D ' Huart

Pharm Resident

PharmD Vigneron Jean

Ph Clarot Igor

Demore D²

Ph.D Béatrice

Elise D'huart
email: dhuartelise@gmail.com

Physicochemical stability of Norepinephrine bitartrate in Polypropylene Syringes at High Concentrations for Intensive Care Units. Étude de stabilité physico-chimique de noradrénaline bitartrate dans des seringues

Keywords: norepinephrine, intensive care unit, HPLC, stability norepinephrine, intensive care unit, HPLC, stability

HAL is

HIGHLIGHTS

-In the literature, to the best of our knowledge, there is no stability data available on norepinephrine at high concentrations up to 1.16 mg/mL.

-This drug is usually used in intensive care units for the restoration of blood pressure.

-These stability data of highly-concentrated solutions provide additional knowledge to assist intensive care services in daily practice.

Objectifs

La noradrénaline est généralement utilisée dans des situations d'urgence, telles que dans les unités de soins intensifs, pour rétablir la tension artérielle. L'objectif était d'étudier la stabilité de solutions hautement concentrées de noradrénaline à 0,50 mg/mL et 1,16 mg/mL, diluées dans du glucose à 5% (G5%) dans des seringues en polypropylène, protégées ou non de la lumière, jusqu'à 48 heures de conservation.

Matériels et méthodes

La stabilité chimique a été analysée par chromatographie liquide à haute performance couplée à un détecteur à barrette de diode, à chaque moment de l'analyse. La méthode a été validée conformément à l'International Conference on Harmonisation Q2(R1). La stabilité physique a été évaluée par un examen visuel et subvisuel. Trois seringues pour chaque condition ont été préparées. A chaque moment de l'analyse, trois échantillons ont été analysés pour chaque seringue. Les valeurs de pH ont été évaluées à chaque moment de l'analyse.

Résultats

Les solutions de noradrénaline à 0,50 et 1,16 mg/mL, diluées dans du G5%, avec ou sans protection contre la lumière, ont conservé plus de 95% de leur concentration initiale après une conservation de 48 heures à 20-25 °C. Les solutions sont restées claires, sans changement de couleur ni précipitation pendant la durée de l'étude. Concernant l'examen subvisuel, les valeurs d'absorbance étaient inférieures à 0,010 UA. Aucun produit de dégradation mis en évidence lors de la dégradation forcée n'a été observé au cours de l'étude. Un pic supplémentaire avec une rétention relative à 0,66 a été observé et était constant durant l'étude. Ce pic a été identifié comme étant du 5hydroxyméthylfurfural, un produit de dégradation du glucose.

Conclusion

La noradrénaline diluée dans du G5% à 0,50 mg/mL et à 1,16 mg/mL a été démontrée physiquement et chimiquement stable sur une période de 48 heures à température ambiante. Ces données de stabilité de solutions hautement concentrées fournissent des connaissances supplémentaires pour aider les services de soins intensifs dans leur pratique quotidienne.

INTRODUCTION

Norepinephrine bitartrate is a drug of the catecholamine family. This drug is usually used in emergency situations such as in intensive care units (ICUs) for the restoration of blood pressure in case of acute hypotension. The recommended initial dose for continuous infusion is a flow rate of 0.1 to 0.3 µg/kg/min of norepinephrine. High doses from 3 to 5 µg/kg/min can be used in the treatment of septic shock or hemorrhagic shock [START_REF]Norepinephrine sulphite-free 2 mg/mL. Solution for solution for infusion. Summary of Product Characteristics[END_REF].

To avoid fluid overload, ICUs often use a minimum volume of infusion by using more concentrated drug solutions. High doses of norepinephrine can be administered over 24 hours with an electrical syringe pump. In clinical practice in ICUs, nurses use a final volume of 48 mL in syringes to have an accurate flow rate of 2 mL/h over 24 hours.

In the literature, to the best of our knowledge, no stability studies show the stability of highlyconcentrated norepinephrine syringes at room temperature, only stability studies on highly-diluted solutions of norepinephrine are available. Peddicord et al. [START_REF] Peddicord | Stability of high-concentration dopamine hydrochloride, norepinephrine bitartrate, epinephrine hydrochloride, and nitroglycerin in 5% dextrose injection[END_REF] determined a 24-hour stability for norepinephrine solutions at 0.064 mg/mL diluted in 0.9% sodium chloride (0.9% NaCl) or 5% glucose (G5%) in PVC bags, stored at 23°C, protected from light. Baumgartner et al. [START_REF] Baumgartner | Norepinephrine stability in saline solutions[END_REF] determined a 24-hour stability for norepinephrine solutions at 0.004 and 0.008 mg/mL diluted in 0.9% NaCl or G5% in PVC bags, stored at 25°C, without protection from light. Walker et al. studied the stability of norepinephrine at 0.064 µg/mL in 0.9% NaCl and in G5% at 4°C for up to 61 days [START_REF] Law | Stability of Norepinephrine Solutions in Normal Saline and 5% Dextrose in Water[END_REF]. Brustugun et al. observed that light is a factor of instability for norepinephrine solutions [START_REF] Brustugun | Photostability of sympathomimetic agents in commonly used infusion media in the absence and presence of bisulfite[END_REF]. Alwood et al. demonstrated the influence of a pH superior to 6.0 on the stability of norepinephrine. They determined a higher stability with G5% as solvent in comparison with 0.9% NaCl [START_REF] Allwood | The stability of four catecholamines in 5% glucose infusions[END_REF].

The objective was to study the stability of norepinephrine solutions at 0.50 mg/mL and 1.16 mg/mL, diluted in G5% in polypropylene syringes, protected or not from light, after the preparation and after a 6-hour, 24-hour and 48-hour storage at room temperature. The concentration at 0.50 mg/mL corresponds to a one-day syringe for a dose at 0.24 µg/kg/min for a patient with an average weight of 70 kg. The concentration at 1.16 mg/mL corresponds to a dose at 0.56 µg/kg/min.

MATERIALS AND METHOD

Stability study of norepinephrine

Chemicals and reagents

For the mobile phase, heptane sulfonic acid sodium salt (VWR Chemicals, batch: 18B134102), orthophosphoric acid 85% (VWR Chemicals, batch: 15D200503) and acetonitrile (Carlo Erba Reagents, batch: D8E017238E) were used. Hydrochloric acid 1M (VWR Chemicals, batch: 17110005) and 0.1M (VWR Chemicals, batch: 17110001), sodium hydroxide 1M (VWR Chemicals, batch: 17110003) and 0.1 M (VWR Chemicals, batch: 17070001), hydroxide peroxide 30 % (Merck, batch: K48743810713) and 5-hydroxymethyl-2-furaldehyde (5-HMF) 99% (Aldrich Chemistry, batch: MKAA1503) were used for the forced degradation and the validation of the analytical method. Water for chromatography was obtained from a reverse osmosis system (Millipore Iberica, Madrid, Spain). Norepinephrine 8 mg/mL sulphite-free, solution for solution for infusion (Mylan, batch: H2051), G5% Lavoisier 500 mL in a glass vial (Chaix et du Marais, Lavoisier, batch : 8F006) were used for test solutions.

Preparation of test solutions

Stability study in polypropylene syringes: for the preparation of the concentration at 0.50 mg/mL, three ampoules of norepinephrine 8 mg/4 mL were used. For the preparation of the concentration at 1.16 mg/mL, seven ampoules of norepinephrine 8 mg/4 mL were used. These solutions were stored in polypropylene syringes (BD Plastipak TM , 50 mL BD Luer-lok TM syringe, Ref: 300865, batch: 1808208) or in amber polypropylene syringes (BD Plastipak TM , 50 mL BD Luer-lok TM syringe, Ref:

300869, batch: 1807228). The final volume of the syringe was completed with G5% to obtain 48 mL.

The syringes were stored at room temperature (20-25°C), protected or not from light. Three syringes were prepared for each condition.

HPLC Assay

Norepinephrine solutions were analysed by a stability-indicating reversed-phase high-performance liquid chromatography (RP-HPLC) method with photodiode array detection [START_REF] Law | Stability of Norepinephrine Solutions in Normal Saline and 5% Dextrose in Water[END_REF].

The HPLC system consisted of an ELITE LaChrom VWR/Hitachi plus autosampler, a VWR photodiode array (PDA) detector L-2455 and a VWR L-2130 HPLC-pump. Data was acquired and integrated by using EZChrom Elite (VWR, Agilent). The column used was LiChrospher® 100 RP-18, LiChroCART® 125-4, 12.5 cm in length, 5 µm particle size (Analytical Chromatography, Merck).

The mobile phase was constituted of a mixture of acetonitrile (acetonitrile HPLC gradient grade, VWR, France) and 0.05M phosphoric acid containing 1 mg/mL of heptane sulfonic acid sodium salt.

The flow rate was set at 1.0 mL/minute, with an injection volume of 10 µL. The detection wavelength was set at 280 nm. The temperature of the injector was set at 20°C and the temperature of the column oven at 25°C. The calibration curve was constructed from plots of peak area versus concentration. The linearity of the method was evaluated with five concentrations (80, 100, 120, 140 and 160 µg/mL). mL. This solution at 400 mg/mL was used to realise standard curves by dilution with ultrapure water.

The intra-day reproducibility was evaluated as recommended by the International Conference on Harmonisation (ICH) Q2 (R1) [START_REF]International Conference on Harmonisation: Validation of Analytical Procedures: Text and Methodology Q2(R1)[END_REF], using three determinations for each concentration at 80, 120 and 160 µg/mL. For interday precision, three determinations for each concentration at 80, 120 and 160 µg/mL of norepinephrine solutions were assayed daily on three different days. To demonstrate the specificity of the method and the absence of interaction between norepinephrine and the solvent, a solution of G5% was analysed by HPLC.

The evaluation of the stability in the autosampler was performed. Solutions of norepinephrine diluted in ultrapure water were stored in the autosampler at 25°C. Chemical stability was evaluated at different times up to 24 hours.

The Diode Array Detector allows the evaluation of the UV spectrum of the chromatographic column effluent every 0.4 second, thus allowing evaluation of the UV purity of an eluting peak. Variations in the UV spectrum over the elution profile of the peak of interest would indicate that the peak is contaminated, that the analytical method does not separate norepinephrine from its degradation products, and that the method is therefore unsuitable [START_REF] Metha | Practice research: Strategies for stability studies on hospital pharmaceutical preparations[END_REF].

The stability-indicating capability was evaluated by analysing forced degraded norepinephrine solutions.

Acidic conditions: one mL of a 480 µg/mL norepinephrine solution was diluted with 1.0 mL HCl 0.5 M, stored at 20-25°C for 6 hours, neutralised by 1.0 mL of NaOH 0.5 M and diluted with 1.0 mL of ultrapure water to obtain a theoretical concentration of 120 µg/mL. Alkaline degradation: one mL of a 480 µg/mL norepinephrine solution was diluted with 1.0 mL NaOH 0.01 M, stored at 20-25°C for 5 minutes, neutralised by 1.0 mL of HCl 0.01 M and diluted with 1.0 mL of ultrapure water to obtain a theoretical concentration of 120 µg/mL.

