDI reflects the polydispersity of the emulsion ranging from 0 to 1, with lower values indicating a more monodispersed suspension. pH was determined using a pH meter 210 (MeterLab, Copenhagen, Denmark). A short term stability of the optimized formulations over a period of 30 days was accomplished at room temperature (+20  2°C) and at +4°C by evaluating the above mentioned parameters.

Measurements were performed in triplicate.

For the determination of the fisetin concentration, nanoemulsions were diluted

(1/2000) in methanol, vortexed, and 100 µL were injected onto a reversed-phase HPLC system (Shimadzu CLASS-VP ® , version 5.3), equipped with an octadecylsilane column (Beckman Ultrasphere ODS, 5 µm; 4.6  250 mm) thermostated at 20°C, and a UV detector set at 360 nm. The mobile phase was composed of 25% acetonitrile and 75% acidified water (2% v/v glacial acetic acid), at a flow rate of 1 mL/min. In these conditions the retention time of fisetin was 8 min. The area of the fisetin peak was reported to a calibration curve to determine the concentration of fisetin. Calibration curves were linear with correlation coefficients near unity.

Effect of fisetin nanoemulsion on endothelial cells

A c c e p t e d M a n u s c r i p t

Immortalized human endothelial cells (EAhy 926) [START_REF] Edgell | Permanent cell line expressing human factor VIII-related antigen established by hybridization[END_REF] were grown in DMEM containing 2 mM L-glutamine, 10% foetal bovine serum, 100 U/mL penicillin and 100 µg/mL streptomycin (37°C, 5% CO 2 ). Cells were plated onto 96-well plates at 5000 cells per well in 100 µL of culture medium. The fisetin-free nanoemulsion or the fisetin nanoemulsion were added to the cells for a 2 h exposure time, and the cell morphology was assessed under microscopy at a magnification of X100 (Nikon Diaphot, Nikon Corp. Japan).

Fisetin pharmacokinetics in mice 2.6.1. Mice and treatments

Female 8 weeks old C57BL/6J mice (body weight 18-22 g), were purchased from Janvier (Le Genest-St-Isle, France). After an overnight fasting period, mice were administered the various treatments as described hereafter. For the intravenous (i.v.) administration into the tail vein, 21 mice received the free fisetin formulated in 20% DMSO, 20% PEG 200 and 60% saline at a final concentration of 1.3 mg/mL (hereafter referred as "free fisetin"). The total volume injected i.v. was 200 µL for a 20 g mouse, which corresponds to a volume of DMSO of 40 µL. It should be noted that an undiluted DMSO volume of 50 µL can be administered safely i.v. to mice without toxicity (Willson et al., 1965), and that in our studies, the final DMSO dose per mouse corresponds to 40 µL (for a 20 g mouse), which was further diluted in saline and injected slowly over 1 minute. We did not encounter any acute mortality using this formulation in our studies.

A second group of 21 mice received the fisetin nanoemulsion in a final volume of 50 µL, previously sterilized by filtration through 0.22 µm filters. The fisetin dose was 13 mg/kg for the free fisetin or its nanoemulsion. Mice were sacrificed at 5, 10, 15, 30 min, 1, 2, 4 h, the A c c e p t e d M a n u s c r i p t 9 blood was obtained by cardiac puncture onto heparinized syringes, centrifuged (10,000  g, 10 min), and the harvested plasma was kept frozen at -20°C until HPLC analysis. For the intraperitoneal (i.p.) administration, the free fisetin (prepared as described above for the i.v.

route) was injected at the maximum tolerated dose by this route (223 mg/kg). The fisetin nanoemulsion dose was 112.5 mg/kg corresponding to an injected volume of 450 µL i.p.

Three mice per time point were sacrificed at 0, 0.25, 0.5, 1, 2, 4, 8, 15 and 24 h to harvest the blood by cardiac puncture (heparinized syringe). Plasma was obtained by centrifugation (10,000  g, 10 min), and frozen at -20°C until HPLC analysis. All animal experiments have been carried out in accordance with institutional and French regulations concerning the protection of animals, and with the European Commission regulations.

Determination of fisetin concentration in plasma

Fisetin concentration in plasma was determined by HPLC as followed: to 100 µL of plasma was added 60 µL of a morin methanolic solution at 0.5 mg/mL (internal standard), and 200 µL of cold acidified methanol (methanol:perchloric acid 70%, 200:1, v:v) to precipitate proteins. After vortexing for 5 min the samples were kept on ice for 15 min, and centrifuged at 10,000  g at 4°C. The supernatant (100 µL) was injected onto a reversed-phase HPLC system as described above with the UV detector set at 360 nm. The ratio of the area of the fisetin peak divided by the internal standard peak area was reported to a calibration curve to determine the concentration of fisetin. Calibration curves were linear with correlation coefficients near unity. The quantification limit of the system was 0.1 µg/mL.

Pharmacokinetic parameters determination

The following non compartmental pharmacokinetic parameters were calculated using standard methods [START_REF] Gibaldi | Noncompartmental analysis based on statistical moment theory[END_REF]: maximum concentration (C max ) extrapolated to was calculated as the MRTi.p. minus MRTi.v. [START_REF] Gibaldi | Noncompartmental analysis based on statistical moment theory[END_REF]. Relative bioavailability (F REL ) comparing the free fisetin and its nanoemulsion for the same route of administration was determined by the following formula: F REL = (AUC NE x dose FREE )/(AUC FREE x dose NE ).

Evaluation of antitumour activity in mice

Female 8 weeks old C57BL/6J mice (body weight 18-22 g) (Janvier, Le Genest-St-Isle, France) were used for antitumour evaluation. Lewis lung tumour fragments (about 2 mm diameter) were implanted subcutaneously (s.c.) bilaterally into mouse flanks using a 12 gauge trocar. Four days after tumour implantation, the mice were submitted to the following i.p.

treatments (4 mice per group) for 12 consecutive days (day 4 to 15): 4 control mice received a fisetin-free nanoemulsion; 4 mice received the fisetin nanoemulsion corresponding to 18.3 mg/kg of fisetin; and, 4 mice were injected the fisetin nanoemulsion at 36.6 mg/kg. Tumour growth was assessed using caliper bi-dimensional measurements (in mm) and the tumour volume (mm 3 ) was calculated according to the following formula: (width 2  length/2).

Statistical analysis

Data are presented as the mean  SEM. Comparison between tumour volumes was assessed by the Student t test.

A c c e p t e d M a n u s c r i p t 11

Results

Fisetin solubility in various solvents

Table 1 presents the solubility of fisetin in various solvents. Fisetin was not soluble in water (<1 mg/g) and was weakly soluble in ethanol (<14 mg/g). Fisetin was also found weakly soluble for all the oil phases tested, with a maximum solubility value < 6 mg/g for triacetin and < 5 mg/g for propylene glycol monocaprylate.

Table 2 shows fisetin solubility in frequently used surfactants. In lipophilic surfactants (low HLB value), fisetin solubility was low, whereas its solubility was markedly increased in hydrophilic surfactants with high HLB values. The best surfactant was found to be Labrasol ® which could solubilize up to 54 mg/g of fisetin.

