Renaud Descourt
email: renaud.descourt@chu-brest.fr

Maurice Perol

Gaëlle Rousseau-Bussac

David Planchard

Bertrand Mennecier

Marie Wislez

Alexis Cortot

Florian Guisier

Loïck Galland

Pascal Do

Pascal Dô

Roland Schott

Eric Dansin

Jennifer Arrondeau

Jean-Bernard Auliac

Christos Chouaid

Renaud Docteur

Descourt

Brigatinib in patients with ALK-positive advanced non-small-cell lung cancer pretreated with sequential ALK inhibitors: A multicentric real-world study (BRIGALK study)

Keywords: Advanced non-small cell lung cancer, ALK rearrangement, brigatinib, crizotinib, ALK inhibitor, Early access program

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

INTRODUCTION

Rearrangements of anaplastic lymphoma kinase gene (ALK) are an oncogenic driver reported in 3-5% of patients with non-small cell lung cancers (NSCLC) [START_REF] Barlesi | Routine molecular profiling of patients with advanced non-small-cell lung cancer: results of a 1-year nationwide programme of the French Cooperative Thoracic Intergroup (IFCT)[END_REF]. Patients with ALK rearrangement (ALK-positive) NSCLC are generally younger, are never or light smokers, with a histology of adenocarcinoma and a high risk of central nervous system (CNS) metastases at diagnosis and progression [START_REF] Shaw | Clinical features and outcome of patients with non-small-cell lung cancer who harbor EML4-ALK[END_REF][START_REF] Toyokawa | Insights into brain metastasis in patients with ALK+ lung cancer: is the brain truly a sanctuary?[END_REF].

Crizotinib was the first ALK tyrosine-kinase inhibitor (TKI) approved for ALK-positive NSCLC. In first-line treatment, crizotinib achieved objective responses rates from 61 to 74% with a median progression-free survival (PFS) of 8-11 months [START_REF] Shaw | Ceritinib versus chemotherapy in patients with ALK-rearranged non-small-cell lung cancer previously given chemotherapy and crizotinib (ASCEND-5): a randomised, controlled, open-label, phase 3 trial[END_REF][START_REF] Camidge | Activity and safety of crizotinib in patients with ALK-positive non-small-cell lung cancer: updated results from a phase 1 study[END_REF][START_REF] Solomon | First-line crizotinib versus chemotherapy in ALK-positive lung cancer[END_REF]. Mechanisms of acquired resistance to crizotinib (alterations in ALK gene or activation of signaling pathways bypassing ALK) explain disease progression in most patients treated with crizotinib [START_REF] Castellanos | Re-Evaluating Progression in an Era of Progress: A Review of First-and Second-Line Treatment Options in Anaplastic Lymphoma Kinase-Positive Non-Small Cell Lung Cancer[END_REF]. The limited CNS penetration of crizotinib is also responsible for intracranial disease progression, which is the most frequent site of relapse. Second-generation ALK inhibitors, namely ceritinib and alectinib, achieved median PFS of 5.7-6.9 months and 8.1-8.9 months, respectively, in patients pretreated with crizotinib [START_REF] Shaw | Ceritinib in ALK-rearranged non-small-cell lung cancer[END_REF][START_REF] Kim | Activity and safety of ceritinib in patients with ALK-rearranged non-small-cell lung cancer (ASCEND-1): updated results from the multicentre, open-label, phase 1 trial[END_REF][START_REF] Crino | Multicenter Phase II Study of Whole-Body and Intracranial Activity With Ceritinib in Patients With ALK-Rearranged Non-Small-Cell Lung Cancer Previously Treated With Chemotherapy and Crizotinib: Results From ASCEND-2[END_REF][START_REF] Shaw | Alectinib in ALK-positive, crizotinibresistant, non-small-cell lung cancer: a single-group, multicentre, phase 2 trial[END_REF][START_REF] Ou | Alectinib in Crizotinib-Refractory ALK-Rearranged Non-Small-Cell Lung Cancer: A Phase II Global Study[END_REF]. However, due to secondary ALK kinase domain resistance mutations (e.g., G1202R mutation) in patients treated by ceritinib or alectinib and previously treated with crizotinib, new ALK inhibitors are required.

Brigatinib is a new ALK inhibitor with a potent activity on many ALK resistance mutations including G1202R mutation, which is inhibitable in vitro and in some patients but perhaps not in all [START_REF] Zhang | The Potent ALK Inhibitor Brigatinib (AP26113) Overcomes Mechanisms of Resistance to First-and Second-Generation ALK Inhibitors in Preclinical Models[END_REF]. A phase II randomized trial included patients with ALK-positive advanced NSCLC and disease progression after crizotinib treatment (69% with brain metastases and 74% with prior chemotherapy) and compared brigatinib 90 or 180 mg/day after a first week at 90 mg/day [START_REF] Kim | Brigatinib in Patients With Crizotinib-Refractory Anaplastic Lymphoma Kinase-Positive Non-Small-Cell Lung Cancer: A Randomized, Multicenter Phase II Trial[END_REF]. Brigatinib yielded confirmed objective response rates (ORRs) of 51% in arm A (112 patients treated at 90 mg daily) and 55% in arm B (110 patients treated at 180 mg daily with a 7-day lead-in at 90 mg, the recommended dosing regimen) per independent 4/18 review committee (IRC). Median progression-free survival (PFS) was 9.2 months in arm A and 16.7 months in arm B, per IRC respectively [START_REF] Ahn | Brigatinib (BRG) in crizotinib (CRZ)refractory ALK+ non-small cell lung cancer (NSCLC): Updates from ALTA, a pivotal randomized phase 2 trial[END_REF]. In patients with measurable brain metastases at baseline, ORR was 42% in 90-mg group and 67% in 180-mg group. Based on the results of the phase II study, the US Food and Drug Administration granted in April 2017 accelerated approval to brigatinib in patients with locally-advanced or metastatic ALKpositive NSCLC who have progressed on or are intolerant to crizotinib. In France, brigatinib was available through an early access program since August 2016 for patients with ALKpositive NSCLC refractory to crizotinib and ceritinib. This program closed on January 21 st , 2018 after the approval of brigatinib by the European Medicines Agency. The primary objective of the BRIGALK study was to assess the efficacy of brigatinib after at least two ALK inhibitors in patients who benefited from this early access program.

PATIENTS AND METHODS

Study design and patients

The BRIGALK study (GFPC 07-2017) was a national retrospective non-interventional study that included patients treated with brigatinib in the French early access program. The objective was to describe in real-world setting the efficacy of brigatinib in patients with advanced NSCLC harboring ALK rearrangement.

The inclusion criteria were the followings: at least 18 years old; advanced NSCLC; ALKpositive NSCLC assessed with in situ hybridization (FISH) and/or immunohistochemistry; previous treatment with at least one ALK inhibitor including crizotinib; treatment with brigatinib in the setting of the early access program from September 1 st , 2016 to January 1 st , 2018.

5/18

The primary objective was to evaluate the investigator-assessed PFS from brigatinib initiation, defined as the time from first dose of brigatinib to first documentation of objective disease progression or to death from any cause. The main secondary objectives were the followings: ORR to brigatinib, overall survival (OS) from brigatinib initiation and from diagnosis of NSCLC; treatment duration and reasons for discontinuation of brigatinib treatment.

The study was conducted in accordance with the Declaration of Helsinki and was approved by the French Advisory Committee on Information Processing in Health Research (CCTIRS). If patients were alive, they received a written information on the study. For deceased patients, an exemption of information was obtained from CCTIRS.

Data collection

Patient data were obtained retrospectively from medical files and included demographics, characteristics of NSCLC, number and localization of metastatic sites, previous treatments, tumor response to brigatinib, resistance mutation before brigatinib initiation or after progression and treatments after progression. Patients were included consecutively in each center according to inclusion criteria without selection.

Statistical analysis

The Kaplan-Meier method was used to estimate PFS and OS for the entire cohort and in defined subgroups according to the number of lines of treatments. Best response to treatment was assessed according to RECIST 1.1 criteria [START_REF] Eisenhauer | New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1)[END_REF]. The statistical analyses were performed with SAS 9.4 software (SAS Institute, Cary, NC, USA).

6/18

RESULTS

Characteristics of patients at diagnosis of NSCLC

A total of 104 patients were included in 42 centers. Their mean (SD) age was 56.6 (11.5) years and 61.5% were never-smokers (Table 1). NSCLC was adenocarcinoma in 98.1% of patients with stage IV disease in 88.5%. At diagnosis, there was a mean of 3.3 (1.3) metastatic sites; CNS metastases were reported in 28.9% of patients. Overall, patients were in good condition with performance status at 0-1 for 91.6%.

The median time from NSCLC diagnosis and result of ALK translocation test was 21 (range, 0-515) days. Diagnosis of ALK translocation was based in 16% of cases on a positive immunohistochemistry with score 3+, on FISH in 84% of cases (78% of these patients had also positive immunohistochemistry with score 2+/3+). Diagnosis was established mainly on biopsy of the primary tumor (62.0%), mediastinal lymph nodes (11.5%), pleural metastases (9.6%) or extra-thoracic metastases (16.9%).

Treatments before brigatinib initiation

Before brigatinib, patients received a median of 3 lines of treatment. Among the different therapeutic sequences presented in Table 2, the most frequent were two (n= 29) or three lines (n = 43). For the 29 patients who received two lines of treatment before brigatinib, the most frequent sequence was crizotinib-ceritinib (n=24; 82.7%); for the 43 patients who received 3 lines before brigatinib, the most frequent sequence was chemotherapy-crizotinib-ceritinib (n=32; 74.4%). Almost all patients received at least two anti-ALK TKIs before brigatinib (97/104; 93.3%).

First-line treatment was chemotherapy for 62.5% of patients or crizotinib for 36.5% (Table 3). All patients with chemotherapy treatment received a platinum/pemetrexed combination.