Oxidative degradation: one mL of a 480 µg/mL norepinephrine solution was diluted with 1.0 mL H2O2 30% stored at 20-25°C and diluted with 2.0 mL of ultrapure water to obtain a theoretical concentration of 120 µg/mL. Heat degradation: a solution of 120 µg/mL norepinephrine was exposed to a temperature of 100°C for 6 hours.

UV degradation: a solution of 120 µg/mL norepinephrine was exposed for 4, 6, 25 and 30 hours under a sun-like spectrum lamp at 254 nm (Vilbert Lourmat®).

Sample dilution for analysis by RP-HPLC

At each time of the analysis, 5 mL were removed from each syringe. The solutions were diluted before analysis with ultrapure water to obtain a concentration at 120 µg/mL (central point of the standard curve) for syringes at 0.50 mg/mL and a concentration at 116 µg/mL for syringes at 1.16 mg/mL. Samples were prepared in triplicate for each syringe. After dilution, each sample was analysed by RP-HPLC. This process was repeated after 6, 24 and 48 hours. Total run time was set at 20 minutes.

Chemical stability was defined as not less than 90% of the initial norepinephrine concentration and in relation with the evolution of potential degradation products [START_REF]International Conference on Harmonisation: Validation of Analytical Procedures: Text and Methodology Q2(R1)[END_REF][START_REF] Sautou | Methodological guidelines for stability studies of hospital pharmaceutical preparations[END_REF]. pH measurement pH measurement was performed using a Bioblock Scientific pH meter. Analysis was carried out for each concentration after preparation and after 6, 24 and 48 hours. pH values were considered to be acceptable if they did not vary by more than 1.0 pH unit from the initial measurement [START_REF] Sautou | Methodological guidelines for stability studies of hospital pharmaceutical preparations[END_REF]. We measured pH for each syringe, for each condition.

Determination of physical stability

Physical stability was defined as the absence of particle formation, haze, colour change and gas evolution [START_REF] Bardin | Guidelines for the practical stability studies of anticancer drugs: A European consensus conference[END_REF]. The samples were visually inspected against a white/black background with the unaided eye by two different technicians at each analysis time. The subvisual aspect was assessed by using a Safas Monaco UV mc 2 spectrophotometer. The absorbance light was scanned at 350, 410 and 550 nm [START_REF] Bardin | Guidelines for the practical stability studies of anticancer drugs: A European consensus conference[END_REF].

RESULTS

Stability study of norepinephrine

Reversed Phase HPLC

The calibration curve was linear, the determination coefficient was 0.9997. The equation of the calibration curve was y= 18940.8 x -61132.9. The intra-day precision expressed as relative standard deviation (RSD) was between 0.07% and 2.65%. The intermediate precision expressed as relative standard deviation (RSD) was 1.43% at 80 µg/mL, 0.32% at 120 µg/mL and 0.66% at 140 µg/mL.

For the evaluation of the stability in the autosample, solutions were stable with a degradation rate less than 0.3%.

The UV spectral purity of the norepinephrine peak in chromatograms of the degraded samples was compared with the spectrum of the undegraded sample of norepinephrine obtained at time 0. These procedures were in accordance with the acceptance standard.

Stability-indicating capacity was proved by using various stressed conditions. The retention time of norepinephrine was around 5.8 minutes. The chromatogram of norepinephrine without stressed degradation is presented in figure 1 and after alkaline stressed condition is presented in figure 2 for example.

After acidic, alkaline and heat stressed degradations, 9.2 %, 24.7 % and 10.9 % of norepinephrine were degraded respectively. In oxidative and UV conditions, norepinephrine was not degraded after adding H2O2 30% or after exposition under a sun-like spectrum lamp. The mass balance and the relative retentions calculated versus the norepinephrine peak are presented in table 1.

Chemical stability of solutions

HPLC Assay

For specificity evaluation, the chromatogram of a solution of G5% is presented in figure 3. A peak with a retention time of 3.8 minutes and a relative retention at 0.66 is observed.

The percentages of norepinephrine diluted in G5% at 0.50 mg/mL and 1.16 mg/mL after storage at 20-25°C for various time points are shown in table 2. The chromatogram of norepinephrine at 0.50 mg/mL after preparation is presented in figure 4. An additional peak was observed for each sample with a retention time of 3.8 minutes (relative retention at 0.66). No degradation product that appeared during stressed degradation was observed during the stability study.

pH measurements

During the stability study, no modification of pH measurements was observed. Table 3 summarizes the pH values obtained for the 3 syringes, at each condition, after preparation and after 48 hours.

Physical stability of solutions

Visual aspect

No visual modification was observed during all the stability study. Solutions remained limpid, without change of colour, precipitation or gas formation.

Subvisual evaluation

Concerning turbidity assays, no change was observed. Whatever the storage conditions and the concentrations, values of absorbance at 350, 410 and 550 nm remained inferior to 0.010 AU.

DISCUSSION

This study demonstrated that solutions of norepinephrine at 0.50 and 1.16 mg/mL, diluted in G5%, with or without protection from light, retained more than 95.0 % of the initial concentration after a 48hour storage at 20-25°C. This result is in accordance with a poster realised by Velghe et al. [START_REF] Velghe | Stabilité de la noradrénaline conditionnée en poche et en seringue aux concentrations thérapeutiques usuelles[END_REF]. They determined a 28-day stability for norepinephrine at 0.2 and 0.5 mg/mL in 0.9% NaCl and G5%. Walker et al. observed a better stability with solutions of norepinephrine protected from light in comparison with solutions not protected [START_REF] Law | Stability of Norepinephrine Solutions in Normal Saline and 5% Dextrose in Water[END_REF]. In our stability study, we have not observed differences between results obtained for solutions protected or not from light. Light seems to have an impact less important for high concentrations of norepinephrine solutions than for diluted solutions.

During the evaluation of the specificity, a G5% solution was analysed by HPLC and a peak was observed on the chromatogram with a retention time of 3.8 minutes. This peak was absent during the evaluation of the linearity, repeatability and the intermediate precision of the analytical method because all samples of norepinephrine were diluted in ultrapure water. During the stability study, norepinephrine was diluted in G5% and an additional peak with a retention time of 3.8 minutes was equally observed on chromatograms.

For the identification of this additional peak, a solution of 5-hydroxymethylfurfural (5-HMF) at 0.05 mg/mL in ultrapure water was prepared and analysed by HPLC. 5-HMF is a product formed during thermal processing or heat sterilization of glucose. The maximum absorption of 5-HMF was obtained at a wavelength of 284 nm, which is close to the wavelength used by our analytical method for the determination of norepinephrine (280 nm) [START_REF] Zhang | Determination of Furfural and Hydroxymethyl furfural by UV Spectroscopy in ethanol-water hydrolysate of Reed[END_REF]. The analysis by HPLC of this solution showed a peak of 5-HMF with a retention time of 3.8 minutes as observed in figure 5. The spectrum of 5-HMF was identical to the spectrum of the peak at 3.8 minutes observed in chromatograms of norepinephrine solutions diluted in G5% and the chromatogram of the G5% solution. The additional product with a retention time of 3.8 minutes observed on the chromatogram of norepinephrine diluted in G5% during the stability study is identified as 5-HMF.

In solutions of norepinephrine at 0.50 and 1.16 mg/mL diluted in G5%, 5-HMF was stable in time. For this stability study, a glass vial of G5% was used. We analysed by HPLC solutions of G5% contained in different containers (Ecoflac®, BBraun, batch: 18101450; Easyflex®, MacoPharma, batch: 17A11F; Viaflo®, Baxter, batch 18F13E4B). In all cases, we observed the peak of 5-HMF on chromatograms in different proportions. We prepared a solution of glucose at 5g/100 mL with 5 grams of glucose precisely weighed (D (+)-Glucose anhydrous, VWR Chemicals®, batch : 16K104113).

After analysis by HPLC of this solution, no peak with a retention time of 3.8 minutes was observed. This difference confirms that the peak of 5-HMF was in relation with the sterilization process of the G5%, used to prepare the syringes.

CONCLUSION

Norepinephrine diluted in G5% at 0.50 mg/mL and 1.16 mg/mL was physically and chemically stable over a period of 48 hours at room temperature. These stability data of highly-concentrated solutions provide additional knowledge to assist intensive care services in daily practice. Table1: Mass balance of norepinephrine solutions after various stressed degradations.

Tableau 1 : Bilan massique de solutions de noradrenaline après des dégradations forces.

Figure 1 :

 1 Figure 1 : Chromatogram of norepinephrine 120 µg/mL in ultrapure water without stressed conditions.

Figure 1 :

 1 Figure 1 : Chromatogramme d'une solution de noradrenaline à 120 µg/mL diluée dans de l'eau ultrapure sans conditions de degradation.

Figure 2 :Figure 2 :Figure 3 :

 223 Figure 2 : Chromatogram of norepinephrine 120 µg/mL in ultrapure water after alkaline stressed conditions (NaOH 0.01M, 5 minutes) Figure 2 : Chromatogramme d'une solution de noradrénaline à 120 µg/mL, diluée dans de l'eau ultrapure dans des conditions de stress alcalines (NaOH 0,01 M, 5 minutes)

Figure 3 :

 3 Figure 3: Chromatogramme d'une solution de glucose 5%.

Figure 4 :

 4 Figure 4 : Chromatogram of a norepinephrine solution at 0.5 mg/mL diluted in glucose5% after preparation.

Figure 4 :

 4 Figure 4 : Chromatogramme d'une solution de noradrenaline à 0,5 mg/mL diluée dans du glucose 5%, après preparation.

Figure 5 :

 5 Figure 5: Chromatogram of 5-HMF at 50 mg/L in ultrapure water.

Figure 5 :

 5 Figure 5 : Chrommatogramme d'une solution de 5-HMF à 50 mg/mL dans de l'eau ultrapure.

Table 2 :

 2 Stability of norepinephrine diluted in glucose 5% (G5%).Tableau 2 : Stabilité de la noradrenaline diluée dans du glucose 5% (G5%) Drug concentrations in samples taken at time zero were designated as 100%. *SD=Standard Deviation. Samples were prepared in triplicate.

	Mean % of initial concentration ± SD*%

Table 3 :

 3

	pH measurements
	Tableau 3 : Mesures de pH
				pH values
	Light conditions	Concentration (mg/mL)	Syringe	0 hour Mean ± SD* 48 hours
			S1
	Not protected from light	0.5 1.16	S2 S3 S1 S2	3.62 ± 0.03 3.5 ± 0.01	3.63 ± 0.03 3.50 ± 0.01
			S3
			S1
		0.5	S2	3.61 ± 0.00	3.61 ± 0.01
	Protected		S3
	from light		S1
		1.16	S2	3.49 ± 0.00	3.49 ± 0.01
			S3
	*SD: standard deviation

Mots clés : noradrénaline, soins intensifs, CLHP, stabilité

ACKNOWLEDGMENTS

Assistance with the study: Thank you to Jacques Kuhnlé for reading through it all and making corrections. Thank you to Franck Blaise, Nathalie Sobalak and Hubert Zenier for their technical assistance and their help during this study.