Finally, the solubility values of fisetin using several combinations of oils and surfactants in various proportions are presented in Table 3. It was found that the mixture composed of Tween ® 80 and Labrasol ® (20/80) could solubilize fisetin most efficiently up to a maximal concentration of 45 mg/g.

Development of a fisetin nanoemulsion

Emulsification capacity

Several nanoemulsion formulations were thereafter tested for their emulsification capacity and 3 cases could be observed, as follows (Table 4): a) no emulsification was obtained for certain formulations lacking Lipoid E80 (e.g., formulations 3 to 7) or containing soybean oil and Capmul MCM, as in formulation 2; b) formation of a

Fisetin incorporation into nanoemulsion formulations

We have next evaluated the maximum quantity of fisetin that could be incorporated into each nanoemulsion (formulations 1, 8, 9), or solution (formulation 10). Table 4 shows that formulation 1 could incorporate only 1 mg/g of fisetin, whereas formulations 8 and 9 allowed the incorporation of 5 mg/g of the flavonoid. These formulations were found visually stable on day 1. Although formulation 10 allowed to solubilize up to 16 mg/g of fisetin due to its higher content in Labrasol ® and Tween ® 80, it was unfortunately found to precipitate on day 1 for concentrations of 8 and 16 mg/g, but was found stable for a fisetin concentration of 4 mg/g.

Choice of final fisetin nanoemulsion formulation

Considering the above results, formulations 8 and 9 were therefore considered a good compromise between fisetin content and nanoemulsion stability. These formulations were further tested for their particle diameter and polydispersity index (PDI) and it was observed that preparation 8 containing soybean oil yielded a nanoparticle diameter of 323  2 nm with a PDI of 0.153, whereas preparation 9 containing Miglyol 812 N showed a nanoparticles diameter of 146  3 nm and a PDI of 0.015. Formulation 9 was therefore chosen for further in vitro and in vivo testing because of its good fisetin content, its nanoparticle size and low PDI value. The final composition of nanoemulsion 9 containing 5 mg/ml of fisetin was therefore as follows: Miglyol 812 N (10%), Labrasol ® (10%), Tween ® 80 (2.5%), Lipoid E80 ®

(1.2%), glycerol (2.25%), NaOH (0.1N) to adjust to pH 7, water to 100%.

A c c e p t e d M a n u s c r i p t 13

Stability of the fisetin nanoemulsion

We next performed short term stability studies of the fisetin nanoemulsion (preparation 9) over a period of 30 days at room temperature (20°C) and at 4°C (Table 5) by evaluating particle diameter, pH, zeta potential and the PDI.

Droplet size of nanoemulsion 9 stored at 20°C increased markedly as a function of time until phase separation occurred on day 30, whereas for the 4°C storage condition, particle diameter remained relatively stable for the 30 day examination period. A slight decrease in pH was noted at both storage temperatures. For the zeta potential, negative values were observed and remained stable over 30 days for the 4°C storage condition. The PDI presented an important increase over time at 20°C, whereas it was found stable and relatively low at 4°C. In order to be administered via parenteral routes, we also checked if the nanoemulsion could sustain standard steam sterilization conditions (121°C for 15 min) but this resulted in phase separation. However, sterilization of nanoemulsion 9 has been successfully carried out using a 0.22 µm filter with preservation of homogeneity and size characteristics.

Effect of fisetin nanoemulsion on endothelial cells

Free fisetin has been reported to exert a distinct morphological effect on endothelial cells that is characterized by the rapid development of pseudopods at non cytotoxic concentrations (Touil et al., 2009). We therefore tested if the fisetin nanoemulsion (preparation 9) could exert the same morphological effects on Eahy 926 endothelial cells to verify if the active principle is indeed released from the pharmaceutical preparation. as the free fisetin on endothelial cells.

Fisetin nanoemulsion pharmacokinetics in mice

The developed fisetin nanoemulsion (preparation 9) was next administered in vivo to evaluate its pharmacokinetics in mice. We first examined the intravenous route (i.v.) by injecting the free fisetin formulation or its nanoemulsion at a dose of 13 mg/kg. We noted a very similar pharmacokinetic profile between the two formulations with plasma concentrations versus time curves almost superimposable (Figure 2). Indeed, similar pharmacokinetic parameters in terms of Cmax, AUC and terminal half-life were observed for both formulations (Table 6). The i.v. route administration of the fisetin nanoemulsion was however found relatively toxic, because we noted a mortality rate of 3/21 mice (14%), apparently due to the rapid administration.

In order to avoid the acute toxicity of the i.v. administration, we next explored the intraperitoneal (i.p.) route. For the fisetin nanoemulsion, a comparison of its pharmacokinetics after i.p. administration with the free fisetin injected by the same route is presented in Figure 3. Compared to the i.p. administration of the free fisetin, it can be seen that the injection of the fisetin nanoemulsion led to a significant increase in fisetin plasma concentrations, even at a fisetin nanoemulsion dose (112.5 mg/kg) half that of the free fisetin dose (223 mg/kg). The pharmacokinetic parameters presented in Table 6 indicate that not only the maximum plasma A c c e p t e d M a n u s c r i p t 15 concentrations reached were higher for the nanoemulsion, but the relative bioavailability was 24-fold higher compared to the free fisetin. This increase in bioavailability with the nanoemulsion appears to be due to a faster absorption with this drug formulation as shown by a shorter mean absorption time (MAT) of 1.97 h compared to 5.98 h for the free fisetin. It is also noteworthy that following the i.p. administration of the fisetin nanoemulsion, no mortality was observed.

Fisetin nanoemulsion antitumour activity in mice

We were next interested to evaluate the antitumour activity of the developed fisetin nanoemulsion (preparation 9) in Lewis lung carcinoma bearing mice. A group of 4 control mice received the empty nanoemulsion and 2 other groups of 4 mice received the fisetin nanoemulsion at an equivalent dose of fisetin of 18.3 and 36.6 mg/kg administered i.p. for 12 days starting on day 4 post tumour implantation. Results depicted in Figure 4 show that the fisetin nanoemulsion can significantly inhibit tumour growth in a dose-dependent manner, even at these relatively low dose levels of fisetin.

A c c e p t e d M a n u s c r i p t

Discussion

The main objectives of this study was to develop a nanoemulsion of the hydrophobic flavonoid fisetin, determine its pharmacokinetics in mice, and evaluate its anticancer activity in vivo. The first problems encountered were the low fisetin solubility in the classical oil phases usually employed in formulation, e.g., soybean oil or medium chain triglycerides [START_REF] Date | Parenteral microemulsions: an overview[END_REF]. The best solubility was observed with triacetin, a short chain triglyceride composed of triester of glycerol and acetic acid. However, triacetin does not exhibit remarkable emulsifying properties [START_REF] Poullain-Termeau | Effect of oil-in-water submicron emulsion surface charge on oral absorption of a poorly water-soluble drug in rats[END_REF]. Fisetin was also found weakly soluble in lipophilic surfactants, but was more soluble in hydrophilic surfactants. As a matter of fact, the maximum solubility was observed with Labrasol ® , a mixture of triglycerides and polyethylene glycol esters possessing a hydrophilic-lipophilic balance of 14. We did not observe any synergistic effect between the oil phase and surfactant, be it hydrophilic or lipophilic. Hence, the fisetin low solubility in lipid phases leads to a weak association of the active principle in this phase and therefore requires more solubilising surfactant, as previously reported for a nanoemulsion of carbamazepine (Kelmann et al., 2007). We finally found that the best mixture was the one composed of Labrasol ® /Tween ® 80 which allowed to achieve an acceptable fisetin concentration of 5 mg/ml.