7/18

Median duration of chemotherapy was 5.8 (range, 0.1-29.6) months and median duration of crizotinib treatment was 9.2 (range, 0.1-22.2) months.

Second-line treatment was chemotherapy (platinum/pemetrexed) for 18.3% of patients and TKI for the others (crizotinib for 52.9% and ceritinib for 26.9%). Median duration of postchemotherapy treatment was 11.5 months for crizotinib and median duration of postcrizotinib treatment was 3.0 months for ceritinib.

A total of 75 patients received a 3 rd line of treatment (chemotherapy, 13.3%; crizotinib, 8.0% and ceritinib, 77.3%); 32, 18 and 11 patients received a 4 th , 5 th , and 6 th line of treatment, respectively (Table 2).

Radiotherapy was administered before brigatinib to 56.7% (59/104) of patients, most frequently during the first treatment line (47.5%; 28/59). Brain was the main target for radiotherapy (64%; 38/59).

Clinical characteristics at brigatinib initiation

At initiation of brigatinib treatment, performance status was 0-1 for 59.1% of patients (Table 4). More than 40% of them had a PS ≥2 and general symptoms (asthenia or weight loss) for 72%; 51.9% of patients had ≥ 3 metastatic sites and 74.5% and 8.8% had CNS metastases and carcinomatous meningitis, respectively. CNS metastases were active in the majority of cases (51.8%).

A tissue rebiopsy was performed for 33.7% (35/104) of patients (Table 3). Detection of resistance mutations was carried out in 18 cases and G1202R mutation was identified in 4 patients; other mutations found are reported in Table 4.

Almost all patients (95.2%; 99/104) received brigatinib at 90 mg/day for 7 days and then 180 mg/day. The 180-mg dose was reduced to 90 mg for 9 (8.8%) patients and 10 (9.6%) had permanent treatment discontinuation due to intolerance or patient request. 8/18

Efficacy of brigatinib in entire population

The date of primary data cut-off was June 30, 2018. Median PFS from initiation of brigatinib was 6.6 (95% CI, 4.8-9.9) months with a median OS of 17.2 (95% CI, 11.0-not reached) months (Figure 1).

For patients who received 2, 3-4 and >4 lines of treatment, median PFS was 4.3 (95% CI, 2.5-8.9), 10.4 (95% CI, 5.9-13.9) and 3.8 (95% CI, 0.8-7.4) months, respectively (Figure 2).

For the 91 evaluable patients, the disease control rate was 78.2% (stable disease, 28.2%; partial response, 45.7%; complete response, 4.3%). Median duration of brigatinib treatment was 6.7 (range, 0.06-20.7) months.

Among the 68 patients with progressive disease after brigatinib, CNS was involved in 29.4% of cases (including new lesions for 5 and carcinomatous meningitis for 5). Brigatinib treatment was continued beyond disease progression in 25% (17/68) of patients in combination with local treatment in 53% (9/17) of them.

During post-brigatinib progression, 15% (12/68) of patients had a rebiopsy (one mutation G1202R and one mutation D1203N were reported). A systemic treatment was administered to 52% (35/68) of patients after brigatinib (lorlatinib, n=25; crizotinib, n=1; chemotherapy, n=7) and 13% (9/68) had a second subsequent treatment line after brigatinib (lorlatinib, n=5, chemotherapy, n=4). Eight patients benefited from stereotactic brain radiation therapy.

Median OS from the diagnosis of NSCLC was 75.3 months (95% CI, 38.2-174.6) (Figure 2).

DISCUSSION

This retrospective study shows that brigatinib exhibits clinical activity in a cohort of patients heavily pretreated with at least two ALK inhibitors. The median PFS and OS from brigatinib initiation were 6.6 months and 17.2 months, respectively. ORR and disease control rate were 9/18 50.0% and 78.2%, respectively, consistently with the results of the 180-mg arm of the phase II study of Kim et al, which included crizotinib-refractory ALK-positive NSCLC patients (ORR, 54%; disease control rate, 84%) [START_REF] Kim | Brigatinib in Patients With Crizotinib-Refractory Anaplastic Lymphoma Kinase-Positive Non-Small-Cell Lung Cancer: A Randomized, Multicenter Phase II Trial[END_REF]. PFS appeared to be lower compared to the 180-mg arm of this clinical trial (6.6 vs. 12.9 months). However, patients in our cohort were more heavily pretreated and had a poorer condition with 40.9% of patients with PS ≥ 2. In the phase II trial, patients with any prior TKI other than crizotinib were not included and only 6% of the patients enrolled had a PS ≥ 2. In our study, PFS under brigatinib differed not consistently according to the number of pre-brigatinib lines. The retrospective design of the study and the small number (n = 29) in the subgroup of patients who received two previous lines of treatment do not allow conclusions to be drawn about the observed results. This subgroup consisted of patients who predominantly received crizotinib and ceritinib (n = 24). The PFS values should be taken with caution because 5 patients had less than one month of brigatinib and 8 are censored because they were still under treatment at the date of analysis.

CNS metastases are frequent in ALK-positive patients. In this cohort, 28.9% of patients had CNS metastases at diagnosis and 74.5% at brigatinib initiation (8.8% had carcinomatous meningitis). Among the 68 patients with disease progression after brigatinib initiation, CNS location was at the forefront in 29.4% of cases, thus suggesting a high efficacy of brigatinib on CNS metastases. This has been demonstrated in an exploratory analysis of brigatinib efficacy in ALK-positive NSCLC patients with brain metastases included in a phase II trial [START_REF] Camidge | Exploratory Analysis of Brigatinib Activity in Patients With Anaplastic Lymphoma Kinase-Positive Non-Small-Cell Lung Cancer and Brain Metastases in Two Clinical Trials[END_REF]. In patients with measurable brain metastases treated with 90 mg/day first week followed by 180 mg/day, a confirmed intracranial ORR of 67% (95% CI, 41-87) and a median intracranial PFS of 18.4 months (95% CI, 12.8-not reached) were reported. About 5% of non-squamous NSCLC are ALK positive and therefore data from large series are rarely reported. To our knowledge, our cohort is the first one to present real-life data on sequential strategy in patient who received at least three ALK inhibitors even though data 10/18 were limited. A previous study of Solomon et al reported a phase II trial in different cohorts of patients with ALK-positive or ROS1-positive advanced NSCLC treated with lorlatinib [START_REF] Solomon | Lorlatinib in patients with ALK-positive non-small-cell lung cancer: results from a global phase 2 study[END_REF]. Thus, the cohort EXP4-5 included 111 ALK-positive patients pretreated with 2 or 3 ALK TKI inhibitors (including the second-generation drugs ceritinib, alectinib and brigatinib) with or without chemotherapy. ORR was 38.7% (intracranial ORR, 48%) and disease control rate was 73%; PFS was 6.9 (95% CI, 5.4-9.5) months [START_REF] Solomon | Lorlatinib in patients with ALK-positive non-small-cell lung cancer: results from a global phase 2 study[END_REF]. These results are also consistent with those of our study.

ALK inhibitors are now considered as the standard of care in patients with ALK-positive NSCLC. Some questions remain unanswered about the optimal sequential therapy with different ALK inhibitors. The benefit of a sequential strategy has been evidenced in different studies. The PROFILE 1014 phase III study compared first-line crizotinib vs. chemotherapy in ALK-positive NSCLC patients [START_REF] Solomon | Final Overall Survival Analysis From a Study Comparing First-Line Crizotinib Versus Chemotherapy in ALK-Mutation-Positive Non-Small-Cell Lung Cancer[END_REF]. The longest OS was obtained for crizotinib-treated patients who received a subsequent ALK TKI, thus indicating the benefit of sequential strategy with ALK TKIs. The French retrospective CLINALK study performed in a large population of unselected patients with ALK-positive NSCLC was also in favor of a sequential strategy. After progression on crizotinib treatment, patients received either best supportive care, subsequent drug other than next-generation ALK inhibitors or next-generation ALK inhibitors. Better survival outcomes were reported for patients with next-generation ALK inhibitors: median OS from progression was 25.0 months vs. 6.4 months for drugs other than next-generation ALK inhibitors and 1.5 months for best supportive care [START_REF] Duruisseaux | Overall survival with crizotinib and next-generation ALK inhibitors in ALK-positive non-small-cell lung cancer (IFCT-1302 CLINALK): a French nationwide cohort retrospective study[END_REF]. It is also important to underline that about half (51.5%) of patients in our cohort with progression received a systemic treatment post brigatinib (most often lorlatinib).

The recent results of the ALEX and ALTA-1L studies have challenged the current sequential strategy with ALK inhibitors [START_REF] Camidge | Brigatinib versus Crizotinib in ALK-Positive Non-Small-Cell Lung Cancer[END_REF][START_REF] Peters | Alectinib versus Crizotinib in Untreated ALK-Positive Non-Small-Cell Lung Cancer[END_REF]. These studies included TKI untreated patients with advanced ALK-positive NSCLC and compared next-generation frontline ALK TKIs to 11/18 crizotinib. In the ALEX study, the rate of PFS at 12 months was significantly higher with alectinib than with crizotinib (68.4% vs. 48.7%, respectively); 12% of patients in the alectinib group had an event of CNS progression and 45% in the crizotinib group [START_REF] Peters | Alectinib versus Crizotinib in Untreated ALK-Positive Non-Small-Cell Lung Cancer[END_REF]. In the ALTA-1L study, the rate of PFS at 12 months was higher with brigatinib than with crizotinib (67% vs. 43%, respectively) [START_REF] Camidge | Brigatinib versus Crizotinib in ALK-Positive Non-Small-Cell Lung Cancer[END_REF]. ORR was 71% with brigatinib and 60% with crizotinib; the rates of intracranial response in patients with measurable lesions were 78% and 29%, respectively. Therefore, the strategy with a next generation ALK inhibitor in frontline showed its clear superiority to crizotinib, especially for the risk of CNS progression. However, data from realworld studies and indirect analyses assessing sequential strategy starting with crizotinib and new inhibitors as first line treatment are needed. In this study, only one patient received brigatinib after alectinib and three patients after lorlatinib and at least after three therapeutics sequences. This did not allow definitive conclusions to be drawn about the efficacy of brigatinib after first-line alectinib.

