Erwan Le Goué

Christian Gardrat
email: christian.gardrat@u-bordeaux.fr

Mélanie Romain
email: melromain@laposte.net

Manuela Rollini
email: manuela.rollini@unimi.itcmoresol@uwaterloo.ca

Christine Moresoli

Véronique Coma
email: veronique.coma@u-bordeaux.fr

Effect of oleic acid on the release of tetrahydrocurcumin in chitosan-based films

Keywords: chitosan, carboxymethyl cellulose, oleic acid, tetrahydrocurcumin, films, blackberries

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Biodegradable polymers represent a promising option to replace polymers produced from petroleum. Among them, chitosan (CH) and carboxymethyl cellulose (CMC) obtained in large amounts from chitin and cellulose respectively, are the most abundant natural biopolymers on earth [START_REF] Gandini | The state of the art[END_REF][START_REF] Peniche | Chitin and chitosan: major sources, properties and applications[END_REF].

CH is a polyaminosaccharide (Fig. S1) consisting of two randomly distributed sub-units, glucosamine and N-acetyl glucosamine, linked by a β- (1,[START_REF]antifungal activity against the growth of B. cinerea. This antifungal activity was observed for CH-CMC-Tween 80-THC films with and without oleic acid[END_REF] bond. The interest of CH in the preparation of food packaging materials, is notably due to its good film forming properties, oxygen barrier properties at low relative humidity, non-toxicity, antimicrobial activity and biodegradability [START_REF] Kong | Antimicrobial properties of chitosan and mode of action: a state of the art review[END_REF][START_REF] Ansorena | Food packaging based on chitosan[END_REF][START_REF] Mujtaba | Current advancements in chitosan-based production for food technology; a review[END_REF]. Nevertheless, weak mechanical properties and high water vapor permeability of chitosan-based films do not meet the requirements for food packaging [START_REF] Dutta | Perspectives for chitosan based antimicrobial films in food applications[END_REF][START_REF] Bonilla | Effect of the incorporation of antioxidants on physicochemical and antioxidant properties of wheat starch-chitosan films[END_REF].

Blending CH with other biopolymers can improve its packaging material properties (van den Broek, [START_REF] Van Den Broek | Chitosan films and blends for packaging material[END_REF]. For example, mixing CH and cellulosic derivatives decreased the hydrophilicity of CH films [START_REF] Moura | Highly stable, edible cellulose films incorporating chitosan nanoparticles[END_REF][START_REF] Vargas | Water interactions and microstructure of chitosan-methylcellulose composite films as affected by water concentration[END_REF]. CMC (Fig. S1) is one of the most important cellulose derivatives, is relatively cheap, biocompatible, biodegradable and non-toxic [START_REF] Paunonen | Strength and barrier enhancements of cellophane and cellulose derivative films: A review[END_REF]Malhotra, Keshawi, & Kharkwal, 2015). CMC is considered to have adequate film forming properties [START_REF] Ghanbarzadeh | Physical properties of edible emulsified films based on carboxymethyl cellulose and oleic acid[END_REF][START_REF] Ebrahimzadeh | Physical properties of carboxymethyl cellulose based nano-biocomposites with graphene nanoplatelets[END_REF], but CMC films are very sensitive to moisture (Moura barrier and mechanical properties [START_REF] Tongdeesoontorn | Effect of carboxymethyl cellulose concentration on physical properties of biodegradable cassava starch-based films[END_REF]. Hydrophobic compounds such as oleic acid or vegetable oils can be added in formulation to improve water vapor permeability of hydrocolloid-based films [START_REF] Vargas | Characterization of chitosan-oleic acid composite films[END_REF][START_REF] Ghanbarzadeh | Physical properties of edible emulsified films based on carboxymethyl cellulose and oleic acid[END_REF]Noshirvani, et al., 2017a;[START_REF] Perdones | Physical, antioxidant and antimicrobial properties of chitosan-cinnamon leaf oil films as affected by oleic acid[END_REF][START_REF] Valenzuela | Quinoa-protein-chitosan-sunflower oil edible film: Mechanical, barrier and structural properties[END_REF][START_REF] Wang | The effect of ultrasonic/microwave assisted treatment on the water barrier properties of soybean protein isolate-based oleic acid/stearic acid blend edible films[END_REF].

Generally, CH alone has limited impact on the common microbial strains involved in food spoilage [START_REF] Dutta | Perspectives for chitosan based antimicrobial films in food applications[END_REF]. The addition of substances isolated from plants, such as essential oils or natural extracts, can improve the antimicrobial activity of the packaging and the shelflife of the food products [START_REF] Ashrafi | Preparation and characterization of biocomposite film based on chitosan and kombucha tea as active food packaging[END_REF][START_REF] Dutta | Perspectives for chitosan based antimicrobial films in food applications[END_REF][START_REF] Grande-Tovar | Chitosan coatings enriched with essential oils : effects on fungi involved in fruit decay and mechanisms of action[END_REF][START_REF] Li | Preparation, characterization and anti-aflatoxigenic activity of chitosan packaging films incorporated with turmeric essential oil[END_REF][START_REF] Ponce | Antimicrobial and antioxidant activities of edible coatings enriched with natural plant extracts: in vitro and in vivo studies[END_REF][START_REF] Xiao | A review of the preparation and application of flavor and essential oils microcapsules based on complex coacervation technology[END_REF][START_REF] Zanetti | Use of encapsulated natural compounds as antimicrobial additives in food packaging : a brief review[END_REF].

To the best of our knowledge, few studies have reported on the release properties and antimicrobial properties of chitosan-carboxymethyl cellulose (CH-CMC) films with oleic acid as a matrix for the encapsulation of bioactive hydrophobic molecules. Previous studies reported on films containing ginger or cinnamon essential oils (Noshirvani et al., 2017a), cinnamon essential oil with cross-linked polysaccharide matrices [START_REF] Valizadeh | Development of bioactive composite films from chitosan and carboxymethyl cellulose using glutaraldehyde, cinnamon essential oil and oleic acid[END_REF] and ZnO particles (Noshirvani, Ghanbarzadeh, Mokarram, & Hashemi, 2017b;Noshirvani, Ghanbarzadeh, Mokarram, Hashemi, & Coma, 2017c). All of these materials exhibited antimicrobial properties against Aspergillus niger (Noshirvani et al., 2017a(Noshirvani et al., , 2017b(Noshirvani et al., , 2017c)), Pseudomonas aeruginosa and Listeria monocytogenes [START_REF] Valizadeh | Development of bioactive composite films from chitosan and carboxymethyl cellulose using glutaraldehyde, cinnamon essential oil and oleic acid[END_REF]. The role of oleic acid in the film formulation remains poorly understood. This could notably be due to differences in the film preparation but also to the volatility and hydrophobicity of the essential oils, creating a dispersion of the active components in the hydrocolloid matrix. These limitations may be addressed by (i) selecting a non-volatile wellidentified hydrophobic antimicrobial agent (ii) defining a protocol to obtain films with and without oleic acid and (iii) investigating the release of the antimicrobial agent in the presence/absence of oleic acid.

In this study, tetrahydrocurcumin (THC, Fig. S2) was selected as the non-volatile hydrophobic active antimicrobial agent. THC is a metabolite of curcumin exhibiting high antioxidant and antifungal activities [START_REF] Coma | In vitro inhibitory effect of tetrahydrocurcuminoids on Fusarium proliferatum growth and fumonisin B1 biosynthesis[END_REF][START_REF] Portes | A comparative study on the antioxidant properties of tetrahydrocurcuminoids and curcuminoids[END_REF][START_REF] Portes | Environmentally friendly films based on chitosan and tetrahydrocurcuminoid derivatives exhibiting antibacterial and antioxidative properties[END_REF][START_REF] Loron | Polysaccharide matrices for the encapsulation of tetrahydro-curcumin -Potential application as biopesticide against Fusarium graminearum[END_REF]. The initial phase consisted in the development of a protocol to create CH-CMC oleic acid films with uniform properties. The second phase focused on the release of THC from the films with and without oleic acid. The in vitro antifungal activity of the FFDs was determined against Botrytis cinerea, selected as target strain being one of the most frequently studied fungal pathogen for more than 500 plant species [START_REF] Williamson | Botrytis cinerea: the cause of gray mould disease[END_REF]. This microorganism has a disastrous economic impact on major crops including grape, strawberry and tomato [START_REF] Dean | The top 10 fungal pathogens in molecular plant pathology[END_REF], can be present inside fruits, seeds and other parts of the plant [START_REF] Hua | Pathogenic mechanisms and control strategies of Botrytis cinerea causing post-harvest decay in fruits and vegetables[END_REF] and can occur before maturity or postharvest [START_REF] Pitt | Primary keys and miscellaneous fungi[END_REF][START_REF] Tournas | Mould and yeast flora in fresh berries, grappes and citrus fruits[END_REF]. The developed FFD and the corresponding films were then assayed on the shelf-life of blackberries. These fruits, grown and harvested inside the garden of one of the authors (M. Rollini, North Italy), were selected to ensure their organic conditions, and as an example of wild-harvested specialty fruits with health attributes and of commercial value but with a limited shelf life due to fungal contamination (mainly Botrytis spp.) [START_REF] Skrovankova | Bioactive compounds and antioxidant activity in different types of berries[END_REF].