Although Labrasol ® has already been employed in injectable preparations [START_REF] Nornoo | Cremophor-free intravenous microemulsions for paclitaxel I: formulation, cytotoxicity and hemolysis[END_REF], no standard preparation has been developed so far, to our knowledge, with this proprietary formulation. Labrasol ® is mostly used in auto-emulsifying systems for oral administration [START_REF] Kommuru | Self-emulsifying drug delivery systems (SEDDS) of coenzyme Q10: formulation development and bioavailability assessment[END_REF] and it has been shown to increase oral absorption of hydrophilic drugs, e.g., gentamicin [START_REF] Hu | A novel emulsifier, labrasol, enhances gastrointestinal absorption of gentamicin[END_REF]. Available toxicity data on Labrasol ®

show that it is relatively non toxic when given orally to rats with a LD50 of 22 g/kg [START_REF] Gad | Nonclinical vehicle use in studies by multiple routes in multiple species[END_REF]. However, acute toxicity data are not available for the intravenous or intraperitoneal In our experiments, a mortality rate of 14% was noted for the i.v. route, whereas no mortality was observed for the i.p. route. The other components of the nanoemulsion are not likely to contribute to acute toxicity because medium chain triglycerides (e.g., Miglyol 812 N) and lecithins are already widely used in injectable preparations. Tween ® 80 can also be ruled out in this toxicity because it is frequently used at high concentrations, e.g., 25% in docetaxel preparation without apparent toxicity [START_REF] Strickley | Solubilizing excipients in oral and injectable formulations[END_REF]. Therefore, a compromise will have to be found between the toxicity of the excipient and the final concentration in fisetin.

With regard to the physico-chemical properties of the fisetin nanoemulsion, it was found that the preparation containing Miglyol 812 N (No. 9) showed acceptable fisetin content, nanoparticle size and PDI. This fisetin nanoemulsion was found to be unstable at room temperature with increasing diameter and PDI values over time, with phase separation occurring on day 30. This relatively slow process could be explained by the Ostwald ripening in which larger particles grow at the expense of smaller ones due to the higher solubility of the smaller particles and to molecular diffusion through the continuous phase [START_REF] Capek | Degradation of kinetically-stable o/w emulsions[END_REF].

This phenomenon finally ends up in the coalescence of the emulsion (Tadros et al., 2004).

Because this phenomenon is temperature sensitive, it was noted that nanoemulsions kept at 4°C were remarkably stable with almost unchanged particle diameter and PDI over the 30 day examination period. PDI smaller that 0.250 are considered acceptable for parenteral preparations [START_REF] Müller | SolEmulsnovel technology for the formulation of i.v. emulsions with poorly soluble drugs[END_REF]). However, it was also noted, as expected, that the nanoemulsion was particularly unstable in steam sterilization conditions (121°C, 15 min) probably due to the non-ionic surface active agents which are not stable at high temperatures [START_REF] Nornoo | Cremophor-free intravenous microemulsions for paclitaxel I: formulation, cytotoxicity and hemolysis[END_REF]. To overcome this problem, sterilization by filtration could therefore be employed for nanoemulsions [START_REF] Floyd | Top ten considerations in the development of parenteral emulsions[END_REF]. The formulated fisetin nanoemulsion presented a We have observed that fisetin nanoemulsion could exert its distinct morphological effects on endothelial cells similar to the free fisetin (Touil et al., 2009) indicating that fisetin could be released from its nanoemulsion formulation. These morphological changes on endothelial cells are attributed to a stabilization of the cytoskeleton as previously shown by increased acetylated alpha tubulin (Touil et al., 2009).

Concerning the i.v. administration of the free fisetin and its nanoemulsion, we did not observe any pharmacokinetic difference, as expected for a classical emulsion, contrary to what is observed for a pegylated formulation which can increase the residence time [START_REF] Reddy | Pharmacokinetics and tissue distribution of etoposide delivered in long circulating parenteral emulsion[END_REF]. As a matter of fact, upon injection of a classical emulsion, the particles interact with the apolipoproteins and are captured by the reticulo-endothelial system leading to their rapid elimination from the blood compartment [START_REF] Kawakami | Disposition characteristics of emulsions and incorporated drugs after systemic or local injection[END_REF]. One possibility to increase the residence time using the i.v. administration would be to pegylate the emulsion that could increase the surface hydrophilic properties by forming a steric barrier that could result in a longer retention time in plasma (Tamilvanan, 2004).

We demonstrated that the i.p. administration of the fisetin nanoemulsion led to a significant improvement in bioavailability compared to the i.p. administration of the free fisetin, with a 24-fold increase in relative bioavailability compared to the free fisetin administered via the same route. This could be due to a faster absorption phase from the peritoneal cavity with the nanoemulsion compared to free fisetin. As a matter of fact, the mean absorption time was shorter with the nanoemulsion (2 h) compared to the free fisetin (6 h). Similar results were reported for the hydrophobic taxoid paclitaxel where i.p.

administration was leading to a significant improvement in bioavailability compared to the i.v. administration [START_REF] Soma | Different tissue distribution of paclitaxel with intravenous and intraperitoneal administration[END_REF]. In addition, this enhanced bioavailability of the fisetin administration which is a favourable property, especially with anticancer drugs that must access lymph nodes which are frequently harbouring metastases [START_REF] Nishioka | Lymphatic targeting with nanoparticulate system[END_REF].

We also demonstrated that the fisetin nanoemulsion could elicit a significant antitumour activity in vivo in Lewis lung tumour bearing mice. It is noteworthy that a relatively low dose of the fisetin nanoemulsion corresponding to 36.6 mg/kg of fisetin was able to reduce by 53% the tumour volume, whereas a 6-fold higher dose (223 mg/kg) was required to obtain a similar tumour growth inhibition with the free fisetin, as recently reported by Touil et al. (Touil et al., 2011). This indicates that the nanoemulsion of fisetin is favourable to its anticancer action in vivo probably by increasing its bioavailability, as shown in this study.