Detection of ALK acquired resistance mutations in patients progressing during ALK TKI treatment is not recommended today in France, outside of clinical trials. For this reason, only a small proportion of patients benefited from tissue rebiopsy or liquid biopsy. However, such information may be valuable in determining the optimal choice of next-generation TKIs, which have different activity against distinct mutations. When rebiopsy of the progressing site is not feasible, comprehensive testing such as a next-generation sequencing panel using circulating tumor DNA can provide information on ALK resistance mutations, but also on other molecular mechanisms of resistance for which the patient may receive treatment either through a clinical trial or expanded access [START_REF] Rolfo | Liquid Biopsy for Advanced Non-Small Cell Lung Cancer (NSCLC): A Statement Paper from the IASLC[END_REF]. Given the small number of patients who had a re-biopsy before brigatinib and the low number of mutations highlighted, it is difficult from the data of this study to assess the relevance of this research on the adaptation of anti-ALK treatments.

12/18

Our study has some limitations. Data were not recorded prospectively, but were obtained from patient medical records. Therefore, we cannot exclude some selection biases. The efficacy of treatment was assessed by the investigator and there was no independent review committee; a bias associated with investigator assessment cannot be discarded. In this real-life study, it was not always possible to have a complete information from medical records in some patients. This was the case, for example, for patients who had a dose reduction or for those who stopped brigatinib before progression. One of the strengths of the study is the absence of stringent criteria for study inclusion. As a consequence, the study population is representative of real-life patients heavily treated for advanced NSCLC.

In conclusion, this study confirms the efficacy of brigatinib in a cohort of patients heavily pretreated for ALK-positive advanced NSCLC. These real-life results are consistent with clinical data reported in randomized trials.

Figure 1 .

 1 Figure 1. PFS (A) and OS (B) since initiation of brigatinib treatment.

Figure 2 .

 2 Figure 2. PFS since initiation of brigatinib according to the number of previous lines of treatment (A) and OS from the diagnosis of NSCLC (B).

Table 1 .

 1 Characteristics of patients at diagnosis of NSCLC.

	N = 104

Table 4 .

 4 Clinical characteristics of patients before brigatinib treatment.

	Characteristics

ACKNOWLEDGEMENTS

We thank the investigators and staffs of all sites who participated: Cécile Dujon (Le Chesnay), Jeannick Madelaine (Caen), Gaelle Jeannin (Clermont Ferrand), Olivier Bylicki (HIA Percy), Catherine Daniel (Institut Curie, Paris), Dominique Spaeth (Polyclinique de Gentilly, Nancy), Benoît Cloarec (Avignon), Alain Vergnenègre (Limoges), Eric Jauffret (Boulogne-Billancourt), Hannah Ghalloussi (Nice), Patricia Barré (Cahors), Laurent Greillier (Marseille), Pierre Fournel (Saint-Priest-en-Jarez), Olivier Molinier (Le Mans), Guillaume Leveillier (Saint Brieuc), Haitham Oweis (Châlons-en-Champagne), Ahmed Azzedine (Montélimar), Marie Coudurier (Chambéry), Anne-Marie Chiappa (Quimper), Arnaud Bedin (Evreux), Hugues Morel (Orléans), Romain Corre (Rennes), Corinne Lamour (Poitiers), Denis Morot-Sibilot (La Tronche), Bachar Chahine (Rouen), Bruno Taviot (Givors) Violaine Giraud (Boulogne-Billancourt), Olivier Herreman (Romans), Laurence Bigay-Game (Toulouse), Anne-Sophie Bugnet (Thonon-les-Bains).

Funding: This work was supported by an academic grant of Takeda.


J-P Méricq

D Bouyer

D Wlodarczyk

L Soussan

C Faur

Modeling of enzymatic chitosan gelation: tools for monitoring chitosan gelation and urea hydrolysis kinetics

Keywords: Chitosan gelation, Gelation pH, Advanced Gelation pH, Gelation rate, Urea hydrolysis rate

A modeling of urea hydrolysis in chitosan medium and global enzymatic chitosan gelation kinetics was developed to better understand the gelation dynamics. The model was validated using experimental results in batch stirred reactor. Comparison between urea hydrolysis reaction rates in water and chitosan media shows that the chitosan has nearly no influence on the enzymatic kinetics parameters. Modeling results allowed decoupling gelation phenomena and urea hydrolysis reaction and pointing out the limitation of urea hydrolysis reaction by urea concentration depletion in chitosan medium. Monitoring pH and d(pH)/dt was useful for determining (i) the gelation time, (ii) the advanced gelation time, (iii) time of the maximum gelation rate and (iv) time of the maximum urea hydrolysis rate.

Introduction

Chitosan is a bio-based N-deacetylated polysaccharide composed of a succession of Dglucosamine (GlcN) and N-acetyl-glucosamine (GlcNAc) units (Figure 1). A given chitosan is characterized by its degree of deacetylation (DDA, expressing the percentage of GlnN units), upper than 60% to differentiate chitosan from chitin, and its weight-average molecular weight (MWchit) between 50,000 and 2000,000 g.mol -1 [1]. Amino-groups of D-glucosamine chitosan units are mostly protoned in acid aqueous medium while they are mostly non-protoned in basic conditions, favoring macromolecular association and resulting in a "physico-chemical" gelation of chitosan in aqueous medium (Reaction 1) when pH becomes higher than the pKa of the chitosan amino-groups (pKa = [6.2 -7.1]) [2 -7]. In order to prepare chitosan gel, chitosan is usually dissolved in dilute acid aqueous solution such as acetic acid or hydrochloric acid to reach a polymer concentration around 2 to 4% w/v. Then, chitosan gelation occurs due to an external supply of sodium hydroxide or ammonia [8 -12]. Recently, an original enzymatic chitosan gelation process has been proposed by Chenite et al. [START_REF] Chenite | Monolithic gelation of chitosan solutions via enzymatic hydrolysis of urea[END_REF] to obtain homogeneous gelation: the OH -ion required for the chitosan gelation (Reaction 1) was produced directly in-situ thanks to the urea hydrolysis by urease enzyme (Reaction 2) and the equilibrium of ammonia/ammonium (Reaction 3). This chitosan enzymatic gelation has been further experimentally investigated and used to produce chitosan hydrogel for different applications such as delivery periodontal ligament cells or membrane preparation [14 -16]. The gelation time of chitosan mainly depends on urease concentration [START_REF] Yan | Enzymatic Control of Chitosan Gelation for Delivery of Periodontal Ligament Cells[END_REF][START_REF] Wlodarzczyk | Enzymatic gelation to prepare chitosan gels: Study of gelation kinetics and identification of limiting parameters for controlled gel morphology[END_REF] and temperature [START_REF] Wlodarzczyk | Enzymatic gelation to prepare chitosan gels: Study of gelation kinetics and identification of limiting parameters for controlled gel morphology[END_REF] while urea concentration was shown to have nearly no influence if this concentration is high enough to prevent urea depletion before the gelation [START_REF] Serrato-Millan | Rheology and gel point of the enzymatic hydrolysis of urea in the presence of urease[END_REF][START_REF] Wlodarzczyk | Enzymatic gelation to prepare chitosan gels: Study of gelation kinetics and identification of limiting parameters for controlled gel morphology[END_REF]. In the aforementioned studies, the gelation kinetics have been monitored by pH measurements [START_REF] Serrato-Millan | Rheology and gel point of the enzymatic hydrolysis of urea in the presence of urease[END_REF][START_REF] Wlodarzczyk | Enzymatic gelation to prepare chitosan gels: Study of gelation kinetics and identification of limiting parameters for controlled gel morphology[END_REF] and compared in both aqueous and chitosan media. The main conclusions of those studies were: (i) presence of chitosan seemed to have a weak influence on enzyme activity during urea hydrolysis [START_REF] Wlodarzczyk | Enzymatic gelation to prepare chitosan gels: Study of gelation kinetics and identification of limiting parameters for controlled gel morphology[END_REF] and (ii) the difference may only be related to the chitosan amino-group equilibrium. However, this assumption has not yet been confirmed in these previous studies. Moreover, pH measurement only allows a partial understanding of the global chitosan enzymatic gelation since kinetics of simultaneous chemical reaction involved in chitosan gelation (Reaction 1) and urea enzymatic hydrolysis (Reaction 2) cannot be dissociated by a pH monitoring only. Investigating and modeling the kinetics of urea hydrolysis reaction in chitosan medium is therefore a key point to deeply understand the global chitosan enzymatic gelation and to predict chitosan gelation time. In this context, the influence of various operating parameters (temperature, urease concentration, urea concentration, pH, product inhibition, substrate inhibition) on urea enzymatic hydrolysis kinetics is well known in aqueous medium [START_REF] Krajewska | Functional, catalytic and kinetic properties: A review[END_REF] and these urea hydrolysis kinetics have been successfully modeled for pH-buffered aqueous medium [START_REF] Fidaleo | Kinetic study of enzymatic urea hydrolysis in the pH range 4-9[END_REF] and buffer-free aqueous medium [START_REF] Qin | Kinetic-Studies of the Urease-Catalyzed Hydrolysis of Urea in a Buffer-Free System[END_REF] but, to our knowledge, the same investigation has never been conducted in chitosan solution.