Financial support and sponsorship: none


Yosra Cherni

David Desseauve

Ph.D Arnaud Decatoire

Nikolaus Veit-Rubinc

Mickael Begon

M.D Fabrice H F Pierre

Ph.D Laetitia Fradet
email: laetitia.fradet@univ-poitiers.fr

M.D Nikolaus Veit- Rubinc

Ph.D Mickaël Begon

Evaluation of ligament laxity during pregnancy

Keywords: Hypermobility, Quantification, Extensometer, Clinical tests

INTRODUCTION

Pregnancy is a unique physiological condition that induces large morphologic changes (e.g. weight and posture) and also modifications of intrinsic parameters like ligamentous laxity [START_REF] Calguneri | Changes in joint laxity occurring during pregnancy[END_REF]. The ligamentous laxity is at its maximum in early childhood after this, it decreases rapidly until adulthood [START_REF] Clinch | Epidemiology of generalized joint laxity (hypermobility) in fourteen-year-old children from the UK: A population-based evaluation[END_REF]. However, in the female population, this gradual decline may be suspended and even reversed during pregnancy. In this regard, some authors explain that some hormones such as relaxine, progesterone or oestradiol which levels increase during pregnancy modify the ligament structure [START_REF] Maclennan | Serum relaxin and pelvic pain of pregnancy[END_REF][START_REF] Charlton | Correlation of estradiol in pregnancy and anterior cruciate ligament laxity[END_REF][START_REF] Reese | Hormonal Influence on the Neuromusculoskeletal System in Pregnancy[END_REF]. These hormonal changes result in physiological relaxation of the ligaments in the pelvic joints in order to enhance the passage of the baby through the birth canal [START_REF] Hakim | The genetic epidemiology of joint hypermobility: a population study of female twins[END_REF]. This loosening would enable the pelvic joint to become more flexible but, as a counterpart, would also affect the pelvic joint and the spine responses to mechanical load. Some authors hypothesize indeed that these modifications of pelvic joint and spine responses favor pelvic dysfunctions and the occurrence of pain in this body region [START_REF] Lindgren | Finger joint laxity, number of previous pregnancies and pregnancy induced back pain in a cohort study[END_REF][START_REF] Ostgaard | Influence of some biomechanical factors on low-back pain in pregnancy[END_REF]. Thus, Ostgaard and colleagues outlined the increase in ligamentous laxity as a potential risk factor for instability of the sacroiliac joints whereas a study by Lindgren and colleagues noted that the peripheral laxity is higher in women with low back pain during pregnancy [START_REF] Lindgren | Finger joint laxity, number of previous pregnancies and pregnancy induced back pain in a cohort study[END_REF]. Many questions remain however open regarding the exact hormonal factors and the mechanisms leading to ligamentous property modifications and pelvic dysfunctions since relaxine alone doesn't seem to be directly related to pelvic girdle pain. During pregnancy, hormonal changes affect not only ligaments in the pelvic joints but more generally collagen of connective tissues [START_REF] Miller | Tendon collagen synthesis at rest and after exercise in women[END_REF] and therefore ligaments and tendons in general (e.g., hand [START_REF] Nygaard | Hand problems in pregnancy[END_REF], wrist [START_REF] Marnach | Characterization of the relationship between joint laxity and maternal hormones in pregnancy[END_REF]). The observations concerning pelvic and spine joints are then extendable to the human body joints in general such as increased laxity may be related to various musculoskeletal disorders and increased pain in various joints of the body during pregnancy [START_REF] Bermas | Musculoskeletal changes and pain during pregnancy[END_REF][START_REF] Castellanos | Poster 279 Radial Neuropathy Exacerbated by Ligamentous Laxity in Pregnancy and by Chemotherapy: A Case Report[END_REF]. Hyperlaxity has also been shown to be linked with the development of pelvic floor disorders pre and post-partum [START_REF] Castellanos | Poster 279 Radial Neuropathy Exacerbated by Ligamentous Laxity in Pregnancy and by Chemotherapy: A Case Report[END_REF]. In the aforementioned studies investigating the relation between ligament laxity and disorders during pregnancy, the evolution of ligament laxity hasn't been assessed throughout pregnancy. Nevertheless, early detection of its increase would be of particular interest in order to develop prevention of various disorders appearing with pregnancy.

In the literature, there is then a wide variety of in vivo laxity assessment devices and methods. The first type of methods try to assess directly the lengthening of a ligament after applying a specific force using medical imaging [START_REF] Abramson | Relaxation of the pelvic joints in pregnancy[END_REF] or knee ligament arthrometers [START_REF] Schauberger | Peripheral joint laxity increases in pregnancy but does not correlate with serum relaxin levels[END_REF][START_REF] Dumas | Laxity of knee cruciate ligaments during pregnancy[END_REF]. This methods allow measurement of small laxity changes with an accuracy up to 0.1 mm [START_REF] Robert | Nouveau système de mesure des laxités sagittales du genou, le GNRB®. Application aux ruptures complètes et incomplètes du ligament croisé antérieur[END_REF] but is quite expensive for a clinical follow up and requires some technical expertise. Moreover, not many ligaments can be isolated.

The second category of devices proposes to evaluate indirectly the ligament laxity through the measurement of articular mobility [START_REF] Calguneri | Changes in joint laxity occurring during pregnancy[END_REF][START_REF] Lindgren | Finger joint laxity, number of previous pregnancies and pregnancy induced back pain in a cohort study[END_REF][START_REF] Ostgaard | Influence of some biomechanical factors on low-back pain in pregnancy[END_REF][START_REF] Marnach | Characterization of the relationship between joint laxity and maternal hormones in pregnancy[END_REF] even though articular mobility results from the mechanical behavior of various ligaments, tendons and capsules [START_REF] Küpper | Measuring knee joint laxity: a review of applicable models and the need for new approaches to minimize variability[END_REF]. Typically, these systems, such as the extensometer of Jobbins [START_REF] Jobbins | A Joint Hyperextensometer for the Quantification of Joint Laxity[END_REF], measure the amplitude reached by a joint of the pregnant woman for a specific torque applied on this joint. Such devices are portable, easy to use, affordable, atraumatic and reliable [START_REF] Calguneri | Changes in joint laxity occurring during pregnancy[END_REF]. The main drawback is that the proposed devices measure flexibility in upper limb joint. Now, upper joints are affected by pregnancy-related changes in laxity but less involved in pathology.

The third category of methods would be clinical tests that estimate joint flexibility. The Beighton score [START_REF] Beighton | Articular mobility in an African population[END_REF] is the most commonly used test to assess the overall ligamentous laxity. It is a nine-point score evaluating laxity in the upper and lower limbs and lumbar and hip flexibility with a score ≥4/9 required to conclude a generalized hyper joint mobility. The reliability and reproducibility of this score was suggested as good to excellent in screening for generalized joint laxity in females [START_REF] Boyle | Intrarater and Interrater Reliability of the Beighton and Horan Joint Mobility Index[END_REF] and children [START_REF] Junge | Inter-tester reproducibility and intermethod agreement of two variations of the Beighton test for determining Generalised Joint Hypermobility in primary school children[END_REF] and has already been used in pregnant women [START_REF] Calguneri | Changes in joint laxity occurring during pregnancy[END_REF][START_REF] Van Dongen | Hypermobility and peripartum pelvic pain syndrome in pregnant South African women[END_REF]. Calguneri and colleagues observed no significant difference in Beighton scores between the third trimester and postpartum [START_REF] Calguneri | Changes in joint laxity occurring during pregnancy[END_REF]. Van Dongen and colleagues used the Beighton scale to study the generalized laxity in pregnant South African women [START_REF] Van Dongen | Hypermobility and peripartum pelvic pain syndrome in pregnant South African women[END_REF]. They showed that hypermobility in this women is low (only 4.9% of the 509 pregnant women), decreasing with age, but not increasing during pregnancy. However, the main limit of the Beighton score would be the computation of the score based on the sum of one and zeros corresponding to the ability to reach or not specific joint postures.

In the literature, other more targeted clinical tests have been suggested to evaluate the lower back flexibility such as the "fingertip to floor test" [START_REF] Perret | Validity, reliability, and responsiveness of the fingertip-to-floor test[END_REF] or the "sit and reach test" [START_REF] Baltaci | Comparison of three different sit and reach tests for measurement of hamstring flexibility in female university students[END_REF]. These tests have been intensively used in epidemiologic studies [START_REF] López-Miñarro | A comparison of the sit-andreach test and the back-saver sit-and-reach test in university students[END_REF] and sport sciences [START_REF] Barbosa | Effects of resistance training on the sit-and-reach test in elderly women[END_REF]. They are easy to administer and require minimal skills training [START_REF] López-Miñarro | A comparison of the sit-andreach test and the back-saver sit-and-reach test in university students[END_REF]. To the best of our knowledge, they have never been used to assess joint laxity in pregnant women. Conversely to the Beighton score, the performance corresponds to a distance, which is continuous measure. As such, they could be more sensitive to small changes in joint laxity and then present a good alternative to assess joint laxity throughout pregnancy. The "fingertip to floor test" is a little bit easier to spread than the "sit and reach test". During the "fingertip to floor test", the distance is indeed measured between the fingertip and the floor. During the "sit and reach test", the distance is measured between the fingertip and a box. One has then to have an adequate box to be able to perform this test. However, a doubt remains about the ability for pregnant women to realize the "fingertip to floor test" at the end of pregnancy. The morphologic changes in terms of abdominal circumference, weight, and modification of the center of gravity position might indeed obstruct the ability to bend forwards as required by the "fingertip to floor test".

In this study, we conducted a longitudinal monitoring of ligament laxity at a wide range of joints in pregnant women by combining an objective measure, the extensometer, that has been proven to be efficient to detect small changes in ligament laxity [START_REF] Calguneri | Changes in joint laxity occurring during pregnancy[END_REF][START_REF] Jobbins | A Joint Hyperextensometer for the Quantification of Joint Laxity[END_REF], and simple clinical tests. The aims of this study were to determine how laxity during pregnancy evolves and to propose a simple clinical methodology that could be used in daily practice helping to prevent pathologies associated with increase in laxity. An objective quantification of laxity could allow us to carry out further studies for a better understanding of the origins of back pain and pelvic floor disorders during pregnancy.

MATERIAL AND METHODS

Population

Eligible participants were women with age over 18 years and with a BMI inferior to 40. None of the participants presented current or previous inflammatory joint disease affecting the integrity of the musculoskeletal system; hypermobility syndrome like Ehlers Danlos or Marfan syndrome. The control group was included to have a baseline value of the laxity before pregnancy. The Ethics Committee of the Poitiers Hospital and the National Agency of Drug Safety reviewed and approved this study protocol and each participant signed a written informed consent.