Conclusion

In conclusion, we have developed a nanoemulsion of fisetin that allowed to solubilize a relatively high concentration of fisetin (5 mg/mL), thanks to the use of two surface active agents, i.e., Tween ® 80 and Labrasol ® . However the latter agent appeared to be relatively toxic when using the i.v. route, but was not found toxic by the i.p. route. The developed fisetin nanoemulsion could also markedly increase the bioavailability of fisetin after i.p.

administration and was also found to significantly improve its antitumoral activity in tumour bearing mice compared to the free fisetin. The developed nanoemulsion of fisetin could therefore advantageously be employed to improve the antiangiogenic and anticancer activities of this flavonoid, as well as other flavonoids sharing the same problems of in vivo administration.

  flavonoids is an area of intense research, solutions are still far from

  were investigated: steam sterilization using 120°C, 15 minutes cycle and filtration through a 0.22 µm filter.

  the i.v. route; area under the plasma concentration versus time curve from time zero to the time of the last measurable concentration (AUC 0-t ) calculated by the trapezoidal method; terminal half-life = ln 2/K el , where K el is the terminal elimination rate constant. The mean residence time (MRT) was calculated as the AUMC/AUC, where AUMC is the area under the first moment curve. Clearance was calculated as the dose/AUC, and the volume of distribution Vss as the CLMRT. The mean absorption time (MAT) after i.p. administration

  Figure 1-A depicts control endothelial cells exposed to 1% DMSO which show typical cobblestone exposure to free fisetin led to the expected pseudopods formation (Figure 1-B). Endothelial cells exposed to control nanoemulsion without fisetin resemble normal control endothelial cells (Figure 1-C), whereas the pseudopods are indeed observed in the cells exposed to the fisetin nanoemulsion (Figure 1-D). This observation indicates that fisetin is indeed released from the nanoemulsion and can exert similar morphological effects

  which is probably due to the anionic fractions of the employed lecithin(Wang et al., 2006).

  probably resulting from the unique lymphatic distribution after i.p.

  ., Pandey, M.K.,Aggarwal, B.B., 2007. Fisetin, an inhibitor of cyclin-dependent kinase 6, down-regulates nuclear factor-kappaB-regulated cell proliferation, antiapoptotic and metastatic gene products through the suppression of TAK-1 and receptor-interacting proteinregulated IkappaBalpha kinase activation.Mol. Pharmacol., 71, 1703-1714. Surh, Y.J., 2003. Cancer chemoprevention with dietary phytochemicals. Nat. Rev. Cancer, 3, 768-780.

Figure 1 .

 1 Figure captions

Figure 2 .

 2 Figure 2. Fisetin pharmacokinetics in mice after intravenous administration of free fisetin

Figure 3 .

 3 Figure 3. Fisetin pharmacokinetics in mice after intraperitoneal administration of free fisetin

Figure 4 .

 4 Figure 4. Antitumour activity of the nanoemulsion of fisetin. Lewis carcinoma bearing mice

  Figure(s)

  The various purified oil phases were provided by the following companies: soybean oil (Société Industrielle des Oléagineux, Saint Laurent Blangy, France);

	carthame oil, ethyl oleate and n-capric acid (Sigma-Aldrich, Saint Quentin Fallavier, France);
	Miglyol ® 812 N and Imwitor

® 742 (Sasol Witten, Germany); Captex ® 355 and Capmul ® PG-8 (Abitec, Janesville, WI, USA); Labrafac lipophile WL 1349 ® (Gattefossé, Saint Priest, France); triacetin (VWR Fontenay-sous-Bois, France).

The surfactants were purchased from the following companies: egg lecithin containing 82.3% phosphatidylcholine (Lipoid E80 ® , Lipoid GmbH Ludwigshafen, Germany); polysorbate 80 (Tween 80), sorbitan trioleate (Span 85 ® ), polyoxyethylene glycol 2000 monostearate (Myrj ® 52) (Uniquema, Everberg, Belgium); polyoxyethylenated ricin oil (Cremophor EL ® , BASF, Ludwigshafen, Germany) ; vitamin E TPGS (Eastman Chemical B.V., Paris, France); a mixture of glycerides and esters of PEG-8 (Labrasol ® ), a mixture of glycerides and esters of PEG 300 (Labrafil M 1944 CS ® ) (Gattefossé Saint Priest, France); glycerol monocaprylocaprate (Capmul MCM ® , Abitec, Janesville, WI, USA). Glycerol was purchased from Labosi (Paris, France). Sterile water for injection was from Fresenius-Kabi (Sèvres, France). Sodium hydroxide 0.1 N was from Carlo Erba Reactif SDS (Peypin, France).

The other chemicals used for drug dissolution, plasma preparation and HPLC analysis were the following: methanol, acetonitrile, perchloric acid (Carlo Erba Reactif SDS, Peypin, France);

DMSO, PEG 200, morin, phosphate buffer (pH 7.4

) and mouse serum (Sigma-

Table 1 . Fisetin solubility in various solvents.

 1 Tadros, T.,Izquierdo, P., Esquena, J., Solans, C., 2004. Formation and stability of nanoemulsions. Adv. Colloid Interface Sci., 108-109, 303-318.

	Mol. Pharm., 5, 1055-1063.
	Tamilvanan, S., 2004. Oil-in-water lipid emulsions: implications for parenteral and ocular
	delivering systems. Prog. Lipid Res., 43, 489-533.
	Touil, Y.S., Fellous, A., Scherman, D., Chabot, G.G., 2009. Flavonoid-induced
	morphological modifications of endothelial cells through microtubule stabilization. Nutr.
	Cancer, 61, 310-321.
	Touil, Y.S., Seguin, J., Scherman, D., Chabot, G.G., 2011. Improved antiangiogenic and
	antitumour activity of the combination of the natural flavonoid fisetin and cyclophosphamide
	in Lewis lung carcinoma-bearing mice. Cancer Chemother. Pharmacol., 68, 445-455.

Tagne, J.B., Kakumanu, S., Nicolosi, R.J., 2008. Nanoemulsion preparations of the anticancer drug dacarbazine significantly increase its efficacy in a xenograft mouse melanoma model.

Table 2 . Fisetin solubility in various surfactants.

 2

	Surfactants

a HLB, hydrophilic-lipophilic balance (a value <10 indicates a majority of lipophilic fractions and a value >10 indicates a majority of hydrophilic fractions).

Table 3 . Fisetin solubility in various mixtures. Mixtures (in percent)

 3 ND, not determined because the phases were not miscible in these proportions.

	Solubility
	mg/g

a

Table 6 . Fisetin pharmacokinetic parameters after intravenous or intraperitoneal administration of free fisetin and fisetin nanoemulsion in mice (preparation 9).

 6 F rel = (AUC NE x dose FREE )/(AUC FREE x dose NE )

	Intravenous	Intraperitoneal

a

Acknowledgements

This research was supported by the Institut National de la Santé et de la Recherche Médicale (INSERM), by the Centre National de la Recherche Scientifique (CNRS) and by a grant from the Institut National du Cancer (INCa, Boulogne-Billancourt, France).