Materials and methods

Materials

Chitosan (CH, low molecular weight, 20-300 cP, 1 wt. % in 1% (v/w) acetic acid, 75-85% deacetylated), sodium carboxymethylcellulose (CMC, high viscosity, art. C5013), diphenylpicrylhydrazyl radical (DPPH), Nile red (9-(diethylamino)-5H-benzo[a]phenoxazin-5-one) and acetone were purchased from Sigma Aldrich (USA and Germany). Analytical grade glycerol, oleic acid (≥ 90 %), acetic acid and Tween 80 were purchased from Merck (Germany) and used as received. Tetrahydrocurcumin (THC, Sabiwhite 95%) was obtained from Sabinsa Corporation (20 Lake Drive, East Windsor, New Jersey).

B. cinerea was obtained from the culture collection of the LCPO Laboratory at the University of Bordeaux (France). B. cinerea was precultured on PDA at 25°C in the dark before use. Sabouraud agar was obtained from Biokar Diagnostics (France) and blackberries were hand picked in a private garden near Milano, Italy.

Elaboration of CH-CMC oleic acid film forming dispersions (FFDs) and films

The following protocol was established by conducting a preliminary study to identify suitable conditions for the preparation of CH-CMC homogenous films. A CH solution: chitosan (0.35 g) was dissolved in distilled water containing acetic acid (50 mL, 0.875% v/v) under magnetic stirring. Tween 80 (0.35 mL) was added as an emulsifier and the solution was stirred overnight. Oleic acid (0.525 mL in 3 mL of ethanol) was added as a hydrophobic carrier under magnetic stirring for 15 min; the pH of the solution was increased to 8.0 using NaOH solution (3M). A CMC solution was prepared separately by dissolving sodium carboxymethylcellulose (CMC, 0.7 g) in hot distilled water (50 mL, 80°C).

FFDs were prepared by mixing the CH solution and the CMC solution followed by homogenization with an Ultra-Turrax (Ika; 5 min) and ultrasound (Bandelin Sonopuls, Germany; 15 min, 31 kJ). After degassing under vacuum, glycerol (0.525 mL; glycerol/CH-CMC 63%, w/w) was introduced as a plasticizer under magnetic stirring (10 min). The FFD was casted (4 x 25 mL) in Petri dishes (diameter 9 cm), which were dried at 40°C for two days and then conditioned for additional three days at 25 °C, 50% RH. The dried films were peeled off and weighed before any characterization.

Different films were prepared with/without oleic acid, with/without THC. In the case of films containing THC (10.5 mg; THC/CH-CMC 10% w/w), THC was incorporated at the same time as oleic acid in the chitosan solution before pH adjustment. The films without oleic acid were produced with the same protocol, omitting oleic acid addition and THC was solubilized in ethanol.

The CH film used for ATR-FTIR analysis was prepared from a chitosan film-forming solution (100 mg) in 100 mL acetic acid solution (0.875% v/v) while the CMC film was prepared from a dispersion of 0.7 g in hot water. The CH-CMC film was prepared following the previous protocol.

Characterization of FFDs

Optical microscopy

Optical microscope Leica DME (Bioblock Scientific, Illkirch, France) was used. A drop of the FFD was deposited on a slide; a x10 magnification was sufficient to generate suitable images. All images were obtained using the software Motic Images plus 2.0 with an image capture format of 1280 x 1024.

Particle size distribution

Analyzes were performed with a Mastersizer 2000 equipment (Malvern Instruments, Orsay, France). Few drops of the FFD were dispersed in an aqueous medium in the measuring cell. 2.4.1 Microscopy 2.4.1.1 Nile Red staining. Nile red was dissolved in acetone (0.1 mg/mL) before film staining according to [START_REF] Kimura | Rapid estimation of lipids in oleaginous fungi and yeasts using Nile red fluorescence[END_REF]. After staining, film was analyzed by confocal microscopy (Leica SP5-AOBS) with a thermostated plate (Linkman).

Characterization of film

Scanning electron microscopy (SEM)

. Surface microstructure analysis of the film was carried out by SEM with a tungsten source in a Quanta 200 device (FEI, USA). Film samples were observed under low vacuum 50 Pa, large field detector, 3 kV and working distance 7-8 mm.

Dynamic penetration of water (PDA)

Measurements were recorded on a PDA.2 C 02 Module HVL (high viscous liquid) from Emtec Electronic Gmbh (Leipzig, Germany). The film was fixed by using a double-sided adhesive tape on the measuring head, which contains the ultrasound receiver. Water (3.5 mL) was deposited on the horizontal area of the measuring cell, which contained the ultrasound transmitter (frequency 1 MHz). The measuring head of the basic device was lowered together with the film sample until in contact with water.

Water contact angle

Water contact angle on the film surfaces was measured by a goniometer Kruss DSA 100 (Germany). A drop of distilled water (≈ 2 μL) was placed on the film surface (10 mm x 60 mm). The initial water contact angle (after 5 seconds) was measured. For each film, three replicates were obtained.

THC release from film and antioxidant activity

THC release was measured for intact film and film after grinding (powder), to evaluate the impact of the physical condition of the film.

Intact film

The films were totally immersed in methanol (100 mL) under magnetic stirring (250 rpm).

The absorption spectra of the solutions were recorded at different time intervals (2, 5.30, 10, 20, 30, 45 and 60 min) and at 280 nm using a spectrophotometer Cary-100 with quartz cells (length 1 cm). These experiments were repeated three times with different films.

The antioxidant activity was evaluated by measuring the DPPH radical scavenging activity. Briefly, a film was immersed in a methanol solution for 5 days (100 mL) in the dark and at room temperature. A 2 mL volume of the methanol solution was mixed with a DPPH solution (150 µM, 2 mL), vigourously shaken and allowed to rest at room temperature for 30 minutes. The absorbance of the methanol solution was measured at 517 nm and the DPPH inhibition (1) was calculated as follows:

I(%)= (Acontrol -Asample)/Acontrol*100 (1)
where Acontrol is the absorbance of the fresh methanol solution and Asample is the absorbance of the methanol solution after 5 days of film immersion.

The presence of THC in the methanol solution was examined by 1 H NMR (Bruker AVANCE III HD 400 MHz spectrometer). The methanol solution was collected after the immersion of a film for 5 days and evaporated. The solid residues were collected with 0.5 mL of deuterated methanol and analysed by NMR.

Grinded film

The film was grinded in liquid nitrogen in order to destroy its structure. The resulting powder was immersed in methanol for 5 days in the dark at room temperature and under magnetic stirring (200 rpm). The antioxidant activity was determined as before, and DPPH inhibition calculated according to equation (1). The presence of THC in the solution was examined by NMR, as previously described.

ATR-Fourier Transformed Infrared spectroscopy (ATR-FTIR)

ATR-FTIR spectra of the films were obtained using a FTIR spectrometer (PIKE technologies/Gladi Atrvertex 70, Bruker, France). Spectra were collected from 4000 to 400 cm -1 (32 scans; resolution 4 cm -1 ).

Antifungal properties of FFDs against B. cinerea 2.7.1. In vitro antifungal activity

The in vitro antifungal activity was obtained from growth inhibition tests performed on Sabouraud medium in Petri dishes (9 cm diameter), inoculated in the centre with 10 µL of B. cinerea spore suspension (10 4 spores/mL). First, the antifungal activity of THC, oleic acid and Tween 80 was evaluated. For that, the medium was supplemented with THC (0.105 g/L), or oleic acid (0.525 g/L), or Tween 80 (0.35 g/L) after dissolution in 1 wt. % of DMSO. The concentration of THC, oleic acid and Tween was similar as for the FFDs. The effect of the solvent DMSO was obtained with the addition of 1 wt. % DMSO in the culture medium.