The metacarpophalangeal joint laxity, hip and lumbar flexibility and generalized joint laxity were measured in pregnant women at the first, second and third trimesters while women of the control group were tested only once.

Metacarpophalangeal joint laxity assessment

An extensometer of the metacarpophalangeal joint of the index was purpose-made (figure 1) according to Jobbins and colleagues principle [START_REF] Jobbins | A Joint Hyperextensometer for the Quantification of Joint Laxity[END_REF]. The extension force applied on this joint was measured by a force sensor while the metacarpophalangeal joint angle and force direction derived from reflective markers captured by an optoelectronical motion system (Vicon Motion System Ltd). Force and kinematic data were synchronized using the Nexus Motion Capture Software (version 1.8.5). Specifically, participants were asked to sit comfortably in front of the extensometer and be relaxed. They put their index of the non-dominant hand in the extensometer interstice. The proximal portion of the metacarpus was immobilized to only measure the movement of the metacarpophalangeal joint of the index. One reflective marker was placed on the base of the finger and a second marker on the tip of the support of the extensometer to measure the metacarpophalangeal joint angle. Then the experimenter turned the rack slowly and continuously until the participant experienced discomfort. Based on the force, its direction and the metacarpophalangeal joint angle, the joint moment was calculated. From the time histories of both joint angle and moment, the angle obtained when the metacarpophalangeal joint underwent a moment of 0.26 Nm was selected [START_REF] Calguneri | Changes in joint laxity occurring during pregnancy[END_REF][START_REF] Jobbins | A Joint Hyperextensometer for the Quantification of Joint Laxity[END_REF].

Hip and lumbar flexibility tests

The first measurement corresponded to the fingertip to floor test (F2F test). From a 20 cm high platform, women were instructed to reach the floor with their fingertips while keeping knees extended and feet together. The distance between the middle finger and the base of the platform was expressed in centimeters. A positive value corresponded to a position of the middle finger above the platform level and a negative value below (figure 2A).

The second measure was an adapted version of the "sit and reach test" that will be later referred to as the horizontal Sit and Reach test (hSR test). The acquisition was done on the delivery bed, where the woman was seated with legs straight. The participant maintained her arms horizontal and bent the trunk as much as possible. For this test, the distance between the middle finger and the vertical plane passing by the soles of the feet was measured (a negative value corresponding to a position of the middle finger that exceeds the passing by the soles of the feet vertical plane, see figure 2B).In both tests, performance was measured by the optoelectronical system using markers placed on the foot soles and the right middle finger tip.

Beighton score

For each participant generalized joint laxity was also assessed using the Beighton score [START_REF] Beighton | Articular mobility in an African population[END_REF] as depicted on Figure 3. The laxity of nine joints was assessed using nine "all-or-nothing" tests (i.e. thumbs opposition, hyperextension of the 5th fingers, recurvatum of the elbows, hyperextension of the knees of the left and right sides, lumbar flexibility). The total score varies from 0 to 9 with high scores denoting more generalized joint laxity.

Statistical methods

For the statistical treatment, the normality of the distributions was confirmed by a Shapiro Wilk test.

Differences for age, height, body mass and BMI between controls and pregnant women in the first trimester were examined using independent samples t-tests. During pregnancy, the trimester effect (three levels: first, second and third trimesters) was analyzed using a repeated measures analysis of variance (ANOVA). In case of significance, Tukey post hoc tests were performed to identify trimesters that were significantly different from each other. The effect size was calculated by dividing the difference between the means for the outcome variable by the pooled SD and was interpreted in accordance with Cohen's guidelines: 0.20 as small, 0.50 as moderate, and 0.80 as large. The correlations between the tests were studied using Pearson correlation coefficients. The significance level adopted was less than 0.05. All analyses were executed using the Statistica software (StatSoft, Tulsa, OK).

RESULTS

Seventeen pregnant women (age: 36 ± 2 years, BMI1: 24 ± 3 kg/m 2 , BMI2: 25 ± 3 kg/m 2 , BMI3:27 ± 3 kg/m 2 at the first, second and third trimesters respectively including 6 primiparous and 11 multiparous) and sixteen controls (age: 31 ± 6 years, BMI: 21.3 ± 1.6 kg/m 2 ) were recruited during a routine gynecological consultation at the University Hospital of Poitiers.

Regarding the anthropometric data, no significant differences between the control group and pregnant women in the first trimester were found for age (p = 0.35) and height (p = 0.7) as shown in Table 1. The body mass and BMI were significantly higher in pregnant women (all p = 0.01).

All women were able to perform all of the laxity tests. However, three have reported discomfort in performing the F2F test. Joint laxity measurements are summarized in Table 2 and Table 3. No significant difference was found for all the laxity tests between the control group and pregnant women at the first trimester, except for the Beighton score (p = 0.02, Cohen's d = 0.80), which was significantly higher in pregnant women (Table 2).

During the pregnancy (Table 3), the extensometer results showed a trimester effect (F ( 2 Pearson correlation coefficients showed a moderate correlation between the results given by extensometer and the Beighton scores in the control group and pregnant women at the first trimester (r = 0.60, p < 0.05). This correlation was low in pregnant women for the second and third trimester (r = 0. 40, p = 0.02). There was no correlation between joint laxity measured by the two hip and lumbar flexibility tests and the extensometer (p > 0.05).

DISCUSSION

Since laxity could be associated with development of different conditions such as back pain [START_REF] Lindgren | Finger joint laxity, number of previous pregnancies and pregnancy induced back pain in a cohort study[END_REF][START_REF] Ostgaard | Influence of some biomechanical factors on low-back pain in pregnancy[END_REF] or pelvic floor dysfunctions [START_REF] Gachon | Changes in pelvic organ mobility and ligamentous laxity during pregnancy and postpartum. Review of literature and prospects[END_REF], it is important to find an assessment as simple as possible to enable its deployment in clinical settings for prevention and patients follow-up regarding the pre-cited conditions.

To the best of our knowledge, this is the first study that confronts data from extensometer for longitudinal monitoring of metacarpophalangeal joint laxity among pregnant women with a series of complementary clinical measures of generalized laxity. Calguneri and colleagues used Jobbin's extensometer for pregnant women only between the third trimester of pregnancy (from 24 th to 40 th week) and after delivery [START_REF] Calguneri | Changes in joint laxity occurring during pregnancy[END_REF].

Our results confirmed Calguneri's findings (1982) that metacarpophalangeal joint laxity evolved during pregnancy [START_REF] Calguneri | Changes in joint laxity occurring during pregnancy[END_REF]. Our results showed that this peripheral laxity measured by the extensometer increased by 11% from the first to the second trimester of pregnancy to keep the same level thereafter which reveals an important evolution of metacarpophalangeal laxity. We believe that these findings are of great interest for the management of conditions associated with increased laxity in pregnant women.

Compared with those of Calguneri and colleagues, the values obtained in the present study are smaller (around 40° in our study and around 65° for Calguneri and colleagues' study (1982)). This difference might be explained by the definition of the torque applied. Indeed, we sought to apply an external torque of 0.26 N.m at the metacarpophalangeal joint. To compute this torque, we took into account the finger weight by measuring the joint torque when horizontal. In the original publication by Jobbins and colleagues, it isn't clear whether the finger weight was taken into account or not and what exactly represents the external torque applied at the metacarpophalangeal joint [START_REF] Calguneri | Changes in joint laxity occurring during pregnancy[END_REF][START_REF] Jobbins | A Joint Hyperextensometer for the Quantification of Joint Laxity[END_REF].

Generalized joint laxity assessed by the Beighton score increased at the second trimester and was moderately correlated with the results given by the extensometer. Therefore, we concluded that laxity evolution is particularly widespread during the two first trimesters of pregnancy by affecting different joints of the body. However, the Beighton score decreased in the third trimester. This result may be due to the inability of women to perform the forward flexion of the trunk in a standing position (only 3/17 participants were able to complete the 5th item) because of the abdominal volume which is higher in the third trimester and which could hamper the execution of this item. Overall, this test is simple to use and evaluates different type of joints. However, we believe that the use of this score to assess the evolution of laxity in pregnant women remains insufficient due to its lack of sensitivity to changes and to the aforementioned problematic of the forward flexion of the trunk with large abdominal volume. The score is indeed based on the sum of ones and zeros corresponding to the ability to reach or not specific joint postures. It is therefore necessary to improve the test interpretation. For instance, the negativity should not allow excluding the diagnosis of hyper laxity.

The Finger to Floor test and the Sit and Reach test are simple clinical tests to evaluate the flexibility in the hip and lumbar joints. These joints are thought to be more exposed to local changes of pregnancy (uterine weight, increased abdominal volume, advancement and lowering of the center of mass [START_REF] Cunningham | Williams Obstetrics 19th Edition[END_REF], balance alterations and increase of the risk of falls [START_REF] Cakmak | Postural balance and the risk of falling during pregnancy[END_REF]). For this reason, we expected that the results from these tests would reflect the changes in joint laxity during pregnancy. However, our results do not confirm this hypothesis. For the Finger to Floor test, the distance measured between the fingertip and the horizontal plane passing by the sole of the feet increased during pregnancy when it should have been the opposite to reflect an increased hip/lumbar joint laxity. None of the pregnant women refused or even mentioned any apprehension to perform the test at the end of pregnancy. However, they often mentioned that the volume and the weight of the abdomen seriously hampered there realization of this test. In our study, the horizontal Sit and Reach was intended to anticipate that some women would refuse to perform the Finger to Floor test. The results indicated a reduction of the distance between the finger and the vertical plane passing by the soles of the feet during the first part of pregnancy compared to control group even though not significant and an increase at the third trimester. The increase in abdominal volume here again and the anterior shift in the location of the center of mass during pregnancy could explain the alteration of the

  , 32) = 4.88, p = 0.018). Tukey post hoc tests highlighted that the laxity was significantly smaller at the first trimester compared to the second (p = 0.024, Cohen's d = 0.5) and the third (p = 0.033, Cohen's d = 0.5). No significant change between the 2 nd and 3 rd trimester was observed (p = 0.917, Cohen's d = 0.0). As regards the results of the flexibility of the hip and lumbar, we observed a significant change during pregnancy for the fingertip to floor test (F (2, 32) = 3.78, p = 0.034) and the hSR test (F (2, 32) = 7.69, p = 0.001). The distance measured between the foot soles and the middle fingers tip was significantly greater at the third trimester of pregnancy compared to the first (p < 0.05) and the second trimester (p < 0.05) according to the results of post hoc tests. No difference between the first and the second trimesters was revealed for these two tests. The Beighton score demonstrated statistically significant changes of generalized laxity during the course of the pregnancy (F (2, 32) = 4.75, p = 0.029). Post hoc tests revealed a significant increase at the second trimester (p = 0.024, Cohen's d = 0.6) compared to the first trimester which decrease after at the third trimester (p = 0.017, Cohen's d = 0.8).