 Cancer Ther.,10,[255][256][257][258][259][260][261][262][263][264][265][266][267][268]Wang, J.J., Sung, K.C., Hu, O.Y., Yeh, C.H., Fang, J.Y., 2006. Submicron lipid emulsion as a drug delivery system for nalbuphine and its prodrugs. J. Control. Release,115,


Chiraz Trabelsi

Samy Meftali

Jean-Luc Dekeyser

Distributed control for reconfigurable FPGA systems: a high-level design approach

Keywords: Distributed control, high-level modeling, UML MARTE, reconfiguration control, partial dynamic reconfiguration, FPGA

Due to their exponential complexity, designing adaptation control for Reconfigurable Systems-on-Chip (RSoC) is becoming one of the most challenging tasks, resulting in longer design cycles and increased time-to-market. This paper addresses this issue and proposes a novel adaptation control design approach for FPGA-based reconfigurable systems aiming to increase design productivity. This approach combines control distribution and high-level modeling in order to decrease design complexity and enhance design reuse and scalability. Control distribution is based on allocating local control aspects (monitoring, decision and reconfiguration) to distributed controllers, while respecting global system constraints/objectives using a coordinator. High-level modeling makes use of Model-Driven Engineering and the MARTE (Modeling and Analysis of Real-Time and Embedded Systems) standard in order to move from high level models to automatic code generation, which significantly simplifies the control design. The proposed design approach is integrated in a model-driven RSoC design flow and allows to model adaptation aspects at different design levels: application, architecture, allocation and deployment, which allows to target a wide range of control requirements. In order to validate our approach, a video processing application was implemented on a reconfigurable system that contained four distributed hardware controllers.

I. INTRODUCTION

Being able to be reconfigured an arbitrary number of times, Reconfigurable Systems-on-Chip (RSoC) are becoming widely used thanks to their flexibility and adaptivity. These systems offer the possibility of being modified after fabrication in order to adapt to changes such as user preferences, application requirements and standard changes. Dynamic reconfiguration has the potential of offering a run-time system modification by replacing reconfiguration data (bitstream). Partial Dynamic Reconfiguration (PDR) is a very powerful mechanism supported by some modern FPGAs [START_REF] Lysaght | Invited paper: Enhanced architectures, design methodologies and cad tools for dynamic reconfiguration of xilinx fpgas[END_REF]. Supporting PDR provides a high platform flexibility allowing to reconfigure only some regions of the FPGA (loading partial bitstreams) without disturbing the operation of the rest of the system. Such a mechanism allows to reduce the total system area while meeting performance constraints. However, adaptivity and reconfigurability implies an additional task for SoC designers, which is adaptation control design whose complexity is increasing with the size of the applications that modern RSoC are able to embed. In this context, designing one controller to handle the adaptation of the whole system is becoming a complex and time-consuming task. Moreover, the rigidity of such a design, due to its independence to the implemented system, represents an obstacle to design reuse. Therefore, increasing autonomy in control design can be viewed as an effective solution to deal with the growing complexity of the reconfigurable systems design [START_REF] Mainsah | Autonomic computing: the next era of computing[END_REF]. Such an autonomy allows to divide the control problem between autonomous controllers having each a local vision of the system, which allows to decrease their design complexity.

Another solution to decrease design complexity is to elevate the low-level technical details using Model-Driven Engineering (MDE) [START_REF] Omg | Portal of the model driven engineering community[END_REF]. MDE is a high-level design approach that is suitable for SoC co-design, allowing to model both software and hardware parts. In order to use the MDE for a high level description of a system in a specific domain such as embedded systems, UML (Unified Modeling Language) [4] profiles are used. A UML profile is a set of stereotypes that add specific information to a UML model in order to describe a system related to a specific domain. Several UML profiles target embedded systems design such as the Modeling and Analysis of Real-Time and Embedded systems (MARTE) [START_REF]UML Profile for MARTE: Modeling and Analysis of Real-Time Embedded Systems[END_REF] profile, a standard profile promoted by the Object Management Group (OMG).

In a previous work [START_REF] Trabelsi | Semi-distributed control for fpga-based reconfigurable systems[END_REF], we proposed a distributed control model for reconfigurable FPGA-based systems. This model is based on autonomous modular controllers and formalismoriented design in order to decrease design complexity and facilitate design reuse and scalability. In this paper, we enhance this work by a high-level modeling approach. This approach allows designers to target dynamic reconfiguration without being expert of modern FPGAs, which simplifies the control design and decreases its complexity. We also integrate this high-level control design into a model-based SoC design framework in order to generate automatically controlled reconfigurable FPGA-based systems, which allows to reduce time-to-market and to enhance design productivity. The rest of the paper is as follows. Section 2 gives a summary of the related works on high-level modeling. Section 3 gives an overview of the proposed control distribution. Section 4 presents the high-level modeling approach through a video processing application case study. Section 5 gives experimental results. The last section presents a conclusion and future works. High-level modeling for reconfigurable systems was proposed in several works. In [START_REF] Vidal | Uml design for dynamically reconfigurable multiprocessor embedded systems[END_REF], the authors present a codesign methodology for reconfigurable MPSoC (Multiprocessor SoC), in order to generate automatically reconfigurable embedded systems using the MARTE UML profile. The proposed solution for control modeling is extremely simplistic, based on a UML state machine handling a repeated sequence of configurations for one reconfigurable co-processor. This solution is not suitable for more complex reconfiguration requirements. Besides, reconfiguration aspects were only handled at the allocation level. In [START_REF] Quadri | Integrating mode automata control models in soc co-design for dynamically reconfigurable fpgas[END_REF], the authors propose a MARTEoriented design approach of dynamic reconfigurable FPGA (Field Programmable Gate Arrays) -based RSoC using mode automata control for the Gaspard2 SoC design framework [START_REF] Labbani | Introducing control in the gaspard2 data-parallel metamodel: Synchronous approach[END_REF]. However, the proposed approach is based on a centralized reconfiguration controller for the whole FPGA, which makes it not suitable for large complex systems. In [START_REF] Quadri | Modeling of configurations for embedded system implementations in marte[END_REF], the authors present a design approach for dynamic reconfigurable FPGAbased RSoC. This approach is based on the MARTE concepts for reconfiguration control such as mode and configuration concepts. However, it has the limit of remodeling the whole system for each possible configuration. There are also several other model-driven approaches for RSoC control that are not MARTE-oriented [START_REF] Pillement | High-level model of dynamically reconfigurable architectures[END_REF] [START_REF] Gumzej | A reconfiguration pattern for distributed embedded systems[END_REF]. Compared to the mentioned works, our approach allows to handle adaptation control design at different design levels (application, architecture, allocation and deployment), which offers a high design flexibility and allows to cover a wide range of control requirements.

III. THE CONTROL DISTRIBUTION APPROACH

The considered reconfigurable systems in this paper are those supporting partial reconfiguration. Figure 1 shows an overview of the targeted architecture. It is mainly composed of a processor, a number of reconfigurable hardware units (RUs), memories, and I/Os. Application tasks are thus composed of software tasks executed by the processor, and hardware tasks executed by the RUs. In order to decrease the control design complexity of such systems, we propose to divide control concerns between distributed controllers in order to decrease their design complexity and facilitates their verification and reuse, resulting in a higher design productivity. Adaptation control is distributed as follows: 1) a software controller (SW-C) handling the software tasks adaptation, 2) a number of hardware controllers (HW-Cs) handling RUs adaptations. Each RU is composed thus of a partial reconfigurable region (PRR), handling a hardware task, and a HW-C controlling it. Software and hardware controllers are coordinated using a coordinator, in order to guarantee that the system configuration respects global constraints/objectives.