To perform the in vitro assays for FFDs, 1 mL of FFD was added to the Sabouraud medium and Petri dishes were then inoculated in the centre with 10 µl of B. cinerea spore suspension (10 4 spores/mL) as previously described.

Growth controls, without any potential inhibitory agents, were conducted in parallel (1 mL H2O pH 7.5 into Sabouraud medium).

All cultures were incubated at 25°C for 5 days and the radial growth of B. cinerea was measured every day. Colony diameters were determined as the average of two perpendicular diameter length measurements (expressed in cm). The tests were performed in triplicate.

All the analyses were performed in triplicate for the FFDs with and without oleic acid.

In vivo antifungal activity of FFD coated blackberries.

Blackberry samples, harvested at the same day of the trials, were dipped for 10 s in the FFD (with and without oleic acid) and subsequently left drying on a paper in sterile conditions (under a biohazard type II hood) for 2 h, avoiding direct contact with each other. Uncoated blackberries were used as control and visually inspected after 0, 3, 4 and 5 days of storage at 4

°C and 20 °C. The coated blackberries were kept in a Falcon tube with open caps at 4 °C and 20

°C and visually inspected after 0, 3, 4 and 5 days. In addition, total bacterial count and yeasts and moulds count were determined according to storage time. For that, blackberry samples (10 g) were transferred aseptically into a Stomacher bag (400 mL PE, Barloworld, France) containing 90 mL of sterile peptoned water (10 g/L bacteriological peptone, Costantino, Italy)

and blended in a Stomacher (Star Blender LB 400, Biosystem, Belgium) at high speed for 3 min. Ten-fold dilution series of the suspension were made in the same solution as for plating.

The following culture media were used: TSA (Merck, Germany) for Total Bacterial Count (TBC), and MEA (Malt Extract Agar, Oxoid) for yeasts and fungi. Colonies were counted after incubation at 30 °C for 24 h for mesophile bacteria and 30 °C for 5 d for yeasts and fungi.

Microbial counts were performed in triplicate and reported as logarithms of the number of colony forming units (log cfu/g fruit), and means and standard deviation (SD) were calculated.

Results and discussion

CH-CMC film preparation

The creation of stable CH-CMC films containing oleic acid required the development and fine tuning of a film forming dispersion methodology as illustrated in Figure 1. The first factor examined was the pH of the CH solution. When CMC solutions (pH 7.40) were added directly to the CH solutions (pH 3.95), physically tight compact hydrogels were obtained, likely due to electrostatic interactions between oppositely charged biopolymers, knowing the anionic character of CMC and cationic character of CH in acidic solutions [START_REF] Farris | Development of polyion-complex hydrogels as an alternative approach for the production of bio-based polymers for food packaging applications: a review[END_REF]. Increasing the pH of the CH solution before mixing with the CMC solution can alter the properties of the films (Noshirvani et al., 2017a). In the present study, the pH of the CH solution was adjusted to 8 before mixing with the CMC solution in order to avoid precipitation. In these conditions, CH was not in its protonated form. To avoid aggregation, which occurred during the increase of the CH solution pH at approximately 5.7, an emulsifier was added, Tween 80.

The addition of oleic acid to the chitosan solution required Ultra-Turrax and ultrasound treatment to obtain a better homogeneous FFD as can be seen in the microscopy images (Fig S3). Large aggregates were observed when using Ultra-Turrax alone (Fig S3a ) but disappeared when ultrasounds and Ultra-Turrax were used (Fig S3b). These aggregates are due to the presence of oleic acid (Fig S3c andd).

The dispersion of oleic acid in the CH solution was improved when Tween 80 was added prior to the addition of oleic acid. In addition, the emulsion stability was improved if Tween 80 and oleic acid were added in the solution before pH adjustment to 8 (Fig. S4 andS5).

Furthermore, if the oleic acid was pre-solubilized in ethanol before its addition to the CH solution, its particle size decreased from 3.5 μm (Fig. S6a) to 0.5 μm (Fig. S6b), leading to a better emulsion stability. The oleic acid partitioning was examined by using Nile red staining according to [START_REF] Kimura | Rapid estimation of lipids in oleaginous fungi and yeasts using Nile red fluorescence[END_REF]. As can be observed from the confocal

Characterization of CH-CMC film

Visual observation

As illustrated in Figure 2, there was a retractation of the film surface without oleic acid, potentially due to direct interactions between CH and CMC, leading to a lower film density. As expected, the addition of oleic acid altered slightly the film transparency but visual transparency remains sufficient for food coatings applications. The CH-CMC films were flexible even when no oleic acid was added (well-known plasticizer for polysaccharide-based materials), wich is not generally the case with pure CH films.

Penetration Dynamic Analysis of water

The impact of oleic acid on the water penetration in films is presented in Fig. S8. The presence of oleic acid leads to a small decrease of ultrasound intensity after the maximum (at 1 s). At the end of the analysis, the increase of the ultrasound intensity is due to the film swelling knowing that the more the water penetration rate, the more the attenuation of the signal.

On the contrary, the initial slope of the curve for film without oleic acid decreased quickly in few milliseconds, followed by an increase of ultrasound intensity, which indicates quick water penetration rate and a swelling of the film.

The presence of oleic acid improved liquid water barrier properties, especially due of its hydrophobic character. Similar observation was reported previously by Noshirvani et al. (2017a). These authors showed that oleic acid addition to CH-CMC films increased the water contact angle, and even more after adding hydrophobic essential oils. In the current study, the water contact angle of CH-oleic acid and CMC-oleic acid films was 44° and 38°, respectively.

For the CH-CMC-oleic acid film, the water contact angle was 48°, indicating that the combination of CH and CMC improved the hydrophobicity of the films. Unexpectedly, the introduction of THC, a hydrophobic compound, reduced the hydrophobicity of the CH-CMColeic acid-THC film with a water contact angle of 33°. It seems that THC led to modifications in the CH-CMC network.

THC release from films and antioxidant activity

In this part, the films containing the following components CH, CMC, Tween 80 and THC with and without oleic acid are called "THC-loaded films". The release of THC from THCloaded films with and without oleic acid was studied over time by measuring the absorbance at 280 nm of the methanol solution (Fig. 3a), as THC has a UV-vis spectrum with a λmax at 280 nm (Fig S9). Oleic acid is soluble in methanol, but its absorbance at 280 nm is too low to have a significant influence. Unfortunately, the commercial Tween 80 is also soluble in methanol and displayed an absorbance between 250 and 290 nm (Fig. S9) [START_REF] Arudi | Purification of oleic acid and linoleic acid[END_REF]. Higher absorbances were observed for THC-loaded films without oleic acid with the maximum absorbance obtained after approximately 1 h. But, because of the unknown detailed composition of Tween 80 and the observed overlap between the absorbance of Tween 80 and THC at 280 nm, the THC released from the films could not be quantified.

An alternative approach to distinguish the release of THC from the potential interferences associated with Tween 80 was adopted and was based on the strong antioxidant activity of THC [START_REF] Portes | A comparative study on the antioxidant properties of tetrahydrocurcuminoids and curcuminoids[END_REF], whereas Tween 80 has minimal antioxidant activity [START_REF] Pérez-Rosés | Antioxidant activity of Tween 20 and Tween 80 evaluated through different in vitro tests[END_REF]. The absence of DPPH antioxidant activity of Tween 80 was confirmed in the current study (0% DPPH inhibition).

The DPPH inhibition of the methanol solution obtained from THC-loaded films are presented Table 1. In order to know if THC remained entrapped in the film matrix, films were reduced to powder and then immersed in a methanol solution for 5 days. The relative inhibition of the film powder with and without oleic acid is presented in Table 1. Higher THC release was obtained for the film powder regardless of the oleic acid presence, indicating that THC could be maintained in the matrix notably by steric hindrance. In addition, higher THC release could also be due to the increase of exchange surface between the film powder and the methanol solution. Without oleic acid present, the THC recovered was double. Similar results were reported previously for THC release from CH-based films [START_REF] Portes | Environmentally friendly films based on chitosan and tetrahydrocurcuminoid derivatives exhibiting antibacterial and antioxidative properties[END_REF] with a plateau reached after 4 hours immersion in methanol and 30% of THC remained in the film, which was fully recovered after film grinding. Another study of CH films containing THC in chitosan particles [START_REF] Bijoux | Films bioactifs à base de chitosane et dérivés de curcumine encapsulés[END_REF] showed that a plateau was reached after two hours and 23% of the THC released in ethanol; and an additional 47% THC was recovered after grinding the film to a powder.