Figure 1 :

 1 Figure 1: Installation of the patient's hand inside of the extensometer which is equipped with

Figure 2 :

 2 Figure 2: (A) Classical version of Finger to floor test; (B) Sit and reach test (hSR test). For

Figure 3 :

 3 Figure 3: The Beighton score. The figure was taken from the Hypermobility Syndromes

results of these tests [START_REF] Dumas | Exercise, posture, and back pain during pregnancy[END_REF]. In summary, evaluating the flexibility of the trunk by these two last tests or by the 5th item of the Beighton score do not seem suitable for pregnant women.

Regarding limitations, our study may be limited by the small sample size. Furthermore, the difference in age between the case group and the control group could have slightly influence on the results of laxity, while it has been cited that laxity changes inversely with age. However, according to the literature [START_REF] Rudolph | Age-Related Changes in Strength, Joint Laxity, and Walking Patterns: Are They Related to Knee Osteoarthritis?[END_REF][START_REF] Zyroul | Knee laxity of Malaysian adults: gender differentials, and association with age and anthropometric measures[END_REF], this difference in age (only four years of average difference) is considered negligible to cause significant changes in laxity. Finally, the laxity measure was here based on the use of an optoelectronical capture system. A simplified version of the extensometer that doesn't require an optical motion capture system is been built.

In conclusion, metacarpophalangeal and generalized laxity increased considerably at the second trimester of pregnancy. According to Carvalho and colleagues low back pain is also more frequent in the second trimester of pregnancy [START_REF] Carvalho | Low back pain during pregnancy[END_REF] and pain in the hand and wrist is known to be the second most prevalent musculoskeletal symptom during pregnancy [START_REF] Nygaard | Hand problems in pregnancy[END_REF]. These concomitant findings indicate the need for prevention strategies that enable better quality of life for pregnant women. The combination of an objective measurement by the extensometer and a global evaluation of the laxity by the Beighton' score may be useful for a daily assessment of laxity in order to establish a specific laxity profile based on which we can choose a special monitoring for pregnant women. These results will be reinvested by two adjacent studies: a first study analyzing the eventual relationship between ligament laxity and pelvic floor disorders in pregnant women and a second one interested in the prevalence of lumbar lordosis in pregnant women.

Conflict of interest statement

The authors have no conflict of interest.


Thomas Secher

Emilie Dalonneau

Marion Ferreira

Christelle Parent

Nicolas Azzopardi

Gilles Paintaud

Mustapha Si-Tahar

Nathalie Heuzé-Vourc'h
email: nathalie.vourch@med.univ-tours.fr

Dr N Heuzé-Vourc'

In a murine model of acute lung infection, airway administration of a therapeutic antibody confers greater protection than parenteral administration

Keywords: Pseudomonas aeruginosa, pulmonary infection, therapeutic antibody, aerosol Airway-administered antibody protects against pneumonia

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

Introduction

Pneumonia (whether either community-or hospital-acquired) is a leading cause of morbidity, qualityadjusted life year loss, and mortality in children, adults, and the elderly. Furthermore, an alarming rise in levels of antimicrobial resistance is compromising the effective prevention and treatment of pneumonia. The opportunistic pneumonia-causing pathogens Pseudomonas aeruginosa (P. aeruginosa) is often associated with devastating, acute disease, and chronic infections [START_REF] Martinez-Solano | Chronic Pseudomonas aeruginosa infection in chronic obstructive pulmonary disease, Clinical infectious diseases : an official publication of the[END_REF][START_REF] Patel | Relationship between bacterial colonisation and the frequency, character, and severity of COPD exacerbations[END_REF].

Pseudomonas aeruginosa is one of the most prevalent infectious agents in intensive care units, and is the leading cause of ventilator-associated pneumonia in patients being treated for trauma or severe viral infections. Moreover, P. aeruginosa-associated lung infections are associated with significantly higher mortality than infections with other pathogens [START_REF] Chastre | Ventilator-associated pneumonia[END_REF][START_REF] Friedland | Antimicrobial susceptibility patterns in Pseudomonas aeruginosa: data from a multicenter Intensive Care Unit Surveillance Study (ISS) in the United States[END_REF]. The prevention and treatment of P. aeruginosa infections are challenging, in view of the bacterium's genetic and biochemical versatility and intrinsic resistance to antibiotics [START_REF] El Solh | Update on the treatment of Pseudomonas aeruginosa pneumonia[END_REF]. Overall, the data show that the significant increase in antibiotic resistance over the last 15 years has been associated with greater premature mortality [START_REF] Bonomo | Mechanisms of multidrug resistance in Acinetobacter species and Pseudomonas aeruginosa, Clinical infectious diseases : an official publication of the[END_REF].

The low number of novel antibiotics coming onto the market means that novel alternative or complementary anti-infectious strategies are urgently needed. One such strategy involves the use of therapeutic antibodies (Abs), which have already proven successful in the prevention or treatment of infectious lung diseases. Three anti-infectious Abs have been approved for clinical use: the humanized IgG1 palivizumab for the prevention of respiratory syncytial virus infection in high-risk infants, and the human IgG1 raxibacumab and the chimeric IgG1 obiltoxaximab for the prophylaxis and/or treatment of pulmonary anthrax. Furthermore, 12 Abs targeting respiratory pathogens (including 4 Abs against P. aeruginosa) are in clinical development [START_REF] Secher | Therapeutic antibodies: A new era in the treatment of respiratory diseases?[END_REF].

In the clinic, most therapeutic antibodies (and, indeed, most therapeutic proteins) are administered parenterally, i.e. via the intravenous (i.v.), subcutaneous (s.c.) or intramuscular (i.m.) route. Oral administration is not indicated because therapeutic proteins are not usually formulated to resist acid denaturation in the stomach or proteolytic degradation in the rest of the gastrointestinal tract. Most Abs for the treatment of respiratory tract infections are administered i.v. [START_REF] Secher | Therapeutic antibodies: A new era in the treatment of respiratory diseases?[END_REF]. However, this route is unlikely to be optimal for therapeutic designed to treat respiratory diseases; the few available literature data indicate that only a small fraction of the injected Ab reaches the lungs/airways [START_REF] Dall'acqua | Properties of human IgG1s engineered for enhanced binding to the neonatal Fc receptor (FcRn)[END_REF][START_REF] Hart | Preclinical efficacy and safety of mepolizumab (SB-240563), a humanized monoclonal antibody to IL-5, in cynomolgus monkeys[END_REF][START_REF] Koleba | Pharmacokinetics of intravenous immunoglobulin: a systematic review[END_REF]. Given that large amounts of Ab are usually required to protect against infections, an Ab's systemic distribution after i.v. injection might limit efficacy while increasing the risk of systemic toxicity (serum thickening, cytokine release syndrome).

As the body's main entry portal for innocuous or harmful environmental antigens, the airways represent an attractive delivery route for drugs designed to treat respiratory diseases. We and others have recently demonstrated that the airway route is feasible for the delivery of Ab-based therapeutics [START_REF] Hansel | The safety and side effects of monoclonal antibodies[END_REF][START_REF] Hacha | Nebulized anti-IL-13 monoclonal antibody Fab' fragment reduces allergeninduced asthma[END_REF][START_REF] Hill | Administration of antibody to the lung protects mice against pneumonic plague[END_REF][START_REF] Koussoroplis | PEGylation of antibody fragments greatly increases their local residence time following delivery to the respiratory tract[END_REF][START_REF] Maillet | Aerodynamical, immunological and pharmacological properties of the anticancer antibody cetuximab following nebulization[END_REF][START_REF] Respaud | Effect of formulation on the stability and aerosol performance of a nebulized antibody[END_REF][START_REF] Respaud | Development of a drug delivery system for efficient alveolar delivery of a neutralizing monoclonal antibody to treat pulmonary intoxication to ricin[END_REF][START_REF] Respaud | Nebulization as a delivery method for mAbs in respiratory diseases[END_REF]. The Abs induce a treatment response, and pass slowly into the systemic circulation [START_REF] Guilleminault | Fate of inhaled monoclonal antibodies after the deposition of aerosolized particles in the respiratory system[END_REF][START_REF] Herve | Heuze-Vourc'h, VEGF neutralizing aerosol therapy in primary pulmonary adenocarcinoma with K-ras activatingmutations[END_REF][START_REF] Maillet | Heuze-Vourc'h, The airways, a novel route for delivering monoclonal antibodies to treat lung tumors[END_REF][START_REF] Mora | Delivery of ALX-0171 by inhalation greatly reduces respiratory syncytial virus disease in newborn lambs[END_REF].

Furthermore, it has been shown that Ab-based therapeutics are sensitive to aerosolization but can retain their physical and immunological properties when delivered with specific devices and after the addition of specific excipients [START_REF] Respaud | Effect of formulation on the stability and aerosol performance of a nebulized antibody[END_REF].

Overall, the results of preclinical studies have shown that inhaled Abs are therapeutically active and thus underpin the growing interest in the pulmonary/airway route [START_REF] Secher | Therapeutic antibodies: A new era in the treatment of respiratory diseases?[END_REF][START_REF] Detalle | Generation and Characterization of ALX-0171, a Potent Novel Therapeutic Nanobody for the Treatment of Respiratory Syncytial Virus Infection[END_REF][START_REF] Lightwood | The discovery, engineering and characterisation of a highly potent anti-human IL-13 fab fragment designed for administration by inhalation[END_REF]. Nevertheless, the literature data on the advantages and disadvantages of parenteral versus local routes of anti-infective Ab administration are scarce. It is probably highly important to take account of situations in which the alveolus-capillary barrier is altered, since this might change the fate of and response to Abs. In the present study (performed in a murine model of acute pneumonia), we investigated the pharmacokinetics (PK) and protective activity of an archetypal anti-P. aeruginosa Ab as a function of its administration route. We found that airway administration of Ab was associated with a favorable PK profile, longer retention in the lung compartment, and a stronger anti-bacterial response by the host (characterized by enhanced neutrophil recruitment and bacterial clearance, and attenuated lung permeability). Local administration of Ab was also associated with a less intense acute inflammatory response, lower levels of pro-inflammatory cytokines.

Materials and methods

Mice and reagents

Adult male C57BL/6jrj (B6) mice (6 to 8 weeks old) were obtained from Janvier (France). All mice were housed under specific-pathogen-free conditions at the PST Animaleries animal facility (France) and had access to food and water ad libitum. All animal experiments complied with the current European legislative, regulatory and ethical requirements and were approved by the local animal care and use committee (reference: APAFIS#7608-2016111515206630). mAb166 was generated using a PTA-9180 hybridoma (LGC Standards, France) and supplied as sterile, pyrogen-free solution in phosphate-buffered saline (PBS), in accordance with good manufacturing practice (BioXcell, USA). The IgG2b isotype control Ab MPC11 (European Collection of Authenticated Cell Cultures, UK), was generated using the same technique. After preliminary experiments, we decided to use a dose of 100 µg of mAb166 in a volume of 50 µL of PBS for airway administration or a volume of 100µL for i.v, i.p and s.c administration. The Ab was injected 2 hours before infection.