As shows Figure 1, each HW-C contains three modules handling monitoring, decision and reconfiguration. Monitored data (monitoring flow in Figure 1) can be related to the internal status of PRRs or detected from external sources (other monitored components in Figure 1), such as battery sensors, user inputs, etc. Monitoring modules detect, from monitored data, information and events that will be used by decision modules for decision-making. Each decision module makes local decisions about whether or not a reconfiguration of the controlled region is required. Due to the local vision of each HW-C, launching a reconfiguration of its controlled region without checking whether it can coexist with the current configurations of the other regions might result in problems such as safety problems or might not respect the control global constraints such as those related to performance, temperature, energy consumption, etc. Therefore, before launching a reconfiguration that it estimates required according to the monitoring data, the Hw-C has to send a reconfiguration request to the coordinator (decision flow).

The role of the coordinator is to check whether the global system constraints allow to launch the requested reconfiguration without adapting other regions. If so, the coordinator authorizes the reconfiguration. Otherwise, it sends reconfiguration suggestions to the HW-Cs of the involved regions. According to the responses of the HW-Cs (acceptance or refusal), the coordinator gives its decision about the requested reconfiguration. This decision can be either an authorization or a refusal of the reconfiguration. More details about the used coordination mechanism can be found in [START_REF] Trabelsi | Semi-distributed control for fpga-based reconfigurable systems[END_REF]. In case of authorization, the coordinator notifies the HW-Cs. Their Fig. 2: Adaptation control integration to the Gaspard2 framework reconfiguration modules launch then reconfiguration through reconfiguration controllers (RCs) in order to load the required partial bitstreams in a parallel way (parallel reconfiguration flow), which reduces reconfiguration time. For a Xilinx platform, these RCs correspond to ICAP controllers. The coordinator notifies also the SW-C so that adapts the application software tasks to the new system configuration.

In order to adapt to technical limitations of current FPGAs, which mostly contain one configuration port (ICAP) and do not allow thus parallel reconfiguration, our control design approach proposes a second reconfiguration flow. In case of a reconfiguration authorization from the coordinator, each reconfiguration module updates a dedicated register indicating which mode is to be loaded in the controlled region. These registers are then read by the SW-C, which communicates with the ICAP port in order to load the required bitstreams in the reconfigurable regions. Then, the SW-C notifies the coordinator and the HW-Cs so that they update their "current configuration" registers. Taking into account the new system configuration, the SW-C updates application software tasks before sending data to reconfigurable regions. This control flow is represented by the sequential reconfiguration flow in Figure 1.

IV. THE HIGH-LEVEL MODELING APPROACH

Gaspard2 [START_REF] Gamatie | A model driven design framework for massively parallel embedded systems[END_REF] is an MDE oriented SoC co-design framework based on the Modeling and Analysis for Real Time and Embedded systems (MARTE) standard. Our contribution in this framework is to integrate reconfiguration aspects in the design flow, by integrating adaptivity aspects at different design levels: application, architecture, allocation and deployment, as shows Figure 2. This is an enhancement to previous control modeling approaches in the Gaspard2 framework [START_REF] Quadri | Integrating mode automata control models in soc co-design for dynamically reconfigurable fpgas[END_REF] [START_REF] Guillet | Modeling and formal control of partial dynamic reconfiguration[END_REF], which targeted centralized control and limit control modeling at one design level. The design flow in Gaspard2 follows several steps: 1)system modeling and deployment, 2)model transformations, 3)code generation, and 4)physical implementation, as shows Figure 2. More details about these steps can be found in [START_REF] Gamatie | A model driven design framework for massively parallel embedded systems[END_REF]. Gaspard2 allows code generation targeting different languages such as Fortran, SystemC, OpenCL, C, VHDL, etc. In Figure 2, only the used languages in this paper are mentioned (C and VHDL). The generation tool generates also platform (FPGA)-dependent files. The generated code can then be integrated into FPGA tools for synthesis, PAR and bitstream generation. It can also be integrated into the IP library in order to be reused in other system designs at the deployment level.

The case study concerned in this paper deals with video scaling, which is considerably important for previews or for streaming for small form factor devices, such as mobile phones. The application is a classical downscaler, which transforms a video signal, which is expressed in Common Intermediate Format (CIF:352x288 pixels), into a smaller size video (132x128 pixels). This application is composed of two main tasks: a horizontal filter and a vertical filter. By applying the horizontal filter on the input frame, its size is reduced to 132x288 pixels. Then, by applying the vertical filter, we obtain the target size (132x128 pixels). Each filter is composed of a repetition of an elementary task. At each iteration of this task, a frame block is treated. In order to guarantee a high performance of the application, both filters are implemented in hardware using hardware accelerators. Each accelerator performs an elementary task of one of the filters. Using different parallelism degrees, each filter can be implemented by more than one accelerator. The input block sizes of the accelerators can also be varied in order to obtain different performance and power results. Indeed, using a bigger input block size for an accelerator reduces the execution time thanks to a higher hardware parallelism, at the cost of a higher resource overhead and thus a higher power consumption.

In our case study, the objective of the control is to adapt the downscaler application to changes in performance and power requirements. Each accelerator is implemented in a reconfigurable region using three different versions, varying the size of the input blocks. In this case study we consider the following block sizes: 35, 19 and 11 pixels for the horizontal filter, and 41, 23 and 14 pixels for the vertical filter. Here, for both filters, the first version corresponds to the highest performance but at the same time the highest power consumption. In order to take performance and consumption requirements into account at runtime, each HW-C monitors two elements. The first element is the required performance level, which is a user input. The second is the battery level sent by a battery sensor.

In the rest of this section, we detail our approach to integrate adaptation control modeling in the Gaspard2 framework, the application, architecture, allocation and deployment levels.

A. Application modeling

An application is composed of hardware and software tasks. Each task is modeled by a component. Inputs and outputs of a task are represented by ports having the FlowPort stereotype of MARTE. More details about application modeling in Gas-pard2 can be found in [START_REF] Gamatie | A model driven design framework for massively parallel embedded systems[END_REF]. In order to integrate adaptation aspects, each reconfigurable task is modeled by a number of components, representing each a version of the related task. In our case study, a hardware task corresponds to an elementary filter task executed by a PRR. Three versions (HwHFilter i /HwV Filter i , i ∈ [1..3]) are possible for both filters, as we said previously. These versions have different input and output block sizes. Software tasks are divided to a number of static tasks and a reconfigurable task. Static tasks perform frame reading and building before and after the downscaler execution, respectively. The reconfigurable task is the task that sends input frame blocks to RUs and retrieves output blocks afterwards. The configuration of this task depends thus on the current configurations of the RUs whose inputs/output block sizes is different from a configuration to another. As we said previously, the coordinator guarantee that the system configuration respects global system constraints. In this case study, we assume that these constraints require that all the RUs implement the same version number. In this case, the reconfigurable software task, has three versions

Downscaler i , i ∈ [1..3],
where each Downscaler i communicates with RUs implementing HwHFilter i and HwV Filter i .