Table 1

Impact of the oleic acid content of THC-loaded films on DPPH inihibition of methanol solution after film incubation during 5 days at ambient temperature. Inhibition percentages are presented as means ± the standard deviations of three replicates.

Sample DPPH inhibition (%)

Film with oleic acid 0

Film without oleic acid 32 ± 6

Film powder with oleic acid 26 ± 6

Film powder without oleic acid 66 ± 7 The addition of oleic acid on the THC release in methanol solution for intact and powder THC-loaded films reduced the THC release. These results are in agreement with the 1 H-NMR spectra of the methanol solution (Fig. S10). This could be due to the encapsulation of THC in oleic acid droplets leading to partial release of THC. As THC is soluble in ethanol but poorly soluble in water, it could be easily incorporated into oleic acid micelles because of its higher affinity for the oleic acid matrix. During the film preparation, the addition of the THC and oleic acid ethanol solution to the acidic CH solution pre-emulsified with Tween 80 may have led to the adsorption of CH on the oleic acid or Tween 80-oleic acid droplets [START_REF] Vargas | Characterization of chitosan-oleic acid composite films[END_REF]. Interactions between oleic acid and CH matrices have been proposed previously [START_REF] Souza | Preparation of biopolymer film from chitosan modified with lipid fraction[END_REF]. As a result, oleic acid could favor the absorption of CH at the droplet interface because of interactions between the amino groups of CH (pKa ≈ 6.5) and the carboxyl groups of oleic acid (pKa ≈ 4.8) (Ham-Pichavant, Sebe, Pardon, [START_REF] Ham-Pichavant | Fat resistance properties of chitosan-based paper packaging for food applications[END_REF][START_REF] Vargas | Characterization of chitosan-oleic acid composite films[END_REF] or by the formation of bonds between the carboxyl group of oleic acid and the polar groups of CH, as suggested recently for basil and thyme essential oils in CHoleic acid matrix [START_REF] Perdones | Properties of film-forming dispersions and films based on chitosan containing basil or thyme essential oil[END_REF]. The pH adjustment of the CH-oleic acid and THC solution before mixing with the CMC solution did not alter the organization of the CH-oleic acid and THC system. THC seems to be embedded in the CH-CMC matrix to give stable FFDs. During the drying of the FFD, the presence of oleic acid in the CH-CMC matrix promoted THC retention as observed previously for CH-based films with cinnamon essential oil [START_REF] Perdones | Physical, antioxidant and antimicrobial properties of chitosan-cinnamon leaf oil films as affected by oleic acid[END_REF][START_REF] Valizadeh | Development of bioactive composite films from chitosan and carboxymethyl cellulose using glutaraldehyde, cinnamon essential oil and oleic acid[END_REF].

Another important observation is the partial release of THC from the THC-loaded film, even without oleic acid. Since no degradation of Tween 80 and THC was observed in methanol for similar duration (UV spectra not shown) and knowing that Tween 80 had no effect on DPPH activity -absence of DPPH activity from intact THC-loaded films with oleic acid-the THC release from fatty acid free films can be evaluated.

By assuming that the release of Tween 80 remained the same with and without oleic acid, the difference of the absorbance at 280 nm for THC-loaded films with and without oleic acid (Figure 3a) could be attributed to the release of THC in methanol (presented in Figure 3b).

The maximum THC release was about 25 % of THC (calibration curve not shown).

ATR-FTIR analysis of films

Potential interactions between the different components of the films on the THC release were examined by ATR-FTIR spectroscopy (Fig. 4). CH films (pH ≈ 4) displayed broad bands between 3100 and 3400 cm -1 due to overlapped O-H and N-H stretching vibrations. Bands at 1635 and 1537 cm -1 were respectively attributed to νC=O (amide I) and δNH3+. The bands between 900 and 1200 cm -1 were characteristic of the carbohydrate structure. CMC films (pH ≈ 7.5) displayed a band between 3100 and 3600 cm -1 corresponding to O-H stretching vibrations. Bands at 1412 and 1585 cm -1 were due to symmetrical and asymmetrical stretching vibrations of the carboxylate groups, respectively.

The bands observed between 900 and 1200 cm -1 may be attributed essentially to the polysaccharide skeleton.

CMC-CH films (pH ≈ 8), showed shift in both bands belonging to the stretching vibrations of the carboxylate groups of CMC, from 1412 cm -1 to 1408 cm -1 and from 1585 cm -1 to 1556 cm -1 with a higher intensity of the former band and a higher width of the latter one. The band corresponding to δNH3+ in chitosan disappeared in agreement with the increase of the pH. The broadening of the band at around 1550 cm -1 could be attributed to an overlap with the bending vibration of the amino groups. In the same time, the amide I band of CH was broader. These modifications indicate strong interactions between CMC and CH in films due to their chemical compatibility. The addition of THC in CH-CMC films (pH ≈ 8) did not alter strongly the overall shape of the infrared spectrum except a broadening of the band centered on 1557 cm -1 , likely due to the overlap with the stretching bands of the aromatic groups of THC. As a consequence, the amide I band of CH gave a smooth shoulder at around 1632 cm - 1 . After addition of Tween 80, a new band with a small intensity appeared at 1734 cm -1 ; as this band has the same wavenumber in the ATR-IR spectrum of Tween 80, it could be concluded that Tween 80 did not strongly interact with the film matrix.

The addition of oleic acid to the CH-CMC films led to some modifications: (i) νC-H stretchings were clearly apparent at 2920 and 2851 cm -1 confirming the presence of oleic acid in the film; (ii) the band at 1736 cm -1 (belonging to Tween 80) and the bands at 1559 and 1409 cm -1 (belonging to the CH-CMC matrix) seemed to have no significant shifts; (iii) the shoulder at ≈1659 cm -1 (chitosan amide I vibration) shifted; (iv) a new shoulder appeared at 1586 cm -1 , due to the transformation of the carboxylic groups of oleic acid into carboxylate ions; (v) two significant modifications appeared in the spectrum of the CH-CMC-Tween 80-Oleic acid-THC film: first, the band at 1559 cm -1 was sharper; secondly, the intensity of the weak shoulder at 1342 cm -1 decreased strongly. This shoulder corresponds likely to complex vibrations belonging partly to the acetamido groups in CH (νC-N, amide III), as suggested from the FTIR spectrum of pure N-acetyl-α-D-glucosamine [START_REF] Kovacs | Vibrational analysis of N-acetyl-α-Dglucosamine and β-D-glucuronic acid[END_REF].

An evolution of this shoulder was observed since it appeared at 1338 cm -1 in the ATR-FTIR spectrum of CH films as described previously [START_REF] Qin | Preparation and characterization of antioxidant, antimicrobial and pH-sensitive films based on chitosan, silver nanoparticles and purple corn extract[END_REF] and at 1342 cm -1 in CH-CMC-THC-Tween 80 films. These observations suggest interactions, respectively, between oleic acid and THC on the one hand, and between CH and oleic acid on the other hand, in agreement with THC entrapment in oleic acid.

Film morphology

SEM images of films with and without THC are presented in Fig. 5 at two magnifications.

The addition of THC led to CH-CMC-oleic acid film with irregular surface whereas the absence of THC led to many small structure similar to shells. These shell-like structures were homogeneously distributed at the CH-CMC-oleic acid film surface and were identified as oleic acid droplets by confocal imaging (Fig. S7). The differences observed in the CH-CMColeic acid film morphology support the alteration of interactions between oleic acid and the CH-CMC matrix due to the presence of THC. During preparation of FFD, the pH of the CH solution increased, lowering the electrostatic interactions between oleic acid and CH. As consequence, without THC, oleic acid droplets rose at the film surface. The presence of THC modified these interactions and fewer oleic acid droplets were observed at the film surface.

The presence of oleic acid droplets at the film surface altered the hydrophobicity of the film and may explain the differences in water contact angle of the CH-CMC-oleic acid films with (33°) and without THC (48°).