Based on prior urea hydrolysis kinetics modeling in aqueous medium, the aim of this paper is to develop a model of both urea hydrolysis in chitosan medium and global enzymatic chitosan gelation. Modeling and experimental results of enzymatic chitosan gelation were compared to validate the numerical model. Modeling results in aqueous medium and in chitosan solution was compared in order to understand the influence of chitosan presence on the urea hydrolysis reaction. Finally, the chitosan gelation and urea hydrolysis kinetics were dissociated and investigated.

Materials and methods

Materials

Chitosan (obtained from France Chitine, France) had a degree of deacetylation ( ) of 80% (supplier's data determined by nuclear magnetic resonance) and a weight-average molecular weight ( ) of 180,000 g.mol -1 (supplier's data determined by size-exclusion chromatography). Urea, urease and hydrochloric acid were purchased from Sigma (France).

Urease (type III, EC 3.5.1.5) from Canavalia ensiformis (Jack bean) was supplied as a glycerol solution and the specific activity of this enzymatic solution was 778,000 U.L -1 [START_REF] Wlodarzczyk | Enzymatic gelation to prepare chitosan gels: Study of gelation kinetics and identification of limiting parameters for controlled gel morphology[END_REF].

Enzymatic urea hydrolysis in buffer-free chitosan solution and in buffer-free water medium

Enzymatic urea hydrolysis in buffer-free chitosan solution and in buffer-free water medium was carried out in a 10 mL glass batch reactor according to the protocol defined in a previous study [START_REF] Wlodarzczyk | Enzymatic gelation to prepare chitosan gels: Study of gelation kinetics and identification of limiting parameters for controlled gel morphology[END_REF]. Osmotic water medium and chitosan solution (2.5 % w/v) were prepared using HCl 0.1 mol.L -1 in order to obtain an initial buffer-free pH of 4.7 ± 0.1. Reactions were performed at different temperatures (274 to 310 K), urea concentrations (0.010 to 0.200 mol.L -1 ) and urease concentrations (500 ± 10 to 10,500 ± 10 U.L -1 ). Reactions were monitored by pH measurements using a Hanna Instruments pH-meter (HI 2214 pH/ORB) and a specific probe for viscous media (HI 2031, Hanna Instruments) in both chitosan solution and water media. For each assay, pH was continuously recorded every 5 seconds until the pH reached a plateau [START_REF] Wlodarzczyk | Enzymatic gelation to prepare chitosan gels: Study of gelation kinetics and identification of limiting parameters for controlled gel morphology[END_REF]. A reference experiment in chitosan solution was reproduced at least five times to determine the uncertainty on pH (± 0.26) [START_REF] Wlodarzczyk | Enzymatic gelation to prepare chitosan gels: Study of gelation kinetics and identification of limiting parameters for controlled gel morphology[END_REF].

Modeling of the chitosan enzymatic gelation kinetics

The mathematical modeling of the chitosan gelation kinetics can be divided in three parts: the chemical equilibrium reaction involved in chitosan gelation (Section 3.1), the urea enzymatic hydrolysis by enzyme urease (Section 3.2) and the chemical equilibrium reactions of the other species in solution (Section 3.3). The modeling algorithm will be described in Section 3.4.

Species are considered in dilute aqueous solutions and activities of the species are assumed to be equal to their concentrations. The activity of the solvent (water) is taken equal to 1. In this study, the whole reactions were modeled in conditions of batch and perfectly mixed reactor.

Chemical equilibrium reaction involved in chitosan gelation

Chitosan gelation results from the chitosan chemical equilibrium reaction (Reaction 1) between the protoned and non-protoned forms of the amino-groups of the chitosan D-glucosamine units. Mass balance on these amino-groups is given at each time t in Equation 1 with the total molar concentration of amino-groups in solution (mol.L -1 ), the total molar concentration of protoned amino-groups in solution (mol.L -1 ) and the total molar concentration of non-protoned aminogroups in solution (mol.L -1 ).

!"#$ % = !"#$ %' !"#$ %' Equation 1
The pKa of these amino-groups of chitosan ranged between 6.2 and 7.2 [START_REF] Rinaudo | Chitin and chitosan: Properties and applications[END_REF][START_REF] Wang | Protonation constants of chitosan with different molecular weight and degree of deacetylation[END_REF] and depends on the chitosan degree of deacetylation. A mean value of 6.7 was retained in this study. The expression of the related equilibrium constant K1 is given with Equation 2.

( ) = * * = * + * , × ) = ) /.1 Equation 2
Chitosan is characterized by its degree of deacetylation (%) and its weight-average molecular weight (g.mol -1 ). The mean molecular weight 234 (g.mol -1 ) of an unit of the chitosan chain can be calculated according to Equation The number 234 (mol.mol -1 ) of moles of D-glucosamine by mole of chitosan can be deduced from Equation 4.

% 789% = ::; ) × 56 !"#$ 56 789

Equation 4

If chitosan is initially in solution at a mass concentration (% w/v) or ′ (g.L -1 ), the concentration of D-glucosamine amino-groups in solution can be calculated with Equation 5.

!"#$ % = ! ! = 56 !"#$ × % 789% = ) ×! ! 56 !"#$ × % 789% Equation 5

Urea enzymatic hydrolysis reaction

Reaction of urea enzymatic hydrolysis by urease is given in Reaction 2 and produced both ammonia and carbon dioxide. The rate of this reaction >? @ (mol.L -1 .min -1 ) is defined as the consumption rate of urea (mol.L -1 ) according to Equation 6.

>? @ = ? ?

Equation 6

Considering an uncompetitive substrate inhibition (by urea at high concentration) and a noncompetitive mechanism for product inhibition (ammonium resulting from the ammonia/ammonium equilibrium in Reaction 3), urea hydrolysis by urease from Canavalia ensiformis (Jack bean) has been modeled for aqueous buffered or buffer-free medium using a similar expression (Equation 7) of hydrolysis reaction rate >? @ . So, the influence of temperature, pH, urea concentration, urease concentration and ammonium concentration on the urea hydrolysis reaction rate was taken into consideration [START_REF] Fidaleo | Kinetic study of enzymatic urea hydrolysis in the pH range 4-9[END_REF][START_REF] Qin | Kinetic-Studies of the Urease-Catalyzed Hydrolysis of Urea in a Buffer-Free System[END_REF].

A "BCAD = E FGH %' !I JK 5 %' !I %' !I ² K M NO) +%' * , K P Q = R S,U' V %' !I JK 5 %' !I %' !I ² K M NO) +%' * , K P Q Equation 7
with the Michaelis constant ( (mol.L -1 ), the substrate inhibition constant ( W (mol.L -1 ), the product inhibition constant ( X (mol.L -1 ) and the enzymatic maximum reaction rate Y Z [ (mol.L -1 .min -1 ) that can be expressed using the temperature and pH-dependent hydrolysis reaction rate coefficient \ ],^ (mol.U -1 .min -1 ) and the urease concentration _ (U.L -1 ).

Unlike previous modeling in water medium, it was preferred in the present study to express the urease concentration in U.L -1 instead of g.L -1 (where one catalytic unit (U) is defined as the enzyme quantity that will liberate 1.0 µmol of NH3 from urea per liter and per minute at pH 7.0 and 298 K) in order to take into consideration a possible loss of enzyme activity.

Hydrolysis reaction rate coefficient \ ],^ depends on temperature and pH and can be expressed according to Equation 8 [START_REF] Fidaleo | Kinetic study of enzymatic urea hydrolysis in the pH range 4-9[END_REF][START_REF] Qin | Kinetic-Studies of the Urease-Catalyzed Hydrolysis of Urea in a Buffer-Free System[END_REF] with the hydrolysis temperature-dependent reaction rate constant \ ] (mol.U -1 .min -1 ) and the pH-dependent correction factor `^ (-).

R S,U' = `^ R S = R aHUb cVG dS e f) ) cU' K VM,) g VM,) K VM, ) cU' g VM, h
Equation 8

Temperature-dependent reaction rate coefficient \ ] was supposed to follow Arrhenius Equation [START_REF] Qin | Kinetic-Studies of the Urease-Catalyzed Hydrolysis of Urea in a Buffer-Free System[END_REF] with the Arrhenius Equation pre-exponential factor \ (mol.L -1 .U -1 . min -1 ), the activation energy _ (J.mol -1 ) and the universal gas constant i (J.mol -1 .K -1 ) as shown in Equation 9.

\ ] = \ [^b _ i]
e Equation 9

pH-dependent correction factor `^ can be expressed according to Equation 10 [18] where ( _W,) and ( _W, are the molecular dissociation constants for the enzyme-substrate complex and j _W,) and j _W, are empirical temperature-independent constants.

`^ = ) f) ) c^ ( _W,) j _W,) ( _W, ) c^ j _W, h

Equation 10

Finally, according to Reaction 2, production rate of ammonia concentration (mol.L -1 ) and carbon dioxide concentration (mol.L -1 ) can be expressed following Equations 11 and 12.

? ? = >? @ Equation 11

? ?

= >? @ Equation 12

Chemical equilibrium reactions in aqueous solution

Since urea enzymatic hydrolysis produces ammonia and carbon dioxide (Reaction 2), two other reaction equilibriums have to be taken into account: the ammonium/ammonia equilibrium reaction (Reaction 3') and the carbon dioxide/hydrogen carbonate/carbonate equilibrium reactions (Reactions 4 and Reaction 5).

↔ Reaction 3' ↔ Reaction 4 ↔ Reaction 5
Equations and constants of these equilibria are presented in Table 1 ( Equations 13,[START_REF] Yan | Enzymatic Control of Chitosan Gelation for Delivery of Periodontal Ligament Cells[END_REF][START_REF] Serrato-Millan | Rheology and gel point of the enzymatic hydrolysis of urea in the presence of urease[END_REF] with also the hydronium-hydroxide equilibrium (Equation 16) using the ammonium (mol.L -1 ), carbonate + , (mol.L -1 ), hydrogen carbonate (mol.L -1 ), hydroxide (mol.L -1 ) and the hydronium (mol.L -1 ) concentrations. The hydronium concentration can be expressed with the pH according to Equation 17 and the water dissociation constant KW (-) is reported in Table 1. Ammonium/ammonia equilibrium constant ( is considered temperature-independent while the influence of the temperature T (K) is considered for the carbon dioxide/hydrogen carbonate/carbonate equilibrium constants k l and ( m .