The model of acute P. aeruginosa infection

Pseudomonas aeruginosa strain PA103 was kindly provided by Professor Teiji Sawa (Kyoto University, Japan). The colonies' uniformity was checked by plating on Pseudomonas isolation agar (PIA) plates. PA 103 has been transformed (using quadriparental mating) with a mini-Tn7T transposon that enables the constitutive expression of the LuxCDABE operon. The mice were infected with a freshly prepared inoculum. Starting from a frozen stock solution at 37°C, an overnight culture was prepared in 10 mL of Luria broth (LB) with shaking at 220 rpm. A 100 µL aliquot was then taken to start a fresh 10 mL culture in LB. The culture was stopped when an optical density (OD) of about 0.4 was achieved; this corresponded to a titer of about 3×10 8 bacteria/mL. The bacteria were washed once in PBS diluted in saline, to obtain a titer of 5×10 5 bacteria/40 µL. Each inoculum was then checked for accuracy (by direct plating on fresh PIA plates) and uniformity in a bioluminescence assay (ChemiDoc TM , Bio-Rad, France). Mice were anaesthetized with 3% isofluorane. An operating otoscope fitted with an intubation speculum was used to maintain tongue retraction and visualize the glottis. A fiber optic wire,threaded through a 20 G catheter and connected to a fiber optic stylet (Harvard Apparatus, France) was inserted into the mouse's trachea. Correct intubation was confirmed using a lung inflation bulb, and 40 µL of the bacterial solution was applied through an ultrafine pipette tip. In all experiments, the animals' mortality and body weight were monitored daily. For ethical reasons, we sacrificed moribund animals and those having lost more than 25% of their initial body weight. The remaining mice were sacrificed 6 hours or 1, 3, 7, 14, 21 or 28 days after infection.

Administration of the therapeutic Ab

mAb166 is a murine monoclonal IgG2b,κ Ab against pcrV, a component of a type three secretion system (T3SS) expressed on the cell surface of P. aeruginosa and that translocates a wide array of toxins into the host cell's cytosol [START_REF] Faure | Effects of monoclonal anti-PcrV antibody on Pseudomonas aeruginosainduced acute lung injury in a rat model[END_REF][START_REF] Frank | Generation and characterization of a protective monoclonal antibody to Pseudomonas aeruginosa PcrV[END_REF].Two hours before the infection, the anti-pcrV Ab (mAb166, 100 µg/animal) or its IgG2b isotype control (MPC11) were administered either via the caudal vein, into dorsal s.c. tissue, intraperitoneally (i.p.), or orotracheally using a MicroSprayer® aerosolizer (Penn-Century, USA), as described previously [START_REF] Maillet | Heuze-Vourc'h, The airways, a novel route for delivering monoclonal antibodies to treat lung tumors[END_REF].

Bronchoalveolar lavage, and organ sampling

At different time-points after P. aeruginosa infection, mice were euthanized using a lethal dose of ketamine/xylazine. Bronchoalveolar lavage fluid was then collected by cannulating the trachea and washing the lung twice with 1.2 mL of PBS at room temperature. The lavage fluid was centrifuged at 400 g for 10 min at 4°C, and the supernatant was stored at -20°C until analysis. The cell pellet was resuspended in PBS, and counted in a hemocytometer chamber. Cytospin samples were prepared using a cytocentrifuge (Shandon TM Cytospin 4 TM , ThermoFisher Scientific, USA; 1000 rpm for 10 min).

After staining with RAL555 reagent (RAL Diagnostic, France), neutrophils, macrophages and lymphocytes were counted. Lungs were homogenized for further analysis.

Pulmonary permeability

An in vivo pulmonary permeability assay was used to assess barrier function, as previously described [START_REF] Chen | Pulmonary permeability assessed by fluorescent-labeled dextran instilled intranasally into mice with LPS-induced acute lung injury[END_REF]. Briefly, animals were injected intranasally with dextran-FITC (FD4; molecular mass: 4 kDa; Sigma, France; 10 mg/kg of body weight) 30 min before drawing blood (via retro-orbital puncture) -at 0 (uninfected controls), 3, 6 and 18 hours after infection -and serum samples were prepared. The fluorescence intensity at 485/525 nm was measured using an Infinite M200 microplate reader (Tecan, Switzerland). The FITC concentrations were determined from standard curves generated by serial dilution of a stock FD4 solution. The FITC recovery was calculated by linear regression of the sample's fluorescence and taking account of the mouse's body weight.

Determination of the bacterial load in the lung

Lung homogenates were prepared in 2 mL of PBS containing broad-spectrum protease inhibitors (Sigma, France), using a GentleMACS tissue homogenizer (Miltenyi Biotec, Germany). Tenfold serial dilutions of homogenates were plated on PIA plates. Plates were incubated at 37°C in a 5% CO2 atmosphere, and the colonies were counted after 24 hours. The colonies' uniformity was checked in a bioluminescence assay (ChemiDoc TM , Bio-Rad, France). The homogenates were centrifuged at 800 g for 10 min at 4°C, and the supernatants were stored at -20°C until analysis. mAb166, cytokine, chemokine and protein assays mAb166 concentrations in broncho-alveolar lavage fluid (BALF) and lung homogenates were measured in an ELISA using purified recombinant pcrV as the coating antigen and HRP-conjugated goat anti-mouse IgG as the detection antibody (Jackson Immunoreseach Laboratories, USA). The Abantigen complexes in the samples were dissociated with 300 mM acetic acid, and the solution was then neutralized with 1 M Tris pH 9, as described previously [START_REF] Patton | An acid dissociation bridging ELISA for detection of antibodies directed against therapeutic proteins in the presence of antigen[END_REF].

Concentrations of CXCL1, TNF-α, and IL-6 in BALF and lung homogenates were measured using specific ELISAs (Duoset Kit; R&D Systems, USA; limit of detection: at 50 pg/mL) according to the manufacturer's instructions. Total BALF protein was determined using a BCA assay (ThermoFischer Scientific, France), according to the manufacturer's instructions (limit of detection: 5 µg/mL).

Pharmacokinetic modeling of mAb166 in mice

The fate of free mAb166 was studied by population pharmacokinetic modelling using Monolix software (2018 R2 version, Lixoft, France). Several structural models were tested and compared using goodness-of-fit and residual plots, and other standard validation procedures. A two-compartment model was found to best describe the concentrations of free mAb166 measured in serum and BALF after airway, s.c., i.p. and i.v. administration. This approach also enabled us to estimate the fraction absorbed following airway and s.c. administration. The model's first compartment corresponds to the blood as reflected by the serum), and the second corresponds to the airways (as reflected by BALF).

The distribution between these two compartments was described by the first-order rate constants k12 (for the transfer from the blood to the airway) and k21 (for the transfer from the airway to the blood).

The absorption rate following s.c. injection was described by the first-order constant ka. Elimination of mAb166 from the blood compartment was described by the first-order constant k10. The area under the concentration versus time curve (AUC) in blood or in airways was estimated between day 0 and day 3, in order to assess each animal's exposure to mAb166 as a function of the administration route.

Statistical analysis

Differences between experimental groups were determined in a one-way analysis of variance (ANOVA) followed by a Newman-Keuls post-test, after confirmation that the data were normally distributed. The log-rank test was used for survival analysis. All statistical tests were performed with GraphPad Prism software (version 4.03 for Windows, GraphPad Software Inc., USA). All data are presented as mean ± standard error of the mean (SEM). The threshold for statistical significance was set to p<0.05.

Results

Acute lung infection by P. aeruginosa PA103 is associated with impairment of the alveoluscapillary barrier

We compared the standard parenteral routes used to administer Abs in the clinic (i.v. and s.c.) with local airway administration. Given that the i.p. route is often used to deliver drugs in preclinical studies, we also included it in our experiments.

To assess the PK, PD and safety profiles of the anti-P. aeruginosa Ab as a function of its administration route, we first established and characterized an acute pneumonia model (using the clinical isolate P. aeruginosa PA103) derived from that described by Frank et al. [START_REF] Frank | Generation and characterization of a protective monoclonal antibody to Pseudomonas aeruginosa PcrV[END_REF]. This model mimics the initial phases of airway colonization by P. aeruginosa in patients with ventilator-associated pneumonia or cystic fibrosis [START_REF] Kukavica-Ibrulj | Animal models of chronic lung infection with Pseudomonas aeruginosa: useful tools for cystic fibrosis studies[END_REF]. First, we determined the dose of PA103 needed to induce a robust (but not extreme) infection. As shown in Figure 1A, orotracheal administration of 5×10 6 and 5×10 5 cfu killed all the animals within 18 hours and 36 hours, respectively. However, all the mice infected with a dose of 5×10 4 cfu survived for 7 days (Figure 1A).To characterize the time course of PA103-induced pneumonia, we measured the bacterial load in the lungs at 1, 3, 6 and 18 hours post-infection. The load increased significantly at 1 hour post-infection, rose further at 3 hours, and reached a plateau after 6 hours (Figure 1B). Discernible signs of infection (such as ruffled fur, a hunched posture, and motor impairment) appeared between 1 and 3 hours post-infection and gradually intensified over time.

Surviving animals showed a noticeable reduction in signs of disease after 48 hours (data not shown).

It is known that in the late stages of infection, P. aeruginosa can disrupt the pulmonary-vascular barrier and thus lead to systemic dissemination, which is associated with a poor clinical prognosis [START_REF] Magret | Bacteremia is an independent risk factor for mortality in nosocomial pneumonia: a prospective and observational multicenter study[END_REF]. Accordingly, we observed a lasting, time-dependent increase in alveolar-capillary permeability after infection, as assessed in vivo by the passage of a fluorescent marker from the airways into the blood (Figure 1C). This increase in the pulmonary epithelium's permeability might have facilitated the observed time-dependent increases in the level (Figure 1D) and prevalence (Figure 1E) of P. aeruginosa translocation into the blood of infected animals.