In vitro antifungal activity

In vitro antifungal activities of the main substances of FFD and the FFD with and without oleic acid, were evaluated against B. cinerea. To examine the putative influence of the FFDs with and without oleic acid on a food product, in vivo experiments were conducted with blackberries.

In vitro antifungal activity of FFD components and FFD with and without oleic acid

In vitro results of the growth of B. cinerea in presence of pure DMSO as the solvent, and THC, oleic acid and Tween 80 in DMSO are reported in Table 2. Plates containing DMSO showed a reduced fungal development comparatively to the control plate (-16% after 4 days of incubation), confirming the low antifungal effect of DMSO as previously observed [START_REF] Elad | The use of antioxidants (free radical scavengers) to control grey mould (Botrytis cinerea) and white mould (Sclerotinia sclerotiorum) in various crops[END_REF]. THC in DMSO reduced the fungal growth up to 36% after 4 days incubation, while the other FFD substances did not show any strong effect compared to the DMSO plate.

Table 2

In vitro antifungal activity of DMSO, THC, Oleic acid and Tween 80 on the radial growth of B. cinerea during incubation time (expressed in cm), presented as means ± the standard deviations of three replicates. The anti-fungal activity of the FFD, with and without oleic acid is reported in Table 3.

Lower fungal growth was observed during the first 4 days for the FFDs. No noticeable difference between the FFD with and without oleic acid was observed. FFDs reduced the growth up to 20-25%. The lower anti-fungal activity of the FFD without oleic acid may be related to limitation for the diffusion of THC inside the solid culture medium due to the lipophilic nature of oleic acid.

Table 3

In vitro antifungal activity of film forming dispersion (FFD) with and without oleic acid on the radial growth of B. cinerea during incubation time (expressed in cm) presented as means ± the standard deviations of three replicates.

In vivo antimicrobial activity of CH-CMC-THC FFD coating on blackberries

The in vivo antimicrobial activity, measured as total bacterial count and total yeast and mould count, of non-coated (control) and CH-CMC-THC FFD coated blackberries, during cold storage (4°C) and room temperature storage (20°C) is presented Fig. 6. As expected, the microbial count was higher for storage at room temperature for coated and uncoated blackberries. The CH-CMC-THC FFD coating did not reduce the microbial count for these storage conditions. This result was unexpected due to the partial inhibition of the FFD on B. cinerea for in vitro conditions, but may be explained by the lack of THC activity for the strains present in the blackberries. FFD may also act as a carbon source, explaining the higher counts observed at 4°C. Oleic acid addition had no significant effect on the antimicrobial and antifungal activity of the CH-CMC-THC FFD coatings (Fig. 6).

Visual observations of blackberries revealed a loss of consistency due to natural decay and water loss, which was more important when stored at room temperature and for the control and the CH-CMC-THC FFD-coated blackerries with oleic acid (Table 4). off-flavour generation was observed for all blackberries likely due to the presence of oleic acid as reported previously for strawberries [START_REF] Vargas | Quality of cold-stored strawberries as affected by chitosan-oleic acid edible coatings[END_REF]. In summary, the CH-CMC-THC free of oleic acid offered the best visual observation.

Fig. 1 .

 1 Fig. 1. Experimental protocol to obtain homogeneous film forming dispersion (FFD).

Fig. 2 .

 2 Fig. 2. Visual observations of CH-CMC films with (left side, A) and without oleic acid (right side, B)

Fig. 3 .

 3 Fig. 3. Absorbance at 280 nm of the methanol solution versus time (a) Absorbance after dipping the THCloaded CH-CMC films with or without oleic acid (b) Absorbance difference corresponding to THC effectively released from the films. Error bars represent the standard deviation of three measurements on three different films.

Fig. 4 .

 4 Fig. 4. ATR-FTIR spectra of films (a. CH-CMC-Tween 80-Oleic acid-THC; b. CH-CMC-Tween 80-THC; c. CH-CMC-THC; d. CH-CMC; e. CMC; f. CH) and pure components (g. Oleic acid; h. Tween 80; i. THC).

Fig. 5 .

 5 Fig. 5. SEM image of film surfaces: CH-CMC-oleic acid-THC (a, x1000, b, x4000) and CH-CMC-oleic acid (c, x1000, d, x4000).

Fig. 6 .

 6 Fig. 6. Total bacteria count (TBC) and Yeast and moulds count (log CFU/g) of CH-CMC-THC coated blackberries after 5 days of incubation at 4°C and 20°C. Errors bars represent the standard deviations of three replicates.

  Percentage of reduction respect to the physiological growth at day 4.

	Time (day)	Control	FFD with oleic acid FFD without oleic acid
			Radial growth (cm)
	0	0	0	0
	1	0.80 ± 0.00	0	0.25 ± 0.42
	2	2.80 ± 0.14	2.17 ± 0.14	1.92 ± 0.42
	3	4.70 ± 0.14	3.65 ± 0.16	3.70 ± 0.28
	4	7.05 ± 0.07	5.27 ± 0.27	5.62 ± 0.31
			-25%*	-20%*
	*

Acknowledgments

The authors thank Chiara Sari for some experiment work.

Imaging was performed on the Bordeaux Imaging Center, member of the France BioImaging national infrastructure (ANR-10-INBS-04).

Declaration of competing interest

The authors declare that there are no conflicts of interest.

CRediTauthorship contribution statement

Conceptualization


Rémi Pelloux-Prayer

Philomène Schiele

Stéphane Oudard

Gwenaëlle Gravis

François Kleinclauss

Gilles Crehange

Christophe Hennequin

Alicia K Morgans

Lionel Geoffrois

Samuel Limat

Antoine Thiery-Vuillemin

Virginie Nerich
email: v1nerich@chu-besancon.fr

PhD Virginie Nerich -Pharmd

Cost-effectiveness Analysis of Innovative Therapy for Patients with Newly Diagnosed Hormone-Sensitive Metastatic Prostate Cancer

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

ABSTRACT:

Background: The optimal therapeutic strategies for patients with metastatic hormonesensitive prostate cancer (mHSPC) followed by metastatic castrate-resistant prostate cancer (mCRPC), in terms of cost and effectiveness remains unknown. This study aims to compare the cost-effectiveness of various potential strategies, from the start of first-line treatment in mHSPC to the death of the patients.

Methods: Two Markov decision-analysis models were developed, one for cohort A "asymptomatic/mildly symptomatic patients in mCRPC" and one for cohort B "symptomatic patients in mCRPC". Each strategy reflects daily practice for mHSPC until progression in mCRPC from the start of first treatment regimen with either docetaxel or abiraterone acetate plus prednisone (AA) in mHSPC to the death of the patient. The cost-effectiveness analysis was performed from the French public healthcare system perspective. Only direct medical costs were included. Survival data were extracted from results of published randomized clinical trials.

Results:

For cohort A, docetaxel followed by AA is the most cost-effective therapeutic strategy (€96,925 for 4.24 life-years). For cohort B, docetaxel followed by docetaxel is the most cost-effective therapeutic strategy (€81,463 for 4.05 life-years). Sensitivity analyses confirmed the robustness of our results except for a price reduction of 70% for AA or enzalutamide.

Conclusions: Our approach is innovative to the extent that our analysis takes into account various potential strategies for mPC. Our economic evaluation suggests that a price reduction of AA or enzalutamide impacts on the results. This approach must continue, including new drugs for patients with mPC. KEY-WORDS: cost-effectiveness analysis; hormone-sensitive; castrate-resistant; metastatic; prostate cancer INTRODUCTION Prostate cancer is the second most common cancer with an estimated 1.1 million new cases worldwide in 2012 and the third most common cause of cancer death in men with 307,000 deaths in developed countries [START_REF] Haas | The Worldwide Epidemiology of Prostate Cancer: Perspectives from Autopsy Studies[END_REF][START_REF] Ferlay | Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012[END_REF][START_REF] Mottet | European Association of Urology (EAU) -European Association of Nuclear Medicine (EANM) -European SocieTy for Radiology and Oncology[END_REF][START_REF] Wu | No improvement noted in overall or cause-specific survival for men presenting with metastatic prostate cancer over a 20-year period[END_REF] .