= ) ^

Equation 17

Table = >? @ = ? ?

1: Equilibrium equations and constants in aqueous solutions

Equilibrium

Equation 19

The initial concentrations of ammonium, ammonia, carbon dioxide, hydrogen carbonate and carbonate are considered null and, as no external transfer is considered, the mass balance for ammonium/ammonia equilibrium and dioxide/hydrogen carbonate/carbonate equilibrium can be written at each time t (min) according to Equation 20 and 21 where (mol.L -1 ) is the initial urea concentration:

= Equation 20 + , =

Equation 21

Finally, the electronic balance is given in Equation 22with the chloride concentration 3 (mol.L -1 ).

= + , 3

Equation 22

Modeling numerical resolution

Figure 2: Modeling numerical resolution strategy

Numerical time-resolution of the modeling was performed using finite difference method with MATLAB-software. Time-step dt = 0.5 min (30 s) was found to be a good compromise between the resolution time and results precision for modeling in chitosan medium while a time-step dt = 0.001 min (0.06 s) was required for modeling in water medium due to faster reactions involved. For each time-step, simulation resolution was performed in two stages.

The first stage was the enzymatic hydrolysis reaction: the urea hydrolysis rate was calculated using Equations 7 and 8 at the pH and urea concentration at the time t. Urea Concentrations of the other species were calculated at this time t + dt using the same set of equations. This routine of two stages was performed for each time-step until the end of the reaction.

Results

Determination of the kinetics parameters for the reaction involved in enzymatic gelation

Enzymatic hydrolysis reaction rate r opqrs reaction depends on several parameters, the Michaelis constant ( , the ammonium enzyme inhibition constant ( X , the urea enzyme inhibition constant ( W and the hydrolysis reaction rate coefficient \ ],^ which depends itself on the molecular dissociation constant for the enzyme-substrate complexes ( _W,) and ( _W, (and their respective associated empirical factor j _W,) and j _W, ), the Arrhenius Equation preexponential factor \ and the activation energy _ . Chitosan may have an influence on these parameters and they have to be determined in chitosan medium using specific experimental results: two sets of experiments were thus used for a 2.45% w/v chitosan concentration, a 3,500 U.L -1 urease concentration and an initial pH equal to 4.7 ± 0.1: (ii) A second set of experiments for a temperature of 298.15K and 0.075, 0.100, 0.150 and 0.200 mol.L -1 for the urea concentration. Experiment at 0.200 mol.L -1 is from the present study while others are from [START_REF] Wlodarzczyk | Enzymatic gelation to prepare chitosan gels: Study of gelation kinetics and identification of limiting parameters for controlled gel morphology[END_REF].

(i) A first
For each experiment, data of pH versus time were used. At each reaction time, urea concentration was calculated from pH using the chitosan gelation and chemical equilibrium reactions, i.e. the set of Equations 1, 2, 13, 14, 15, 16, 17, 20, 21 and 22 using build-in fsolve MATLAB®software function (least square method). Experimental hydrolysis reaction rate A aHU (mol.L -1 .min -1 ) was then calculated using Equation 23:

[^= ?

?

Equation 23

The values of the enzymatic hydrolysis reaction rate parameters for this study were determined using experimental data and reported in Table 2. This table also presents, for comparison, parameters from literature for both buffer-free water medium [START_REF] Qin | Kinetic-Studies of the Urease-Catalyzed Hydrolysis of Urea in a Buffer-Free System[END_REF] and buffered water medium [START_REF] Fidaleo | Kinetic study of enzymatic urea hydrolysis in the pH range 4-9[END_REF], i.e. without chitosan. In this table, urease mass (g) has been converted in urease activity (U) using the enzyme activity-mass conversion factor k' = 1.15×10 4 U.g -1 determined from supplier data for the enzyme used in the present study. Furthermore, \ was calculated from T* (i.e. the temperature in K at which \ ] = 1 mol.g -1 . min -1 ) and _ for [START_REF] Fidaleo | Kinetic study of enzymatic urea hydrolysis in the pH range 4-9[END_REF]. Michaelis constant ( has been reported to be ranged between 2.9×10 -3 and 3.6×10 -3 mol.L -1 according to the purity of the enzyme and the conditions for its determination like temperature, buffered or buffer-free conditions, pH and concentration [START_REF] Krajewska | Functional, catalytic and kinetic properties: A review[END_REF]. An intermediate value of 3.21×10 -3 mol.L -1 was retained for the present work (Table 2) and it was considered independent of the temperature and pH [START_REF] Fidaleo | Kinetic study of enzymatic urea hydrolysis in the pH range 4-9[END_REF].

An uncompetitive inhibition mechanism of urease by substrate urea can be generally considered and values of urea inhibition constant ( W from 3 to 6.4 mol.L -1 have been reported in literature [START_REF] Krajewska | Functional, catalytic and kinetic properties: A review[END_REF][START_REF] Qin | Kinetic-Studies of the Urease-Catalyzed Hydrolysis of Urea in a Buffer-Free System[END_REF][START_REF] Moynihan | Urea Hydrolysis by Immobilized Urease in a Fixed-Bed Reactor -Analysis and Kinetic Parameter-Estimation[END_REF]. In the same way, a non-competitive inhibition mechanism of urease by product ammonium have been reported in literature with inhibition constant ( X from 2×10 -3 to 118×10 -3 mol.L -1 [START_REF] Krajewska | Functional, catalytic and kinetic properties: A review[END_REF][START_REF] Fidaleo | Kinetic study of enzymatic urea hydrolysis in the pH range 4-9[END_REF][START_REF] Hoare | The Molecular Kinetics of the Urea-Urease System .2. The Inhibition by Products[END_REF]. Due to the small urea and ammonium concentrations in this study, these inhibitions were neglected and both ( W and ( X were thus considered infinite.

The temperature-dependent reaction coefficient \ ] was used as fitting parameter, at each reaction time, between modeling value of hydrolysis rate >? @ (Equation 8) and experimental value of hydrolysis rate [^ (Equations 23) using least square minimization for the results of the first set of experiments (temperature between 274.15 and 310.15K). An

Arrhenius pre-exponential factor k0 of 4.14×10 -3 mol.U -1 .min -1 was found, which is higher than the one reported by Qin et al. 1994 [19] but the comparison is difficult since the mass/activity conversion factor u = was established for the present work though it should be different in their work. Activation energy _ of 22,221 J.mol -1 was found (Table 2), which is slightly lower than results reported in literature for buffer-free medium (29,440 J.mol -1 [START_REF] Qin | Kinetic-Studies of the Urease-Catalyzed Hydrolysis of Urea in a Buffer-Free System[END_REF] or 27,781 J.mol -1 [START_REF] Dixon | Jack Bean Urease[END_REF]). This might be due to the larger temperature range used in the present work (a temperature range of 293.15 to 313.15 K have been generally used in other studies).

The pH correction factor `^ was considered temperature independent as reported by Fidaleo et al. 2003 [START_REF] Fidaleo | Kinetic study of enzymatic urea hydrolysis in the pH range 4-9[END_REF] and unlike Qin et al. 1994 [START_REF] Qin | Kinetic-Studies of the Urease-Catalyzed Hydrolysis of Urea in a Buffer-Free System[END_REF]. At each reaction time, `^ was used as fitting parameter between modeling value >? @ (Equation 8) and experimental value [^ (Equations 23) using least square minimization for results of the second set of experiment.

Considering j _W,) =j _W, = ), values for the molecular dissociation constant were found to be ( _W,) = (1.04 ±0.01)×10 -6 and ( _W, = (2.98 ±0.01)×10 -9 mol.L -1 .

Comparison between modeling and experimental results in chitosan aqueous solution

The simulation results of the enzymatic chitosan gelation were compared to experimental results from our previous work [START_REF] Wlodarzczyk | Enzymatic gelation to prepare chitosan gels: Study of gelation kinetics and identification of limiting parameters for controlled gel morphology[END_REF] for a buffer-free 2.5% w/v chitosan solution at initial pH = 4.7. Figure 3 presents pH time-variation during the chitosan enzymatic gelation [START_REF] Wlodarzczyk | Enzymatic gelation to prepare chitosan gels: Study of gelation kinetics and identification of limiting parameters for controlled gel morphology[END_REF]: i)

for different urea initial concentrations with a temperature of 298 K and an urease concentration of 3,500 U.L -1 (Figure 3a); ii) for different values of the temperature T with an urea initial concentration of 0.075 mol.L -1 and an urease concentration of 3,500 U.L -1 (Figure 3b); iii) and for different urease concentrations _ with a temperature of 298 K and an urea initial concentration of 0.075 mol.L -1 (Figure 3c). Figure 3 shows a fairly good concordance between experimental and modeling results, thus validating the model and allowing the determination of its associated kinetics parameters (Table 2) for a large range of operating conditions: an urease concentration _ between 500 and 10,500 U.L -1 , an urea concentration between 0 and 0.200 mol.L -1 , a pH between 4.0 and 9.0 and a temperature between 274 and 310 K. However, some differences can be still raised between modeling and experimental data that should be investigated more deeply.