B. Architecture modeling

The architecture model contains the hardware system implemented on FPGA, on which the application will be running.

More details about architecture models in Gaspard2 can be found in [START_REF] Gamatie | A model driven design framework for massively parallel embedded systems[END_REF]. Here, we focus on the adaptation control integration at the architecture level. For this, we integrate RUs modeling at this level. For our case study, we choose to implement the application with two RUs for horizontal filter and two for the vertical filter. The architecture follows the concepts presented in Figure 1. The reconfigurable part of the system corresponds to 4 PRRs. Each PRR is controlled by a HW-C forming thus 4 RUs. HW-Cs are connected to a coordinator. The inputs of the HW-Cs are the battery level sent by the battery sensor, the bus signals, and the coordination information. In order to offer the possibility for the user to introduce the required performance level, the system contains push buttons and a interruption controller to handle the user commands as interruptions. The system contains a SysACE controller for accessing the partial bitstreams placed in a Compact Flash, an ICAP controller to carry out partial reconfiguration using the read-modify-write mechanism, and the DDR3 to store the input and downscaled frames. The compact Flash will also be used to store the images to be downscaled. This can be replaced by a camera connected to the FPGA in order to downscale its input video. The system contains also a TFT (Thin Film Transistor) controller in order to display images before and after downscaling, and a UART allowing the user to follow the application and the control progress.

Figure 3 shows the structure of one of the RUs used for the horizontal filter. The rest of the RUs follow the same concept. Each HW-C is composed of four components: monitoring, decision-making, reconfiguration and interface. The interface component allows the communication of the processor with In order to model the behavior of the HW-C, we associate to its decision component a mode-automaton, where each mode corresponds to a given configuration/mode of the controlled RU. Each mode represents thus a version of the hardware tasks mentioned previously. The mode-automaton is modeled using the MARTE stereotypes. Inspired from reactive mode automata formalism [START_REF] Maraninchi | Mode-automata: a new domain-specific construct for the development of safe critical systems[END_REF], a mode in MARTE is an extension of the UML state, and mode behavior is an extension of the UML state machine. The mode-automaton has the modeBahavior stereotype, and each mode has the mode stereotype. Transitions have the modeTransition stereotype, and give the events triggering the transition and the activity that has to be carried out to move from a mode to another. Provided that all reconfigurable regions have three configuration possibilities, each Hw-C uses a decision module modeled by a three-mode automaton. In order to simplify the modeling of the Hw-C decisions, we added extensions to the MARTE profile. These extensions allow to facilitate the modeling the decision module activities (requests, suggestion acceptance and suggestion refusal), by making them implicit in transitions. Figure 4 shows the mode-automaton of the HW-C modeled in Figure 3. Three stereotypes are added and are applied to transitions as extensions of the ModeTransition stereotype. The Transition-Request stereotype indicates that the decision module sends a request to the coordinator asking for a reconfiguration to the target mode if the transition condition is valid. Likewise, TransitionAcceptance and TransitionRefusal stereotypes indicate that the decision module accepts or refuses the received reconfiguration suggestion to the target mode. Although these stereotypes are applied to transitions, at the implementation level (the decision mode-automaton to be generated), they are not actual transitions to the target mode since, in the targeted control implementation, the actual transition is only done after the loading of the corresponding configuration (partial bitstream). However, using these extensions allows to hide many implementation details (which are handled automatically by the model transformation and code generation tools) for designers. This simplifies significantly the control design. The designer has only to mention the transition conditions, since the transition activities are implicit (request, acceptance and refusal). In our case study, the decision making process of each decision module is mainly based on the following rules, which are represented by the transition conditions in Figure 4:

• Being at H/V Filter mode j1 , a controller decides that a reconfiguration to a less consuming mode H/V Filter mode j2 is required, only if the user requires a lower performance level, or the consumption constraints do not allow to stay at H/V Filter mode j1 , which is the case when the following condition is valid battery level/H j1 < T j1, j2 /H 1 [START_REF] Lysaght | Invited paper: Enhanced architectures, design methodologies and cad tools for dynamic reconfiguration of xilinx fpgas[END_REF] where H j (V j for the vertical filter) is the energy consumption per cycle of the controlled region's mode H/V Filter mode j . This constraint allows to check whether the available energy is under a threshold (determined by T j1, j2 ) that allows to stay at H/V Filter mode j1 , taking as a reference the highest consumption (H 1 ). • In order to move from a H/V Filter mode j2 to a H/V Filter mode j1 that consumes more, it is necessary that the user requires a performance level that is higher than the previous one and that the consumption constraints allow to move to the target mode, which is the case when battery level/H j1 >= T j2, j1 /H 1 (2)

• If the controller receives a reconfiguration suggestion from the coordinator it treats it as follows. If the suggestion requires to move to a less consuming mode, the controller accepts directly. Otherwise, the controller checks the consumption constraints in (2) in order to accept or refuse. As for the coordinator modeling, it can be done by defining global constraints using a contract-based language as presented in [START_REF] Guillet | Modeling and formal control of partial dynamic reconfiguration[END_REF]. However, contracts cannot be modeled using MARTE. Therefore, MARTE has to be extended in order to handle further control needs. This extension is out of the scope of this paper. In the current implementation, the coordinator is written directly in VHDL and stored in the IP library in order to be attached later to the design at the deployment level.

In order to model the SW-C, we associate a mode-automaton to the processor. Each mode of the automaton corresponds to one configuration of the system. Here, we speak about a system configuration because in our case, in addition to software adaptation, the processor has to have a vision of the PRR configurations in order to launch the PRRs reconfiguration correctly. This global vision will be modeled later at the allocation model. As we said previously, we assume that global system constraints require that all the RUs implement the same mode number. Therefore, there are three possible system configurations, which correspond to a three-mode automaton. Transitions from a mode to another is handled by the SW-C that the processor will implement. The determination of the required system configuration will be handled by the code generation tool. This tool will generate the necessary code allowing the processor to communicate with the HW-Cs in order to extract the required system configuration and launch partial reconfiguration accordingly. As for the required partial bitstreams, they will be indicated later at the deployment level, so that the code generation tool inserts them in the SW-C generated code.

C. Allocation modeling

The allocation model allows to allocate application tasks to architectural components (the processor and the PRRs). For this, Gaspard2 uses the MARTE allocate stereotype, which is an extension of the UML abstraction. In order to integrate adaptation aspects at this level, we use the configuration MARTE stereotype, in order to model the different configurations of the PRRs as well as the reconfigurable software tasks. Figure 5 shows a part of the allocation model. It gives, as example, the configuration corresponding to the first mode of a PRR implementing the horizontal filter, following the mode-automaton in modeled in Figure 4). This configuration indicates the allocation of a version of a hardware task (HwHFilter1) to the PRR (HFilter PRR1). For this, the property nature of the allocate stereotype is set to timeScheduling in order to indicate to the generation code tool that the PRRs will be reconfigured in order to implement different hardware tasks. In order to enhance design reuse, our approach reuses the PRR configurations when modeling system configurations, as shows Figure 6. System modes are also modeled by configurations. Each configuration combines the allocation of the software tasks to the processor, and the PRRs configurations corresponding to the considered system mode. Reconfigurable software tasks are allocated using timeScheduling for the nature property of the allocate stereotype, as shows Figure 6.