Treatment of metastatic prostate cancer (mPC) can be divided into two different stages: (1) metastatic hormone-sensitive prostate cancer (mHSPC), i.e. naive from androgen deprivation therapy (ADT); (2) metastatic castrate-resistant prostate cancer (mCRPC), when the cancer has progressed on ADT. Following drug labels and clinical trial design, patients are also treated according their symptomatic state (asymptomatic/mildly symptomatic or symptomatic) when their cancer becomes castrate resistant [START_REF] Sartor | Metastatic Prostate Cancer[END_REF] .

For men with an initial diagnosis of mHSPC, continuous ADT represented the standard of care until 2015. In 2015, chemohormonal combination therapy with docetaxel became a new standard of treatment based on results of CHAARTED and STAMPEDE arm C randomized clinical trials (RCTs) [START_REF] James | Addition of docetaxel, zoledronic acid, or both to first-line long-term hormone therapy in prostate cancer (STAMPEDE): survival results from an adaptive, multiarm, multistage, platform randomised controlled trial[END_REF][START_REF] Sweeney | Chemohormonal Therapy in Metastatic Hormone-Sensitive Prostate Cancer[END_REF] . In 2017, abiraterone acetate plus prednisone (AA) has shown survival benefit in terms of overall survival (OS) and progression-free survival (PFS) in mHSPC setting [START_REF] James | Addition of docetaxel, zoledronic acid, or both to first-line long-term hormone therapy in prostate cancer (STAMPEDE): survival results from an adaptive, multiarm, multistage, platform randomised controlled trial[END_REF]8 . Adding AA to ADT in newly diagnosed mHSPC is an alternative standard of care to docetaxel with ADT 3 . After disease progression, multiple additional treatments have shown to improve OS, including docetaxel, AA, enzalutamide, radium-223, sipuleucel-T, and cabazitaxel after docetaxel 9-15 . Despite these benefits, the use of androgen directed therapies in mHSPC and mCRPC strategies are likely to increase the already substantial economic burden associated with the management of mPC. Furthermore, the optimal strategy of treatment for these patients, in terms of cost and effectiveness, is unknown.

In the context of rational decision making in health care, a major challenge in economic evaluation (EE) is to provide cost-effectiveness data than are relevant to daily practice and that may optimize the consumption of healthcare resources. Thus, the purpose of this study was to compare the cost-effectiveness of various sequential strategies, from the start of firstline treatment in mHSPC to patient death.

MATERIAL AND METHODS

Two Markov decision-analysis models were developed to compare the cost and effectiveness of strategies, according to the presence or absence of symptoms in the mCRPC stage: cohort A with "asymptomatic or mildly symptomatic patients" and cohort B with "symptomatic patients" [START_REF]Methodological Guidance 2020 -Choices in methods for economic evaluation[END_REF] . The rational is based on the eligibility criteria of the registration trials and the European label of AA and enzalutamide. All strategies reflect daily practice for mHSPC until progression in mCRPC from the start of first treatment regimen with either docetaxel plus prednisone or AA in mHSPC to the death of the patient 3 (Figure 1). Radium-223 dichloride has been excluded because in Europe, this treatment is currently only for patients with progression after at least two prior lines of systemic therapy for mCRPC, or who are ineligible for any available systemic mCRPC treatment.

MARKOV DECISION-ANALYSIS MODEL

For all strategies compared (SA1DOCE SA1ABI, SA2DOCE SA2ENZA, SA3ABI SA3ENZA, SA4ABI SA4DOCE, SB1DOCE SB1CABA, SB2DOCE SB2DOCE and SB3ABI SB3DOCE), three potential mutually exclusive health states defined disease history: mHSPC, mCRPC and death. All patients started in the mHSPC state, with the introduction of ADT and were followed up, with the possibility to move to the mCRPC state or dying (death state) (Figure 2). Cycle length was set at 1 month to facilitate patient monitoring and the use of published survival data.

MODEL ESTIMATES: CLINICAL PARAMETERS

A systematic literature search was conducted in PubMed to identify published RCTs until February 2020 based on selected Medical Subject Heading (MeSH) (Appendix 1). Articles were chosen according inclusion criteria: phase III RCT in mHSPC or mCRPC with treatment regimens matching with treatment regimens used in our EE. Concerning the first-line chemotherapy in mCRPC setting, we used data from the FIRSTANA phase III RCT because it is a more recent dataset than the TAX327 and TROPIC RCTs 9,10 .

We retained (1) OS, defined as the time from initiation of first-line systemic therapy to death from any cause or to last monitoring for survivors, and (2) PFS, defined as the time from treatment initiation to one of the first of the following: death, biochemical clinical or radiological progression according to RCTs [START_REF] James | Addition of docetaxel, zoledronic acid, or both to first-line long-term hormone therapy in prostate cancer (STAMPEDE): survival results from an adaptive, multiarm, multistage, platform randomised controlled trial[END_REF][START_REF] Sweeney | Chemohormonal Therapy in Metastatic Hormone-Sensitive Prostate Cancer[END_REF][8][9]14 . Based on selection of the survival data published in the literature, transition probabilities were calculated using the Declining Exponential Approximation of Life Expectancy (DEALE) method for each health state (Table 1) [START_REF] Beck | A convenient approximation of life expectancy (the "DEALE"). I. Validation of the method[END_REF][START_REF] Beck | A convenient approximation of life expectancy (the "DEALE"). II. Use in medical decision-making[END_REF] .

MODEL ESTIMATES: COSTS

The cost-effectiveness analysis (CEA) was performed from the perspective of the French public healthcare system. Only direct medical costs were included, from the start of treatment until death of patient (lifetime horizon), including cost related to treatment (hospitalization, in-and outpatient treatments), monitoring (radiological, clinical and biological), medical consultations and management of adverse events (appendix 2, 3, 4, 5 and 6). Minor costs and costs considered independent of the treatment regimen were not taken into account. Costs are expressed in Euros (reference year 2020).

A healthcare resource utilization model was built based on data from summaries of product characteristics of selected drugs, international guidelines, RCTs and consensus conferences (Appendix 5) [START_REF] Mottet | European Association of Urology (EAU) -European Association of Nuclear Medicine (EANM) -European SocieTy for Radiology and Oncology[END_REF][START_REF] James | Addition of docetaxel, zoledronic acid, or both to first-line long-term hormone therapy in prostate cancer (STAMPEDE): survival results from an adaptive, multiarm, multistage, platform randomised controlled trial[END_REF][START_REF] Sweeney | Chemohormonal Therapy in Metastatic Hormone-Sensitive Prostate Cancer[END_REF][8][9]14,[START_REF] Gillessen | Management of Patients with Advanced Prostate Cancer: Report of the Advanced Prostate Cancer Consensus Conference 2019[END_REF][START_REF]Network -Evidence-Based Cancer Guidelines, Oncology Drug Compendium[END_REF] . A committee of 8 experts (medical oncologists, radiation oncologists, urologists) then validated it.

The cost of a treatment administrated in hospital (consultation, drug and administration) is included in a French public Diagnosis-Related Group (DRG), with the exception of drugs deemed as innovative and expensive such as cabazitaxel (http://atih.sante.fr). For instance, the DRG "28Z07Z" for outpatient chemotherapy session is estimated at €384.0.

For each reimbursed drug administered in ambulatory settings, the exact used number of units per patient has been determined according to the posology and multiplied by its unit purchase price (Appendix 2). Unit prices of drugs were obtained from wholesale price lists from official tariffs (http://ameli.fr). Because of the large number of options available to administer ADT, an average cost of €124.6 has been applied to represent ADT use. All drugs were reimbursed at 100%. The cost of radiological, clinical and biological monitoring for each strategy during mHSPC and mCRPC were based on official tariffs (http://ameli.fr) (Appendix 3, Appendix 4).

Each medical consultation was valued according the specialty of physician: €23 for general practitioner, €28 for specialist physician such as medical or radiation oncologist or urologist (http://ameli.fr) (Appendix 5).

Based on the selected phase III RCTs, common adverse events for grade 3 and 4 (≥2%) have been identified and associated with a principal diagnosis for the event according to the International Classification of Diseases 10 th revision code (ICD-10 code) (Appendix 6). Each ICD-10 code is associated with one DRG and then one cost.