First, except for the experiments where urea is a limiting parameter due to its too low concentration (below 0.050 mol.L -1 ), the modeling results always underestimate the plateau of pH at the end of the experiments (Figure 3a). This plateau indicates the end of the experiment, i.e. the depletion of urea. It would indicate that the depletion of urea occurred later and that there was initially more urea in the solution than expected (which allows reaching a higher pH value). In Section 4.1, urea concentration during the reaction, used for the determination of kinetics parameters of the reaction involved in enzymatic gelation, was estimated using pH value. However, uncertainty may exist on chitosan characteristics such as the pKa of aminogroups, the degree of deacetylation and the molecular weight used for the calculation of the amino-group concentration which thus could induce slight uncertainty on urea concentration.

Secondly, modeling and experimental results are less concordant at low temperatures (Figure 3b) like 283 K or less. It might be related to a divergence from Arrhenius Equation at such low temperatures. Indeed, in water medium, enzyme activity has only been modeled for temperature higher than 293 K (Table 2) and a significant decrease of enzymatic activity has been considered in some studies for temperature lower than 278 K [START_REF] Chenite | Monolithic gelation of chitosan solutions via enzymatic hydrolysis of urea[END_REF].

Finally, modeling and experimental results diverge for intermediate pH values (around 7-8) in some experiments (for example experiment at an urea concentration of 0.150 mol.L -1 in Figure 3a). This phenomenon was also observed in water medium [START_REF] Qin | Kinetic-Studies of the Urease-Catalyzed Hydrolysis of Urea in a Buffer-Free System[END_REF]. A possible explanation could be a transfer of CO2 at the interface between the solution and the external environment around this pH, and thus experimental pH could be higher than predicted by the modeling. Another explanation is the possible inhibition by the product ammonium that would no longer be negligible. Indeed, around these intermediate pH values, ammonium concentration tends to be higher while urea concentration is still high and hence is not yet a limiting factor.

Even if slight differences may be found between the modeling and experimental results of the pH variations versus time, these uncertainties have only a weak influence on the prediction of the gelation time tg and the advanced gelation time tag. Gelation time is the time when chitosan elastic modulus G' and its viscous modulus G'' are equals in rheological measurements [START_REF] Serrato-Millan | Rheology and gel point of the enzymatic hydrolysis of urea in the presence of urease[END_REF][START_REF] Wlodarzczyk | Enzymatic gelation to prepare chitosan gels: Study of gelation kinetics and identification of limiting parameters for controlled gel morphology[END_REF][START_REF] Mi | Fabrication and characterization of a sponge-like asymmetric chitosan membrane as a wound dressing[END_REF]. The advanced gelation time is the time when a completely rigid gel is obtained [START_REF] Wlodarzczyk | Enzymatic gelation to prepare chitosan gels: Study of gelation kinetics and identification of limiting parameters for controlled gel morphology[END_REF]. From our previous work [START_REF] Wlodarzczyk | Enzymatic gelation to prepare chitosan gels: Study of gelation kinetics and identification of limiting parameters for controlled gel morphology[END_REF], gelation time corresponded to pH 6.5 while advanced gelation time corresponded to pH 7.35. Figure 4 presents a comparison between experimental and predicted values of gelation time and advanced gelation time (when the experiment allows reaching its value).

Figure 4: Comparison of the experimental and modeling gelation and advanced gelation times

Figure 4 shows a very good agreement between predicted and experimental characteristic times. An average difference of 20% is observed for the gelation time and of 16% for the advanced gelation time. Enzymatic chitosan gelation modeling can thus be a good tool to predict gelation and advanced gelation time.

Urea enzymatic hydrolysis reaction kinetics in water and chitosan media

One of the aims of the enzymatic chitosan gelation modeling was to determine the possible influence of chitosan on the urea enzymatic hydrolysis kinetics. The same enzymatic chitosan gelation modeling was applied in water medium (i.e. for a chitosan concentration vw equal zero) without modifying the enzyme and kinetic parameters determined in Section 4.1 for chitosan medium. Figure 5 presents experimental [START_REF] Wlodarzczyk | Enzymatic gelation to prepare chitosan gels: Study of gelation kinetics and identification of limiting parameters for controlled gel morphology[END_REF] and modelling pH time-variation during urea enzymatic hydrolysis in non-buffered aqueous medium at initial pH 4.7 for different urea initial concentrations with a temperature of 298 K and an urease concentration of 500 U.L -1 (Figure 5a); and for different temperatures T with an urea initial concentration of 0.075 mol.L -1 and an urease concentration of 500 U.L -1 (Figure 5b). It must be noted that an urease concentration of 500 U.L -1 was used here (instead of 3,500 U.L -1 in chitosan medium) in order to slow down pH increase, hence to visualize an effect of the operating conditions. Important differences concerning the pH time-variations were observed between water medium and chitosan medium [START_REF] Wlodarzczyk | Enzymatic gelation to prepare chitosan gels: Study of gelation kinetics and identification of limiting parameters for controlled gel morphology[END_REF]: for example a pH 6.5 was reached in less than one minutes for an urea concentration of 0.075 mol.L -1 , a temperature of 298 K and an urease concentration of 500 U.L -1 in water medium (Figure 5a) while a pH of nearly 6 was only reached after 200 minutes in chitosan medium. In water medium, pH increased very fast before reaching an "apparent" plateau around 8.5 -9. This fast increase of pH can be directly related to the ammonia produced by urea hydrolysis reaction (Reactions 2 and 3) and thus to the urea enzymatic hydrolysis reaction kinetics.

In chitosan medium, pH increased more slowly but reached a plateau as well. However, in this case the pH could no more be only related to the ammonia produced by urea hydrolysis reaction (Reactions 2 and 3) because of the presence of the chitosan reaction equilibrium (Reaction 1). pH variation is related to the reaction kinetics of the global enzymatic chitosan gelation (Reactions 1, 2 and 3). The chitosan equilibrium reaction is thus the most suitable explanation to understand why the variation of pH is so different between water and chitosan media. Indeed, a good concordance between modeling and experimental results was observed in water medium using enzyme and kinetics parameters established for chitosan medium.

At last, it was interesting to focus on the difference between modeling and experimental results when urea concentration is considered a limiting factor (for 0.025 mol.L -1 in Figure 5a) while a very good concordance was observed in chitosan medium in the same condition (Figure 3a). The difference could be related to a possible product inhibition and may be more visible for low initial urea concentration when there is no product consumption unlike in the case of chitosan medium.

In both chitosan medium and water medium, pH seemed to reach a plateau at the end of the reaction. However, explanations of this behaviour are totally different for chitosan and water media since a "real" plateau was obtained in chitosan medium though only an "apparent" plateau was reached in water medium. Figure 6 presents simulated urea concentration versus time for chitosan medium (Figure 6a, experiments from Figure 3a) and for water medium (Figure 6b, experiments from Figure 5a) Figure 6a shows that urea concentration dropped to zero at the end of the urea enzymatic hydrolysis reaction in chitosan medium. This depletion of urea occurred when the pH reached a "real" plateau (Figure 3a). The level of this plateau, i.e. the final value of pH, is only related to the final cumulated production of the urea hydrolysis reaction and thus to the urea initial concentration (higher pH plateau level corresponding to higher urea initial concentration).

a b
However, the time to reach this plateau depends on the reaction kinetics and thus mainly on temperature and urease concentration [START_REF] Yan | Enzymatic Control of Chitosan Gelation for Delivery of Periodontal Ligament Cells[END_REF][START_REF] Wlodarzczyk | Enzymatic gelation to prepare chitosan gels: Study of gelation kinetics and identification of limiting parameters for controlled gel morphology[END_REF] as seen on Figures 3b and3c.

On the contrary, for water medium (Figure 6b), urea concentration stayed nearly unchanged when the "apparent" plateau of pH was reached at the end of the experiment (after 4 minutes).

To explain the "apparent" plateau reached in water medium, a longer experiment (150 min) was simulated for an initial urea concentration of 0.150 mol.L -1 , an initial pH = 4.7, and with a temperature of 310 K for an urease concentration of 3,500 U.L -1 (in order to simulate faster kinetics). Results of this simulation (not shown here) confirmed that it is an "apparent" plateau since a low pH increase rate of 0.006 min -1 is maintained. Urea hydrolysis rate >? @ was calculated in water and was reported in Figures 7a (versus the time) and 7b (versus the pH). For comparison, the same experiment was also simulated in 2.5% w/v chitosan medium and urea hydrolysis rate >? @ in chitosan medium is also represented in on Figure 7. versus pH (with also the urea concentration)

As expected from the pH measurement in water medium (the fast increase of the pH at the beginning of the reaction), Figure 7a exhibits a peak of the hydrolysis reaction rate at 2.3×10 -3 mol.min -1 at the beginning of the reaction (for time less than one minute) then it reaches a value below 5.0×10 -4 mol.min -1 and exhibits a very slow decreasing. On the contrary, the hydrolysis rate has a bell-shape around a maximum value of 2.3×10 -3 mol.min -1 in chitosan medium. Figure 7b shows variations of reaction rate (with also the variation of urea concentration) in water and chitosan media versus pH. The dependence of the hydrolysis rate on the pH is very similar up to a value of 7.2 which corresponds to the optimum pH for enzyme activity. Until this optimum, pH is reached (few seconds in water medium and about 50 minutes in chitosan medium), urea is still at a high concentration and the reaction limiting parameter is the low pH. After this optimum pH value, a large amount of urea quantity was consumed in chitosan medium because of longer experiment time corresponding to a concentration below 0.075 mol.L -1 : thus urea concentration becomes the limiting parameter for the hydrolysis reaction rate and then the depletion of urea induces the final drop of the hydrolysis rate. On the contrary, the limiting parameter in water medium is always the pH since urea concentration is maintained high and never becomes the limiting parameter. The "apparent" plateau of pH reached in water medium is thus due to the influence of pH that slowdown the hydrolysis reaction at higher value but does not completely stop it. It is also important to note that the pH at the "apparent" plateau is close to the ammonia/ammonium equilibrium (pKa = 9.3) which also contributes to slow-down the pH increasing by a buffer effect. Finally, it is interesting to point out that the surprising trend of the curve representing the urea concentration versus pH is easily explained by the fact that only few urea is consumed to reach a pH of 9.2 (very short experiment duration) and then urea concentration decreases for a longer period of time (at a rate of around 0.464×10 -3 mol.L -1 .min -1 ) while the pH exhibits a very slow variation (at a rate of only 0.006 min -1 ) during this same period.