Passage and retention of an anti-P. aeruginosa Ab in the lung compartment depends on the administration route

To determine the impact of the presence of the antigen and pneumonia-associated inflammation on the fate of Abs after administration by different routes, we measured the free and total mAb166 concentrations in both the serum (Figure 2A) and BALF (Figure 2B) of animals infected with P. aeruginosa. During the acute phase of the infection (6 h, and 1 and 3 days post-infection), we found that the proportion of free mAb166 in the serum was higher for systemic administration than for delivery to the airways (Figure 2A). Conversely, the free mAb166 concentration in BALF was higher in animals treated locally (Figure 2B). When comparing free and total mAb166 levels in the serum (Supplementary Figure 1A) and the BALF (Supplementary figure 1B), we observed that a significant proportion of the Ab was complexed with bacteria in the systemically treated groups; this is doubtless explained by the greater level of bacteremia in these groups (Figures 1D and1E). In both compartments, mAb166 could no longer be detected between 7 and 14 days after infection. A compartmentalized PK analysis described the measured concentrations to a satisfactory extent (Table 1). The model suggested that 8% of the Ab was absorbed following airway administration. If one assumes that the entire dose reached the BALF, this means that 92% of mAb166 was able to reach pcrV in the airways following pulmonary administration. The animals' local exposure to mAb166 (as indicated by the airways/blood AUC ratio) was 10 times higher after airway administration than after other types of administration (Figure 2C).

mAb166's therapeutic efficacy against P. aeruginosa pneumonia depends on the administration route

The use of an Ab from the same species as the experimental model may avoid confounding immunogenicity and nonspecific stimulation of the immune system. The murine mAb166 has been shown to prevent infection in both mice and rat lungs infected with P. aeruginosa, following either prophylactic administration or concurrent administration with the infectious agent [START_REF] Faure | Effects of monoclonal anti-PcrV antibody on Pseudomonas aeruginosainduced acute lung injury in a rat model[END_REF][START_REF] Frank | Generation and characterization of a protective monoclonal antibody to Pseudomonas aeruginosa PcrV[END_REF]. In our experiments, mAb166 was administered 2 hours before the dose of P. aeruginosa; this was done to avoid the generation of immune complexes that can arise during concomitant administration and thus obliterate PA103's virulence prior to contact with the host (Figure 3A). The Ab was delivered by each of the four tested routes, and the survival of the mice in each group was monitored for 1 week. To be noted, no additional death was observed during a 100-days follow-up. All mice treated with 100 µg of mAb166 exhibited displayed a higher survival rate (Figure 3B) than untreated mice. The survival rate after airway administration of mAb166 (80%) was significantly higher than after parenteral administration (40% to 55%) (Figure 3B). The mAb166 response was specific for PA103, and was not due to unrelated anti-inflammatory effect of IgG2b; the survival rate for mice treated with an irrelevant IgG2b isotype Ab was essentially the same as for untreated animals (Supplementary Figure 2). Taken as a whole, these data demonstrate that the airway administration of an anti-infectious Ab conferred a higher level of specific protection against acute pneumonia than parenteral administration.

The clearance of P. aeruginosa and neutrophil recruitment in the lungs depend on mAb166's delivery route

To gain insight into the mechanisms that contributed to the Ab's beneficial effect on P. aeruginosainduced pneumonia, we analyzed the host's antibacterial responses 24 hours after infection. Mice treated parenterally with mAb166 exhibited a slightly (but not significantly) lower lung bacterial load (~0.2-0.4 log10 cfu) than untreated controls, whereas delivery of mAb166 to the airways was associated with a significantly lower P. aeruginosa load in the lungs -about 1.3 log10 cfu (Figure 4A). Beyond 24 hours, the P. aeruginosa load in surviving mice gradually diminished, and could not be detected 7 days post-infection (data not shown). Given that the exacerbation of pneumonia is intimately associated with lung remodeling and damage, we evaluated protein egress into the BALF as a surrogate marker for leakage of the alveolus-capillary barrier. Only animals treated with aerosolized mAb166 had significantly lower levels of protein in the BALF (Figure 4B), relative to untreated controls.

In airway-treated animals, the blood-air barrier was therefore maintained for at least 7 days after the infection (Supplementary Figure 3A).

To determine which cellular components might be involved in the enhanced clearance of P. aeruginosa, we analyzed the BALF's immune cell counts 1 and 3 days post-infection. In all groups, total cell counts were higher in mAb166-treated mice than in untreated controls (Figure 4C). On day 1 post-infection, the cellular influx was significantly higher in animals treated with aerosolized mAb166 than animals in which mAb166 had been administered by other routes. Most of the recruited cells were neutrophils, which are essential for quickly controlling invading pathogens [START_REF] Lavoie | Innate immune responses to Pseudomonas aeruginosa infection[END_REF] (Figure 4D). On day 3 post-infection (i.e. after neutrophil exhaustion), monocytes/macrophages became preponderant for the mediation of neutrophil phagocytosis, inflammation resolution, and repair. As expected, pulmonary administration of mAb166 led to a significantly higher macrophage count in the BALF, relative to the other routes (Figure 4E). At later time points (i.e. in surviving animals), there were no intergroup differences (Supplementary Figures 3B, 3C and3D). Although lymphocyte recruitment was similar in all groups at early time points (Figure 4F), we observed a significantly higher lymphocyte counts in the BALF of animals treated with aerosolized mAb166 21 and 28 days post-infection (Supplementary Figure 3E). Taken as a whole, our results suggest that the early, sustained presence of the Ab in the lung compartment post-infection enhances the lung's innate immune response to the pathogen, which in turn is correlated with better control of the bacterial load and a higher survival rate.

In mice treated with mAb166, the modulation of lung inflammation after P. aeruginosa infection depends on the Ab's administration route

Although inflammation is critical for the host's defense against invading pathogens, a protracted, uncontrolled reaction can cause tissue damage and organ dysfunction [START_REF] Wiersinga | van der Poll, Host innate immune responses to sepsis[END_REF]. We therefore sought to determine the effect of mAb166 treatment on levels of inflammatory mediators in the BALF on day 1 after P. aeruginosa infection. Levels of the prototypal pro-inflammatory cytokines TNFα (Figures 5A and5B) and IL-6 (Figures 5E and5F) were significantly lower in mice treated with aerosolized mAb166 than in animals treated via other routes or in untreated controls. Low levels of CXCL1 (the main chemokine responsible for neutrophil recruitment) were observed in all mAb166-treated groups (Figures 5C and5D) but the difference was greater in the lungs of animals that had received aerosolized mAb166 (Figure 5D). At later time points (i.e. in surviving animals), there were no intergroup differences (Supplementary Figure 4). Our results indicate that an enhanced innate immune response was associated with a weaker pro-inflammatory response, which protected the host against harmful lung dysfunction.

Discussion

Although many different P. aeruginosa antigens have been investigated as parenterally administered immunotherapeutics or vaccine candidates (e.g. lipopolysaccharide, flagellin, and pilins), their efficacy remains limited [START_REF] Doring | Vaccines and immunotherapy against Pseudomonas aeruginosa[END_REF][START_REF] Sharma | Recent developments for Pseudomonas vaccines[END_REF]. This may be because the parenteral administration route is not optimal. Since the main pathophysiological mechanisms underlying P. aeruginosa infection act within the lungs, matching the Ab's delivery route to the pathogenic target's location may produce a higher concentration in the vicinity of the target while limiting systemic exposure and reducing the likelihood of side effects. To the best of our knowledge, only a few literature studies have compared the local vs. parenteral delivery of anti-infectious Abs in the treatment of respiratory diseases [START_REF] Leyva-Grado | Direct administration in the respiratory tract improves efficacy of broadly neutralizing anti-influenza virus monoclonal antibodies[END_REF][START_REF] Ye | Intranasal delivery of an IgA monoclonal antibody effective against sublethal H5N1 influenza virus infection in mice[END_REF], and none of these characterized the Ab's PK/PD profiles in depth, in pathophysiological conditions which impact on lung remodeling and inflammation and thereby may affect the fate of Ab. Here, we showed for the first time that airway delivery of Ab is more efficacious than other delivery routes for containing and resolving P. aeruginosa pneumonia. There are several possible explanations for this greater efficacy.

Firstly, we found that the anti-infectious Ab accumulated in the infected airways after local administration. Most of the studies published to date characterized the fate of Ab at steady-state conditions, and found that IgG distribution to the airways was low after systemic delivery [START_REF] Dall'acqua | Properties of human IgG1s engineered for enhanced binding to the neonatal Fc receptor (FcRn)[END_REF][START_REF] Hart | Preclinical efficacy and safety of mepolizumab (SB-240563), a humanized monoclonal antibody to IL-5, in cynomolgus monkeys[END_REF][START_REF] Respaud | Development of a drug delivery system for efficient alveolar delivery of a neutralizing monoclonal antibody to treat pulmonary intoxication to ricin[END_REF][START_REF] Secher | Anti-Pseudomonas aeruginosa serotype O11 LPS immunoglobulin M monoclonal antibody panobacumab (KBPA101) confers protection in a murine model of acute lung infection[END_REF] or even nasal delivery [START_REF] Leyva-Grado | Direct administration in the respiratory tract improves efficacy of broadly neutralizing anti-influenza virus monoclonal antibodies[END_REF]. The lung inflammation and remodeling that occur during pneumonia might modulate this situation by increasing alveolus-capillary permeability, inducing proteolysis, modifying albumin homeostasis and thus altering the deposition, distribution and clearance of IgG. Accordingly, our PK model revealed a complex, infection-related mechanism of Ab distribution between the blood and the airways. Even though the mouse model of acute pneumonia likely corresponds to the worst possible scenario of alveolus-capillary barrier leakage and thus may be poorly representative of the clinic situation, the lungs' exposure to Ab was markedly higher after airway administration than after parenteral delivery. After local administration, the free mAb166 concentration was 10 times higher in the airways than in the blood. As previously shown under steady-state conditions or in models with an intact lung barrier [START_REF] Guilleminault | Fate of inhaled monoclonal antibodies after the deposition of aerosolized particles in the respiratory system[END_REF][START_REF] Magret | Bacteremia is an independent risk factor for mortality in nosocomial pneumonia: a prospective and observational multicenter study[END_REF], we observed that airway-delivered Ab passed slowly and poorly into the systemic circulation and accumulated at high levels in the respiratory tract for a few days. It is possible that the rapid, efficacious control of the bacterial load and the lower level of inflammation associated with airway-delivered Ab might limit damage to the lung and this could explain (at least in part) the PK profile.

Indeed, protection against pneumonia after the airway delivery of mAb166 was associated with a lower bacterial load in the lung parenchyma. After airway delivery, the higher concentration of the Ab in the airways during the acute phase of the infection results in better access to the target antigen and thus better bacterial neutralization. The translocon protein pcrV is located within the T3SS' cap required for the injection of toxins into the host cell's cytosol [START_REF] Schoehn | Oligomerization of type III secretion proteins PopB and PopD precedes pore formation in Pseudomonas[END_REF], and is indispensable for P. aeruginosa's virulence [START_REF] El-Solh | Clinical outcomes of type III Pseudomonas aeruginosa bacteremia[END_REF]. Thus, the Ab has a direct anti-infective action through antigen inhibition.

Moreover, Ab delivered through the airways provides greater protection against pneumonia by modulating the immune response. The clearance of P. aeruginosa from the airways results from the complex, coordinated involvement of many different cell types, including respiratory epithelial cells, resident and actively recruited myeloid cells, and lymphocytes. In the present study, we observed greater accumulation of neutrophils in the BALF of animals treated with locally administered mAb166, when compared with parenterally treated mice and untreated mice. The early, massive recruitment of lung neutrophils is the main component of the innate immune system's response against P. aeruginosa infection [START_REF] Lavoie | Innate immune responses to Pseudomonas aeruginosa infection[END_REF], and is essential for controlling P. aeruginosa infections [START_REF] Sun | Effect of IL-10 on neutrophil recruitment and survival after Pseudomonas aeruginosa challenge[END_REF].