BASE-CASE PRESENTATION AND SENSITIVITY ANALYSES

The Markov decision-analysis model and sensitivity analyses were conducted using TreeAge Pro Suite software (TreeAge Software, Inc., Williamstown, MA, USA). Effectiveness and direct medical costs were measured over 12 months, discounted according to 2.5% rate according to the French recommendations [START_REF]Methodological Guidance 2020 -Choices in methods for economic evaluation[END_REF] . Strategies were ranked using an efficiency frontier. To do this, four strategies in cohort A and three strategies in cohort B were ranked from the least expensive to the most one, respectively. Each dominated strategy (i.e. more expensive and less effective than the previous one) and each extended dominated strategy (i.e. with higher ICER, which is greater than that of a more effective strategy) were excluded from further analysis. Incremental Cost-Effectiveness Ratios (ICERs) were calculated using the following formula: (CostStrategyA-CostStrategyB)/(EffectivenessStrategyA-EffectivenessStrategyB).

The robustness of base-case analysis was assessed through a series of deterministic one-way sensitivity analyses. Firstly, the potential impact of STAMPEDE indirect comparison of docetaxel versus AA results was assessed by applying a new transition probability based on one-quarter decrease in PFS of first-line docetaxel [START_REF] Sydes | Adding abiraterone or docetaxel to long-term hormone therapy for prostate cancer: directly randomised data from the STAMPEDE multi-arm, multi-stage platform protocol[END_REF] . Secondly, the potential cross-resistance between AA and enzalutamide were taken into account by applying the new transition probability based on a 50% decrease in PFS of second-line enzalutamide for the strategy SA3ABI ENZA [START_REF] De Wit | Cabazitaxel versus Abiraterone or Enzalutamide in Metastatic Prostate Cancer[END_REF][START_REF] Khalaf | Optimal sequencing of enzalutamide and abiraterone acetate plus prednisone in metastatic castration-resistant prostate cancer: a multicentre, randomised, open-label, phase 2, crossover trial[END_REF] . Thirdly, the introduction of generic cabazitaxel, AA and enzalutamide into the European market in the near future will be associated with a price reduction assumed to be between 30 and 70%. We investigated such a reduction from the original price of 30, 50 and 70%.

BASE-CASE ANALYSIS

For cohort A, the least costly strategy is SA1DOCE ABI, at a cost of €96,925 for 4.24 life-years (LYs) (Table 2). With the same effectiveness (4.25 LYs), the strategy SA2DOCE ENZA is more expensive, at a cost of €102,090. Strategies SA4ABI DOCE and SA3ABI ENZA are dominated due to the greater cost and lower effectiveness.

For cohort B, the least costly strategy is SB2DOCE DOCE, at a cost of €81,463 for 4.05 LYs (Table 3). With the same effectiveness (4.07 LYs), the strategy SB1DOCE CABA is more expensive, at a cost of €105,748. Strategy SB3ABI DOCE is dominated due to the greater cost and lower effectiveness.

SENSITIVITY ANALYSES

The robustness of the base-case result was confirmed for cohort A when either a 50% decrease of PFS of enzalutamide, or a cost reduction of AA and enzalutamide of 30% or 50% was applied (Table 2). However, a cost reduction of AA and enzalutamide of 70% influenced the base-case results; SA3ABI ENZA become efficient at the threshold value of social willingness to pay of €50,000/LY in comparison with SA4ABI DOCE.

It is also confirmed for cohort B (Table 3). Only, a cost reduction of AA and cabazitaxel of 70% influences the base-case result; the strategy SB3ABI DOCE is then the least costly and effective and SB2DOCE DOCE and SB1DOCE CABA are not efficient at the threshold value of social willingness to pay of €50,000/LY in comparison with SB3ABI DOCE and SB2DOCE DOCE respectively.

DISCUSSION

Cost-utility analysis is the preferred EE method whether health-related quality of life is identified as an important health effect of interventions. Unfortunately, not all health state utility values (HSUV) essential to develop one were available in the literature. In addition, the choice of population used to elicit HSUVs is heterogeneous, and none are reported for French patients specifically. As a consequence, we conducted a CEA.

The chosen model dividing into two cohorts based on symptoms when patient becomes castrate resistant can be criticized, when currently majority of patients receive either AA or enzalutamide in first-line castrate resistant. From that perspective our model is covering this possibility as shown in figure 1a, therefore the main patients' flow is covered. However, the choice to describe this 2 possible scenarii was deliberate while taking into account the European label of AA and enzalutamide as the first-line in castrate resistant stage. On the same line, the eligibility criteria of the phase III registration trials COUAA302 and PREVAIL selected patient asymptomatic or mildly symptomatic from mPC, without opioid consumption 13,14 .

The results of our CEA suggest that docetaxel for patient with mHSPC is cost-effective in comparison with AA. This result is consistent with the literature [START_REF] Hu | Abiraterone acetate and docetaxel with androgen deprivation therapy in high-volume metastatic hormone-sensitive prostate cancer in China: an indirect treatment comparison and cost analysis[END_REF][START_REF] Sathianathen | A Cost-effectiveness Analysis of Systemic Therapy for Metastatic Hormone-sensitive Prostate Cancer[END_REF][START_REF] Chiang | Cost-effectiveness analysis of Abiraterone Acetate versus Docetaxel in the management of metastatic castration-sensitive prostate cancer: Hong Kong's perspective[END_REF][START_REF] Hird | Abiraterone vs. docetaxel for metastatic hormone-sensitive prostate cancer: A microsimulation model[END_REF][START_REF] Ramamurthy | Cost-effectiveness of abiraterone versus docetaxel in the treatment of metastatic hormone naïve prostate cancer[END_REF] . Strategies SA1DOCE ABI and SA2DOCE ENZA are dominant for cohort A, while SB2DOCE DOCE and SB1DOCE CABA are dominant for cohort B.

For cohort A, the choice of first-line treatment in mCRPC can be either AA, or enzalutamide, but enzalutamide is €5,165 more when compared with AA (€102,090 versus €96,925). However, because enzalutamide does not require corticosteroids, and does not require frequent blood work to assess liver function and electrolytes, it may be easier to use and a more practical choice for some patients.

For cohort B, the choice of first-line treatment in mCRPC can be either docetaxel, or cabazitaxel, but cabazitaxel is €24,285 more when compared with docetaxel (€105,748 versus €81,463). Cabazitaxel was developed for use in patients who had disease progression after treatment with docetaxel, and has significant benefit in the post-docetaxel setting due to lack of complete cross-resistance [START_REF] Calcagno | Safety and efficacy of cabazitaxel in the docetaxeltreated patients with hormone-refractory prostate cancer[END_REF] .

The uncertainty related to clinical estimates and cost estimates and assumptions were taken into account in the sensitivity analyses (only deterministic not probabilistic) to assess the robustness of results. The latter was confirmed. However, a cost reduction of AA, enzalutamide and cabazitaxel by 70% influenced the base-case result. The introduction of generic AA, enzalutamide and cabazitaxel into the European market in the near future is expected to be associated with a price reduction of 70%, and will modify the results of the CEA and may influence daily practice. More recently, trials led to the use of additional androgen receptor directed therapies (enzalutamide and apalutamide) to be used in combination with ADT for mHSPC [START_REF] Chi | Apalutamide for Metastatic, Castration-Sensitive Prostate Cancer[END_REF][START_REF] Armstrong | ARCHES: A Randomized, Phase III Study of Androgen Deprivation Therapy With Enzalutamide or Placebo in Men With Metastatic Hormone-Sensitive Prostate Cancer[END_REF][START_REF] Davis | Enzalutamide with standard first-line therapy in metastatic prostate cancer[END_REF] . These results will also need to be updated by the availability and coverage of new treatments such as apalutamide or enzalutamide in mHSPC setting.

However, our results need to be viewed within its limited context, i.e. a Markov decisionmodel with hypothetical cohort of patients with characteristics derived from selected RCTs. Clinical parameters of both models derived from results of selected phase III RCTs and may not be generalizable to the broader patient population. It was possible to choose between the results of FIRSTANA and PROSELICA to calculate transition probabilities related to cabazitaxel for strategy SB1DOCE CABA. The model incorporated data from FIRSTANA because it allowed homogenization in terms of first-line of chemotherapy in mCRPC whereas PROSELICA included second-line chemotherapy in mCRPC 15 . Moreover, there are no largescale published prospective data detailing the sequence of docetaxel in mHSPC followed by cabazitaxel as first-line therapy for mCRPC.