To conclude this section, pH measurement can be a useful tool for monitoring urea enzymatic hydrolysis reaction rate in water medium, but only for the initial time of the reaction before the "apparent" plateau, while pH measurement might characterize the global chitosan enzymatic gelation rate in chitosan medium.

Monitoring of gelation and urea hydrolysis reaction kinetics in chitosan medium

Figure 8a presents the pH time-variation of the simulated experiment in 2.5% w/v chitosan medium for an initial urea concentration of 0.150 mol.L -1 , an initial pH 4.7, a temperature of 310 K and an urease concentration of 3,500 U.L -1 . Gelation point is usually defined thanks to rheological measurements [START_REF] Mi | Fabrication and characterization of a sponge-like asymmetric chitosan membrane as a wound dressing[END_REF] when the elastic modulus G' (storage modulus which represents the gel solid component) overcomes the viscous modulus G'' (loss modulus which represents the gel liquid component). It corresponds to the formation of inter-molecular non-covalent bonds (hydrogen bonds and van der Waals interactions) because of the deprotonation of chitosan amino-groups. At this gelation point, a gelation time tg and a corresponding gelation pHg can be defined [START_REF] Wlodarzczyk | Enzymatic gelation to prepare chitosan gels: Study of gelation kinetics and identification of limiting parameters for controlled gel morphology[END_REF]. However, at this pH, gel can still flow and in previous study, we defined an advanced gelation time tag with a corresponding advanced gelation pHag where a fully rigid gel (that cannot flow anymore) was obtained.

A complementary approach can be to consider gelation as the chemical equilibrium between the protoned and non-protoned forms of the amino-groups of the chitosan D-glucosamine unit. According to this definition, a gelation degree may thus be defined according to Equation 24and is also shown on Figure 8a (purple dashed line). This gelation degree indicates the ratio of chitosan non-protoned D-glucosamine units over the total chitosan D-glucosamine units (both protoned and non-protoned). Figure 8a shows that if a gelation pHg of 6.5 is considered as in our previous work [START_REF] Wlodarzczyk | Enzymatic gelation to prepare chitosan gels: Study of gelation kinetics and identification of limiting parameters for controlled gel morphology[END_REF] for a time of 27 minutes, it corresponds to a gelation degree of only 34%. Figure 8b represents the time-variation of d(pH)/dt. The local minimum of this curve is obtained at 32 minutes which corresponds to pH of 6.7, i.e. the pKa of the chitosan amino-groups retained for the modeling.

Indeed, the slowest variations of pH obtained in the buffer zone of the chitosan amino-groups.

For the simulations, this pH must thus be considered the "real gelation pH" and correspond to a gelation degree of 50%.

Gelation pH can thus be defined both as the pH when G' and G" modulus are equal and as the pH equal to the pKa of the chitosan amino-groups, but it must be noted that both definitions only concern the "initiation" of the gelation process that is a continuous evolution: if a gel is well obtained, it does not signify that the gel is rigid and does not flow. Moreover, of only little difference is obtained between the gelation times if gelation pH of 6.5 (27 minutes) or 6.7 (32 minutes) are considered, this small variation of pH in this zone induces an important For the advanced gelation time (when a rigid gel is obtained) [START_REF] Wlodarzczyk | Enzymatic gelation to prepare chitosan gels: Study of gelation kinetics and identification of limiting parameters for controlled gel morphology[END_REF], a gelation degree of 83% was obtained which reports on a higher gel rigidity than at the gelation time thanks to the presence of a larger percentage of chitosan non-protoned D-glucosamine units. This point could be considered the end of the gelation process.

Following d(pH)/dt as a function of time seems to be a good way for determining the gelation pH. In order to validate this method, Figure 9 presents variations of d(pH)/dt for the experiment corresponding to 2.5% w/v chitosan medium, 0.150 mol.L -1 urea concentration, 298 K and a 3,500 U.L -1 urease concentration (experiment of Figure 3a). gelation and determine the gelation time and the advanced gelation time without using rheological measurements. Furthermore, a thorough analysis of pH evolution can also give some useful information to differentiate the chitosan gelation kinetics and the enzymatic reaction kinetics.

Figure 10 presents time-variation of the chitosan gelation rate r yz{ (defined according Equation 25), the urea hydrolysis reaction rate r opqrs for the same simulated experiment of

Conclusion

Based on urea hydrolysis kinetics modeling in water medium, an enzymatic chitosan gelation kinetics modeling was developed and successfully validated by experimental measurement in batch stirred reactor. Comparison between urea hydrolysis reaction rates in water and chitosan media exhibited that the chitosan has nearly no influence on the enzymatic kinetics parameters. The differences observed in pH measurement were only due to the presence of the chitosan equilibrium.

Simulations results allowed decoupling and better understanding gelation and urea hydrolysis reaction and pointing out the limitation of urea hydrolysis reaction by urea concentration depletion in chitosan medium and by pH in water medium. Modeling results exhibited also that monitoring pH and d(pH)/dt was a good indicator for the determination of (i) the gelation time, (ii) the advanced gelation time, (iii) time of the maximum gelation rate and (iv) time of the maximum urea hydrolysis rate.

Next step of this modeling will be the addition of internal and external mass and heat transfer modeling in order to deeply investigate fate of dissolved organic carbon and ammonia that can be present in the solution.

Data availability

The raw/processed data required to reproduce these findings cannot be shared at this time as the data also forms part of an ongoing study.

List of Symbols

Figure 1 :

 1 Figure 1: Chitosan N-acetyl glucosamine (GlcNAc) and D-glucosamine (GlcN) units.

Figure 2 3 (

 23 Figure2presents a scheme of the resolution strategy. Initial parameters, i.e. at the beginning of the chitosan enzymatic gelation (for time t = 0 min), are the temperature T (K), ^ = ^ , the urease concentration _ (U.L -1 ), the urea initial concentration

a b c Figure 3 :

 3 Comparison of experimental and modeling pH variations during chitosan enzymatic gelation for a buffer-free 2.5% w/v chitosan solution of a) assay of initial urea concentration with T = 298 K and _ = 3,500 U.L -1 ; b) assay of temperature with = 0.075 mol.L -1 and _ = 3,500 U.L -1 ; and c) assay of urease concentration with T = 298 K and = 0.075 mol.L -1

Figure 5 :

 5 Figure 5: Comparison of experimental and modeling pH variations during enzymatic urea hydrolysis for non-buffered aqueous medium for _ = 500 U.L -1 : a) influence of at T = 298 K and b) influence of T with = 0.075 mol.L -1

Figure 6 :

 6 Figure 6: Variation of urea concentration versus time for different urea initial concentration at T = 298 K during a) chitosan enzymatic gelation for a buffer-free 2.5% w/v chitosan solution with _ = 3,500 U.L -1 and b) during enzymatic urea hydrolysis for a buffer-free aqueous medium with _ = 500 U.L -1

Figure 7 :

 7 Figure 7: Variation of urea hydrolysis rate in water and 2.5% w/v chitosan media for = 0.150 mol.L -1 , T = 310 K and _ = 3,500 U.L -1 : a) versus time and b)

Figure 8 :

 8 Figure 8: Variations of gelation percentage versus time for = 0.150 mol.L -1 , T = 310 K and _ = 3,500 U.L -1 and a non-buffered 2.5% w/v chitosan solution a) with the pH variations and b) with the dpH/dt variations.

Figure 9 :

 9 Figure 9: Variations of dpH/dt for for = 0.150 mol.L -1 , T = 298 K and _ = 3,500 U.L -1 and a non-buffered 2.5% w/v chitosan solution

Figure 8 25 Figure 10 :

 82510 Figure 8 and the variation of d(pH)/dt variations versus time.

(234((

  Carbonate and hydrogen carbonate equilibrium constant -( mCarbon dioxide and hydrogen carbonate equilibrium constant -( _W,)Enzyme-substrate complex 1 molecular dissociation constant mol.L -1 Chitosan unit mean molecular weight g.mol -1 234 Chitosan D-glucosamine unit molecular weight g.mol -1 234 4 Chitosan N-acetyl-glucosamine unit molecular weight g.mol -1 234 Number of moles of D-glucosamine units by mole of chitosan mol.mol -1 pKa pKa of the chitosan amino-groups -Urea enzymatic hydrolysis maximum reaction rate mol.L -1 .min -1 `^ pH-dependent urea hydrolysis reaction rate coefficient -A. Chenite, M. Buschmann, D. Wang, C. Chaput, N. Kandani, Rheological characterisation of thermogelling chitosan/glycerol-phosphate solutions, Carbohydrate Polymers 46 (2001) 39-47 [2] K.M. Varum and O. Smidsrod, Structure-Property relationship in chitosans. Polysaccharides: Structural diversity and Functional Versatility, Second Edition, CRC Press, New-York, 2004, 625-642 correction factor Chitosan total amino-group initial concentration mol.L -1 Chitosan non-protoned amino-group activity -Chitosan non-protoned amino-group concentration mol.L -1