D. Deployment modeling

The deployment level allows to link existing IPs to the modeled components in order to add details allowing to get closer Fig. 6: Modeling of a system configuration to the targeted platform. At this level, we used the Gaspard2 deployment profile. Stereotypes softwareIP and hardwareIP are used to indicate several platform-dependent attributes such as the implementation language and other parameters. The softwareIP stereotype is used for the application software elementary components. The hardwareIP stereotype is used for the application and architecture hardware elementary components. More details about Gaspard2 deployment can be found in [START_REF] Gamatie | A model driven design framework for massively parallel embedded systems[END_REF]. All the elementary components of our design are deployed using the softwareIP and hardwareIP stereotypes, except for the PRRs since they have different implementations. Existing code files are attached to elementary components through the codeFile stereotype, which is an extension of the UML artifact. We associate VHDL codeFiles to hardware IPs, C codeFiles to software IPs. For the PRR deployment, we associate codeFiles to the different PRR configurations. These codeFiles indicate the name of the corresponding bitstreams as shows Figure 7, which gives a part of the deployment model handling the deployment of a software task, a hardware component (the monitoring component), and a PRR configuration. As for the HW-Cs, we associate code files to the interface, monitoring and configuration components, whereas decision modules will be generated automatically from the modeled mode-automata.

V. EXPERIMENTAL RESULTS

After deployment, automatic model transformations are carried out to add some details to the input model and get closer to the targeted technologies. In order to take control aspects into account in model transformation and code generation, we integrated the proposed control aspects in the Gaspard2 metamodels as well as model transformation rules, in order to get at the end of the transformation chain an RTL model that conforms to the extended RTL metamodel, as shows Figure 2. This model contains technical details allowing the code generation related to the targeted FPGA technology. The code generation tool was extended as well in order to take control aspects into account. This tool allows to generate the code to be integrated into the Xilinx tools for physical implementation. The output of the code generation tool are C code, VHDL code and other platform dependent files as shows Figure 2. Using deployment information and code generation rules, the code generation tool generates the following elements: 1) a software project for the processor. This project contains the software application tasks to be executed by the processor as well as the code of the SW-C, handling software adaptation and the communication with distributed reconfiguration modules and the ICAP, 2) the Xilinx MHS (Microprocessor Hardware Specification) file, describing the architecture to be implemented on FPGA, 3) the UCF (User Constraints File) file, which allows to allocate the hardware component external ports to the FPGA pins, 4) a VHDL project for each RU.

Given to the Xilinx EDK tool, the generated files allowed to generate the system netlists. After system synthesis, the Xilinx PlanAhead tool is used to place the PRRs. This tool takes as input the netlist file (an ngc file) generated by the EDK tool, as well as the UCF file. To each PRR, we associate a number of netlist files corresponding the synthesis result of the different hardware tasks to be implemented by it. Bitstreams can then be generated automatically.

In our case study, three partial bitstreams were generated for each PRR, as well as a full bitstream for the initial system configuration. The horizontal and vertical filters partial bitstreams have as size 193Ko and 365Ko respectively. This difference is due to the fact that the horizontal filter treats larger input and output blocks. Reconfiguration times of horizontal and vertical filter PRRS are 4,3ms and 8,1ms respectively. Table I shows the performance and power consumption results for the system configurations. In our case study, the battery sensor was emulated by a hardware module that decrements at each cycle an energy counter depending to the current configuration. This counter was initialized with 1000J. The last column of Table I shows the number of frames that can be treated if the system keeps the same configuration until the battery is flat. Experimental results show that partial reconfiguration allows to make a trade-off between performance and power. Indeed, without partial reconfiguration, if all the hardware accelerators implement the first mode, the battery (1000J) allows to downscale only 2361 frames. Using partial reconfiguration allows to handle user performance requirements and to save, at the same time, power and to treat thus higher numbers of frames (up to 9,4% of increase) for acceptable reconfiguration times. This trade-off can be further refined by exploring different implementations of the PRRs, but in this paper, we focus on the validation of the distributed decision-making. This validation was done by testing different scenarios by varying the values of the user performance level and the moments it is modified, which allowed to test all transition conditions of Figure 4 for all controllers. Due to space limitation, these scenarios are not detailed in this paper. More details about the distributed decision-making mechanism can be found in [START_REF] Trabelsi | Semi-distributed control for fpga-based reconfigurable systems[END_REF].

In order to evaluate the efficiency of our control model compared to the centralized one in terms of design reusability and scalability, we varied the application parallelism in order to implement higher numbers of PRRs for each filter. Experiments were carried out for a number of PRRs up to n = 10 regions, where n/2 regions implement the horizontal filter and the rest the vertical filter. The HW-Cs generated previously were reused in order to target larger control systems. For the coordination scalability, we only modified the number of coordinated controllers given as parameter to the coordinator, as well as the global constraints handled by it. The same systems were designed using a centralized controller. We noticed that adapting the centralized controller to different numbers of regions was more complicated. Indeed, for the centralized controller the decision-making had to be rewritten each time to adapt to the system implementation, which led to longer design phases.

VI. CONCLUSION

This paper presents a novel design approach for RSoC adaptation control. This approach combines distributed control together with high-level design in order to decrease design complexity, enhance design flexibility and reuse, and automate code generation. It is based on splitting the control concerns between a number of distributed controllers. A coordinator is used to guarantee that the local decisions made by distributed controllers respect global system constraints. Using Model-Driven Engineering and the MARTE standard, adaptation aspects were integrated in a whole RSoC design flow at different design levels (application, architecture, allocation and deployment). Generated code was then integrated into FPGA tools for physical implementation. FPGA implementations allowed to validate the distributed control and to show that it is more flexible, reusable and scalable than the centralized one. As future works, we plan to explore different control approaches such as those based on optimization problems in order to target a wider range of control objectives and reconfigurable systems. The MARTE profile can also be extended to cope with further control needs allowing, for instance, to model control in form of contracts and generate the coordinator automatically. The MARTE standard is being reworked is the ANR project FAMOUS [START_REF] Guillet | Modeling and formal control of partial dynamic reconfiguration[END_REF], which is more dedicated to reconfigurable systems compared to Gaspard2, and in which we plan to integrate our work.

Fig. 1 :

 1 Fig. 1: Considered reconfigurable system

Fig. 3 :

 3 Fig. 3: The RU modeling

Fig. 4 :

 4 Fig. 4: The HW-C's mode-automaton

Fig. 5 :Fig. 7 :

 57 Fig. 5: Allocation of a hardware task on a PRR SwHFilterBlock1-C <<softwareIP>> Monitoring-VHDL <<hardwareIP>> << configuration >> HFilter1_Conf1

TABLE I :

 I Performance and power results for the case-study system

VII. ACKNOWLEDGMENT

The authors would like to thank the ANR project FAMOUS for the financial support of this work.