The assessment of PFS differed according to selected RCTs (clinical, radiological or tumor)

at the origin of a potential bias. We can also underline that we expect only limited benefit to treatment with enzalutamide in patients previously treated with AA for mHSPC (SA3ABI ENZA), due to similar mechanisms of cross resistance for these treatments [START_REF] Khalaf | Optimal sequencing of enzalutamide and abiraterone acetate plus prednisone in metastatic castration-resistant prostate cancer: a multicentre, randomised, open-label, phase 2, crossover trial[END_REF] . The choice of DEALE method is questionable as long as other methods exist to extrapolate survival data.

Out-of-pocket, indirect and intangible medical costs were not taken into account as costs for loss of productivity. The cost for each strategy has therefore been underestimated.

Nevertheless, large out-of-pocket costs related to things like missed work and other indirect expenses are relatively unlikely given the mean age of patients with mHSPC.

To our knowledge, no EE has previously been published describing the cost-effective of two successive lines of treatment in patients with mPC. Published EE have been restricted to one line of treatment: either treatment for mHSPC, or first-or second-line treatment for mCRPC [START_REF] Hu | Abiraterone acetate and docetaxel with androgen deprivation therapy in high-volume metastatic hormone-sensitive prostate cancer in China: an indirect treatment comparison and cost analysis[END_REF][START_REF] Sathianathen | A Cost-effectiveness Analysis of Systemic Therapy for Metastatic Hormone-sensitive Prostate Cancer[END_REF][START_REF] Chiang | Cost-effectiveness analysis of Abiraterone Acetate versus Docetaxel in the management of metastatic castration-sensitive prostate cancer: Hong Kong's perspective[END_REF][START_REF] Hird | Abiraterone vs. docetaxel for metastatic hormone-sensitive prostate cancer: A microsimulation model[END_REF][START_REF] Ramamurthy | Cost-effectiveness of abiraterone versus docetaxel in the treatment of metastatic hormone naïve prostate cancer[END_REF][START_REF] Barqawi | Cost-effectiveness model of abiraterone plus prednisone, cabazitaxel plus prednisone and enzalutamide for visceral metastatic castration resistant prostate cancer therapy after docetaxel therapy resistance[END_REF][START_REF] Kreis | Treatment-Related Healthcare Costs of Metastatic Castration-Resistant Prostate Cancer in Germany: A Claims Data Study[END_REF][START_REF] Flannery | Budgetary Impact of Cabazitaxel Use After Docetaxel Treatment for Metastatic Castration-Resistant Prostate Cancer[END_REF][START_REF] Kearns | Cabazitaxel for Hormone-Relapsed Metastatic Prostate Cancer Previously Treated With a Docetaxel-Containing Regimen: An Evidence Review Group Perspective of a NICE Single Technology Appraisal[END_REF][37] . In addition, none of these were developed from a French or European healthcare system perspective. Thus, comparison with our study is limited due to our inclusion of multiple lines of therapy, and the populations assessed. We believe the innovative approach of this study in defining optimal sequencing for patients moving from mHSPC to mCRPC is unique, and the results add useful information to clinician and patients facing treatment decisions in the real world.

In conclusion, the management of mPC has been significantly modified by the availability of effective but expensive treatments. The best therapeutic sequence in terms of cost and effectiveness has not been previously defined. Our CEA suggests that docetaxel in the mHSPC setting is cost-effective, and can be followed by either AA, or a second round of docetaxel. Making choices based on the degree of symptomatology may lead clinicians to use more docetaxel followed by another taxane in patients with a greater symptom burden. In the next version of this analysis, we will incorporate newer treatments such as apalutamide and enzalutamide in patients with mHSPC.

Fizazi

FIGURESFigure 1 : 7 SA2

 17 FIGURES

Figure 2 :

 2 Figure 2: Markov Health states and transitions

  K, Tran N, Fein L, et al. Abiraterone plus Prednisone in Metastatic, Castration-

	37. Pilon D, Queener M, Lefebvre P, Ellis LA. Cost per median overall survival month
	Sensitive associated with abiraterone acetate and enzalutamide for treatment of patients with Prostate Cancer. N Engl J Med. 2017;377(4):352-360.
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	for metastatic castration-resistant prostate cancer progressing after docetaxel treatment: a
	randomised open-label trial. Lancet. 2010;376(9747):1147-1154. doi:10.1016/S0140-
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	10. Tannock IF, de Wit R, Berry WR, et al. Docetaxel plus prednisone or mitoxantrone plus
	prednisone for advanced prostate cancer. N Engl J Med. 2004;351(15):1502-1512.
	doi:10.1056/NEJMoa040720
	11. Parker C, Nilsson S, Heinrich D, et al. Alpha emitter radium-223 and survival in
	metastatic	prostate	cancer.	N	Engl	J	Med.	2013;369(3):213-223.
	doi:10.1056/NEJMoa1213755
	12. Kantoff PW, Higano CS, Shore ND, et al. Sipuleucel-T immunotherapy for castration-
	resistant	prostate	cancer.	N	Engl	J	Med.	2010;363(5):411-422.
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	Therapy for Patients With Metastatic Castration-Resistant Prostate Cancer: A

Table 1 :

 1 TABLES Randomized clinical trials used to determine transition probabilities

	Study

Abbreviations: ADT = Androgen Deprivation Therapy; CI = Confidence Interval; cPFS = clinical Progression-Free Survival, mCRPC = metastatic Castration Resistant Prostate Cancer; mHSPC = metastatic Hormone Sensitive Prostate Cancer; NR = not reached; rPFS = radiological Progression-Free Survival; SA = Strategy of cohort A; SB = Strategy of cohort B; tPFS = tumor Progression-Free Survival; vs = versus *all strategies are described in Figure 1

Table 2 :

 2 Base-case results and deterministic one-way sensitivity analyses for cohort A

	Strategy*	Cost (€)	Effectiveness (LYs)	Incremental cost (€)	Incremental effectiveness (LYs)	ICER (€/LYs)
	Base-case analysis
	SA1DOCE ABI	96,925	4.24	-	-	Reference
	SA2DOCE ENZA	102,090	4.25	5,165	0.01	476,769
	SA4ABI DOCE	149,865	3.97	47,775	-0.27	Dominated
	SA3ABI ENZA	168,383	4.15	18,518	0.17	Dominated
	Sensitivity analysis: 25% decrease of PFS of first-line docetaxel used in the base-case analysis
	SA1DOCE ABI	86,641	3.73	-	-	Reference
	SA2DOCE ENZA	92,485	3.75	5,844	0.01	Ext. dominated
	SA4ABI DOCE	149,865	3.97	57,380	0.23	Ext. dominated
	SA3ABI ENZA	168,383	4.15	18,518	0.17	198,966
	Sensitivity analysis: 50% decrease of PFS of second-line enzalutamide post-abiraterone acetate plus prednisone used in the base-case
	analysis
	SA1DOCE ABI	96,925	4.24	-	-	Reference

Table 3 :

 3 Base-case results and deterministic one-way sensitivity analyses for cohort B

	Strategy*	Cost (€)	Effectiveness (LYs)	Incremental cost (€)	Incremental effectiveness (LYs)	ICER (€/LYs)
	Base-case analysis
	SB2DOCE DOCE	81,463	4.05	-	-	Reference
	SB1DOCE CABA	105,748	4.07	24,285	0.02	1,267,043
	SB3ABI DOCE	149,865	3.97	44,117	-0.10	Dominated
	Sensitivity analysis: 25% decrease of PFS of first-line docetaxel used in the base-case analysis
	SB2DOCE DOCE	69,473	3.53	-	-	Reference
	SB1DOCE CABA	96,228	3.55	26,755	0.02	Ext. dominated
	SB3ABI DOCE	149,865	3.97	53,637	0.43	179,982
	Sensitivity analysis: cost reduction of abiraterone acetate plus prednisone and cabazitaxel of 30%
	SB2DOCE DOCE	81,463	4.05	-	-	Reference
	SB1DOCE CABA	98,629	4.07	17,166	0.02	895,617
	SB3ABI DOCE	115,909	3.97	17,280	-0.10	Dominated
	Sensitivity analysis: cost reduction of abiraterone acetate plus prednisone and cabazitaxel of 50%

Abbreviations: ADT = Androgen Deprivation Therapy; Ext. dominated = Extended dominated; ICER = Incremental Cost Effectiveness Ratio; LYs = Life-Years; SB = Strategy of cohort B

CONFLICT OF INTEREST AND FUNDING STATEMENT

Conflicts of interest: none. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.