In contrast, a protracted, uncontrolled inflammatory response is detrimental for the host, and eventually leads to multiple organ dysfunction [START_REF] Kurahashi | Pathogenesis of septic shock in Pseudomonas aeruginosa pneumonia[END_REF]. Here, we showed, that 24 hours after the infection, the pulmonary administration of mAb166 was associated with lower levels of TNF, IL-6 and CXCL1 production, relative to untreated and parenterally treated mice. At 6 hours TNF and CXCL1 production were similar between all treated groups (data not shown); CXCL1 decreasing over time only in pulmonary treated-animals. As previously shown, early production of CXCL1 preceding neutrophil recruitment is important to handle P. aeruginosa infection [START_REF] Sun | Effect of IL-10 on neutrophil recruitment and survival after Pseudomonas aeruginosa challenge[END_REF], while its accumulation correlates with deleterious inflammation [START_REF] Muhlebach | Quantitation of inflammatory responses to bacteria in young cystic fibrosis and control patients[END_REF][START_REF] Qiu | Biopsy neutrophilia, neutrophil chemokine and receptor gene expression in severe exacerbations of chronic obstructive pulmonary disease[END_REF].

These findings are in line with reports showing that although inflammation is important for controlling P. aeruginosa infection [START_REF] Sun | Effect of IL-10 on neutrophil recruitment and survival after Pseudomonas aeruginosa challenge[END_REF], its maintenance is correlated with a poor outcome [START_REF] Muhlebach | Quantitation of inflammatory responses to bacteria in young cystic fibrosis and control patients[END_REF][START_REF] Qiu | Biopsy neutrophilia, neutrophil chemokine and receptor gene expression in severe exacerbations of chronic obstructive pulmonary disease[END_REF]. Thus, airway delivery of anti-P. aeruginosa Ab may help to better control lung inflammation -an effect that has proven to be of benefit in patients with cystic fibrosis [START_REF] Koehler | Lung inflammation as a therapeutic target in cystic fibrosis[END_REF].

It should be noted that the pneumonia in this murine model is especially aggressive. Usually, untreated animals exhibit signs of severe inflammation within 6-8 hours of infection and die within 24-36 hours of infection. As previously reported [START_REF] Frank | Generation and characterization of a protective monoclonal antibody to Pseudomonas aeruginosa PcrV[END_REF], we gave the anti-infectious Ab prophylactically in order to prevent the excessive loss of animals. Although the prevention of pneumonia may be useful in people at a high risk of P. aeruginosa respiratory infections (such as mechanically ventilated patients), a therapeutic approach would have greater clinical relevance (i.e. the initiation of specific immunotherapy following confirmation of the P. aeruginosa infection). In the light of our present results, it is tempting to speculate that therapeutic administration through the airways may enable better control of P. aeruginosa infection; however, this will need to be confirmed in further research.

After decades of (mis)use of broad-spectrum antibiotics, antibiotic-resistant pathogens have become a real threat for human health and have made the management of pneumonia even more challenging [START_REF] Folgori | Epidemiology and clinical outcomes of multidrug-resistant, gram-negative bloodstream infections in a European tertiary pediatric hospital during a 12-month period[END_REF]. Pseudomonas aeruginosa is on the World Health Organization's priority list of antibiotic-resistant bacteria, and infection-control experts and researchers are making concerted efforts to fight this deadly pathogen [START_REF] Communications | WHO publishes list of bacteria for which new antibiotics are urgently needed[END_REF]. As shown in the present study, passive immunotherapy with an aerosolized anti-pcrV Ab might be a valuable way of preventing and possibly treating P. aeruginosa pneumonia. A PEGylated fragment of mAb166 for i.v. administration was developed by KaloBios Pharmaceuticals Inc. and has been tested in a Phase IIa study in mechanically ventilated patients infected with P. aeruginosa [START_REF] Francois | Safety and pharmacokinetics of an anti-PcrV PEGylated monoclonal antibody fragment in mechanically ventilated patients colonized with Pseudomonas aeruginosa: a randomized,doubleblind, placebo-controlled trial[END_REF]. However, the Ab did not to meet the primary clinical endpoint [START_REF] Jain | aeruginosa-infected mice. B6 mice were treated (or not) with 100 µg of mAb166 via the pulmonary (airway), intravenous (i.v.), intraperitoneal (i.p.) or subcutaneous (s.c.) route 2 hours before being infected by the orotracheal instillation of 40 µL of P. aeruginosa strain PA103 (5×10 5 cfu). The concentrations of TNF (A and B), CXCL1 (C and D) and IL-6 (E and F) in BALF and in lung homogenates were determined 3 and 7 days after the infection. The data are quoted as the mean ± SEM[END_REF]. Based on our findings, airway administration and a prophylactic clinical regimen (rather than therapeutic) might be more effective.

In conclusion, we demonstrated that prophylactic administration of anti-P. aeruginosa Ab to the lungs led to a more efficacious antibacterial and host response than conventional parenteral administration did. Our findings open up perspectives for the development of novel, inhaled, Ab-based prophylactics and therapeutics, which may facilitate the development of novel, inhaled, Ab-based prophylactics and 386 therapeutics for use against respiratory infections.

Table 1 577 578 B6 mice received 100 µg of mAb166 via the pulmonary (airway), intravenous (i.v.), intraperitoneal (i.p.) or subcutaneous (s.c.) route 2 hours before being infected by the orotracheal instillation of 40 µL of P. aeruginosa strain PA103 (5×10 5 cfu). The total mAb166 concentrations in serum (A) and in BALF (B) were determined 6 h and 1, 3, 7, 14, 21 and 28 days after the infection, using an ELISA. The data are quoted as the mean values ± SEM.

Using estimated pharmacokinetic parameters for day 0 and day 3 (Table 1), the airways/blood (C) AUC ratios were determined. The lower and upper hinges correspond to the interquartile range (IQR, the first and third quartiles). The upper whisker extends to the largest value no further than 1.5 × IQR from the hinge. The lower whisker extends from the hinge to the smallest value no further than 1.5 × IQR from the hinge. The results correspond to 7 pooled, independent experiments (n=12-27 mice for each). B6 mice were treated (or not) with 100 µg of mAb166 via the pulmonary (airway), intravenous (i.v.), intraperitoneal (i.p.) or subcutaneous (s.c.) route 2 hours before being infected by the orotracheal instillation of 40 µL of P. aeruginosa strain PA103 (5×10 5 cfu). The bacterial load (cfu) in the lungs (A)

Figure 1 :

 1 Figure 1: P. aeruginosa strain PA103 induces acute lung infection and increases the permeability of the capillary-alveolar barrier.C57/BL6jRj (B6) mice received a 40 µL orotracheal instillation of P. aeruginosa strain PA103 (5×10 4 , 5×105 or 5×10 6 cfu). Survival (A) was monitored for 7 days after infection. In another experiment, B6 mice received a 40 µL orotracheal instillation of P. aeruginosa (5×10 5 cfu) and were injected intranasally with dextran-FITC 30 min before analysis. The bacterial load (cfu) in the lung (B), the translocation of 4 kDA dextran-FITC (FD4) into blood (C), and the bacterial load in the blood (cfu) (D) were measured 0, 1, 3, 6 and 18 hours after the infection. The proportion of mice with P. aeruginosa in the blood (E) was evaluated. The data are quoted as the mean ± SEM. *, ** and ***: p<0.05, p<0.01 and p<0.001, respectively, in a one-way ANOVA with Newman-Keuls's correction for multiple comparisons. The results are representative of two independent experiments (n=5-10 mice for each).

Figure 2 :

 2 Figure 2: Kinetics of and exposure to mAb166 in the serum and the airways after PA103 infection, as a function of the route of administration.

Figure 3 :

 3 Figure 3: Airway administration of a therapeutic antibody is associated with greater resistance against P. aeruginosa pneumonia.B6 mice were treated (or not) with 100 µg of mAb166 via the pulmonary (airway), intravenous (i.v.), intraperitoneal (i.p.) or subcutaneous (s.c.) route 2 hours before being infected by the orotracheal instillation of 40 µL of P. aeruginosa strain PA103 (5×10 5 cfu). Survival was monitored for 7 days after infection. *, ** and ***: p<0.05, p<0.01 and p<0.001, respectively, in a log-rank test). The results correspond to 7 pooled, independent experiments (n=12-27 mice for each).

Figure 4 :

 4 Figure 4: Enhanced P. aeruginosa clearance and neutrophil recruitment in the lungs of airway-treated mice.

Acknowledgements

This work was supported by a public grant overseen by the French National Research Agency (ANR) as part of the "Investissements d'Avenir" program (LabEx MAbImprove, ANR-10-LABX-53-01). TS is funded by a fellowship from ANR-10-LABX-53-01.

We sincerely thank David Fraser PhD (Biotech Communication SARL, France) for copy-editing services.

Competing interests statement

The authors have no competing interests to declare.

Figure 3

Supplementary Figure 1 Supplementary Figure 2 Supplementary Figure 3 26 Supplementary Figure 4 was measured 24 and 72 hours post-infection. Total protein (B), the total cell count (C), and counts of neutrophils (D), macrophages (E) and lymphocytes (F) in the BALF were evaluated 24 h postinfection. The data are quoted as the mean ± SEM. *, ** and ***: p<0.05, p<0.01 and p<0.001, respectively, in a one-way ANOVA with Newman-Keuls s correction for multiple comparisons. The results are representative of three independent experiments (n=5-10 mice for each). Supplementary Figure 1: Kinetics of free mAb166 in the serum and the airways after PA103 infection, as a function of the route of administration. B6 mice received 100 µg of mAb166 via the pulmonary (airway), intravenous (i.v.), intraperitoneal (i.p.) or subcutaneous (s.c.) route 2 hours before being infected by the orotracheal instillation of 40 µL of P. aeruginosa strain PA103 (5×105 cfu). The free mAb166 concentrations in serum (A) and in BALF (B) were determined 6 h and 1, 3, 7, 14, 21 and 28 days after the infection, using an ELISA. The data are quoted as the mean values ± SEM. Supplementary Figure 2: Airway administration of a therapeutic antibody is associated with greater resistance against P. aeruginosa pneumonia. B6 mice were treated (or not) with 100 µg of mAb166 or an IgG2b isotype control via the pulmonary route 2 hours before being infected by orotracheal instillation of 40 µL of P. aeruginosa strain PA103 (5×10 5 cfu). Survival was monitored for 7 days after infection. *: p<0.05 in a log-rank test. The results are representative of 1 experiment (n= 5-8 mice). Supplementary Figure 3: Late leukocyte recruitment in the lungs of P. aeruginosa-infected mice.