Table 2 : Enzymatic reaction kinetics validation range and parameters

 2

		Source	This study	[19]	[18]
		Urease concentration (U.L -1 )	500 -10,500	4.6 -115	230 -2,300
		Urea concentration (mol.L -1 )	0 -0.200	0 -3	0 -0.070
	Validation range	pH	4 -9 (Buffer-free)	5 -9 (Buffer-free)	4 -9 (Buffered)
		Ammonium (mol.L -1 )	0 -0.400	0 -0.040	0 -0.080
		Temperature (K)	274 -310	293 -313	298 -310
		Chitosan concentration (% w/v)	2 -4	0	0
	Modeling parameters	( (mol.L -1 ) ( X (mol.L -1 ) ( W (mol.L -1 ) _ (J.mol -1 ) ] * (K) \ (mol.U -1 .min -1 ) ( _W,) (mol.L -1 )	(3.21 ±0.01)×10 -3 +∞ +∞ 22,221 ±1 -(4.14 ±0.01)×10 -3 (1.04 ±0.01)×10 -6	2.56×10 -3 6.93×10 -2 6.18 29,100 -6.96×10 -4 10 -5.62 (298 K), 10 -5.28 K) (308 K), 10 -5.15 (318	(3.21 ±0.36)×10 -3 (1.22 ±0.11)×10 -2 +∞ 35,300 414.6 2.44 (7.57 ±0.04)×10 -7
		( _W, (mol.L -1 )	(2.98 ±0.01)×10 -9	10 -9.07 (298 K), 10 -9.43 K) (308 K), 10 -9.58 (318	(1.27 ±0.08)×10 -8
		j _W,)	1	0.564 (298 K), 0.717 K) (318 K), 0.769 (318	1
		j _W,	1	0.373 (298 K), 0.489 0.542 (318 K) (308 K),	1


Maxime Coutrot

Matthieu Legrand
email: matthieu.legrand@aphp.fr

Etienne Gayat

Arrêt ou poursuite des antihypertenseurs en peropératoire ? Le point de vue de l'anesthésiste-réanimateur

Keywords: hypertension, antihypertenseur, complication de l'anesthésie hypertension, antihypertensive agent, complication of anesthesia

Introduction

La prévalence de l'hypertension artérielle est actuellement d'environ 30 % dans les populations occidentales adultes. Elle est aujourd'hui la pathologie chronique la plus fréquente au niveau mondial et devrait affecter un tiers de la population dans les années à venir selon l'Organisation Mondiale de la Santé.

L'hypertension artérielle est associée à une augmentation de l'incidence des évènements cardiovasculaires : accident vasculaire cérébral, infarctus du myocarde, ou encore insuffisance cardiaque ou rénale, aigue(s) ou chronique(s). Les bénéfices des traitements antihypertenseurs ont depuis longtemps clairement démontré une efficacité dans la prévention de ces complications. L'hypertension artérielle est donc, depuis plusieurs décennies, un enjeu majeur de santé publique et les patients hypertendus sont aujourd'hui traités par différentes classes d'antihypertenseurs en mono ou polythérapie.

En dehors des règles hygiéno-diététiques, le traitement antihypertenseur comprend l'introduction d'une bithérapie en première ligne par un inhibiteur de l'enzyme de conversion (IEC) ou un antagoniste des récepteurs de l'angiotensine 2 (ARA2), et un inhibiteur calcique, ou un diurétique thiazidique [1].

Les bétabloquants sont recommandés en première ligne, en association aux IEC ou ARA2, en cas d'antécédent d'infarctus du myocarde. Les objectifs tensionnels recommandés par les dernières recommandations européennes sont de 120-130/70-79 mmHg chez la plupart des patients de moins de 65 ans et de 130-140/< 80 mmHg chez les insuffisants rénaux et chez les patients plus âgés, si le traitement est bien toléré [1].

La prise en charge des patients hypertendus et la gestion de ces traitements antihypertenseurs en période péri-opératoire par l'anesthésiste doit permettre d'éviter un déséquilibre cardiovasculaire pouvant favoriser la survenue d'évènements cardiaques, rénaux et cérébro-vasculaires péri-opératoires, mais également de prévenir les effets indésirables potentiellement liés à ces traitements.

Hypertension artérielle et anesthésie

Risque anesthésique chez le patient hypertendu

La littérature concernant le risque péri-opératoire des patients hypertendus chroniques est globalement ancienne, de faible qualité méthodologique, de puissance insuffisante, et centrée sur le risque d'ischémie myocardique et la mortalité [1][2][3][4].

Les études les plus anciennes ne retrouvent pas d'association claire entre hypertension artérielle modérée préopératoire et morbidité cardiovasculaire en postopératoire de chirurgie non cardiaque [1][2]. Une méta-analyse, publiée en 2004, suggère un risque de complications cardiovasculaires péri-opératoires (i.e. mortalité cardiovasculaire, infarctus du myocarde, angor de novo ou de repos, insuffisance cardiaque, arythmies graves, et accident vasculaire cérébral) majoré de plus de 30 % chez les patients hypertendus [4]. Les 30 études inclues dans ce travail présentent cependant une hétérogénéité importante. L'hypertension artérielle sévère ou non traitée est en revanche associée à un risque important de souffrance myocardique, en l'absence de traitement bêtabloquant. Ce risque pourrait être lié à la stimulation adrénergique durant les épisodes douloureux [5][6]. Une étude de cohorte anglaise rapportait une surmortalité chez les patients ayant une pression artérielle diastolique préopératoire supérieure à 84 mmHg (OR 1.07 IC 95 % 1.01-1.13) [7]. L'hypertension artérielle préopératoire apparaît également être un facteur de risque d'insuffisance rénale aigue en postopératoire ou en réanimation [8].

Les données plus récentes apportent quelques éléments pour identifier plus précisément le risque péri-opératoire des patients hypertendus, ou les souspopulations à risque au sein des patients hypertendus. Une augmentation d'allure exponentielle du risque de dommage myocardique avec l'augmentation de pression pulsée en péri-opératoire était observée dans une étude observationnelle prospective [9]. Les pressions artérielles systoliques et pulsées n'étaient quant à elles pas associées à une surmortalité dans la population globale de l'étude (251 567 patients) ou chez les patients âgés de plus de 65 ans. Enfin, les épisodes de baisse de pression artérielle peropératoire par rapport à la pression artérielle préopératoire semblent particulièrement délétères. Une prise en charge hémodynamique individualisée, afin de maintenir avec un objectif de pression artérielle systolique peropératoire de plus ou moins 10 % des chiffres de pression préopératoires, diminuait les complications postopératoires et notamment les épisodes d'insuffisance rénale aiguë [10]. Les données de la littérature sont donc discordantes en ce qui concerne l'association entre hypertension artérielle contrôlée per se et morbi-mortalité cardiovasculaire périopératoire (en dehors du risque rénal). Cependant, les pathologies souvent associées à l'hypertension artérielle, telles que l'insuffisance cardiaque, la coronaropathie, les pathologies cérébro-vasculaires et l'insuffisance rénale chronique, sont clairement associées à un risque de complications cardiovasculaires péri-opératoire [11].

Evaluation préopératoire

Comme précédemment mentionné, une hypertension artérielle non contrôlée est associée à un risque plus élevé de complications postopératoires. En France, 20 % des patients hypertendus ne bénéficient d'aucun traitement antihypertenseur. Il semble donc licite de dépister une hypertension artérielle en consultation d'anesthésie, d'autant plus si le patient n'a pas bénéficié d'un dépistage préalable (par son médecin traitant, la médecine du travail…).

Chez le patient hypertendu traité, il est important d'évaluer, à l'interrogatoire et par sa mesure éventuellement répétée, le contrôle de la pression artérielle et d'évaluer ainsi le risque d'accès hypertensif péri-opératoire. Environ 40 % des patients hypertendus ne sont pas dans les cibles de pressions artérielles recommandées. Une hypertension artérielle non contrôlée (pression artérielle systolique (PAS) > 180 mmHg et/ou pression artérielle diastolique (PAD) > 110 mmHg), ou symptomatique, doit faire discuter le report de l'intervention pour permettre un meilleur contrôle en cas de chirurgie non urgente.

La recherche et l'évaluation de la sévérité des comorbidités associées à l'hypertension artérielle (cardiopathie hypertrophique avec éventuelle insuffisance cardiaque diastolique ou systolique, coronaropathie, insuffisance rénale ou pathologie cérébro-vasculaire) doit-être systématique, y compris en cas de découverte d'une hypertension artérielle en consultation d'anesthésie [1]. La découverte ou le mauvais contrôle des pathologies associées peut motiver des explorations complémentaires selon les recommandations des sociétés savantes [1][12]. Ces examens doivent avoir une finalité : mieux apprécier la sévérité d'une pathologie, son risque de décompensation et donc influencer la stratégie de prise en charge du patient en péri-opératoire, ou bien différer éventuellement l'intervention si une prise en charge spécifique est nécessaire avant chirurgie programmée (coronarographie +/-revascularisation coronaire, prise en charge d'une valvulopathie, équilibration d'un traitement de l'insuffisance cardiaque, chirurgie carotidienne…).

L'évaluation de la capacité fonctionnelle du patient et la recherche des signes cliniques en faveur d'une lésion coronaire méconnue est aussi un élément important dans l'évaluation du risque péri-opératoire du patient hypertendu. Un électrocardiogramme est indiqué pour dépister des stigmates de maladie coronarienne, pour contrôler celui-ci en cas de nouveaux éléments à l'anamnèse, ou si cela n'a pas été fait par le médecin traitant ou le cardiologue dans les 12 mois précédents [12][13].

Enfin, la poursuite ou l'arrêt des traitements antihypertenseurs dépend des classes médicamenteuses utilisées, d'une éventuelle « double » voire « triple » indication pour une autre pathologie que l'hypertension artérielle, et doit donc faire l'objet d'une évaluation du rapport bénéfice/risque à l'échelle individuelle, à la lumière des connaissances actuelles.

Gestion des traitements anti-hypertenseurs

Gestion des bétabloquants

Les bétabloquants sont notamment prescrits pour l'hypertension artérielle, en particulier en cas d'antécédent d'infarctus du myocarde ou d'insuffisance cardiaque. Ils ne sont cependant pas le traitement de première ligne en cas d'hypertension artérielle sans antécédent cardiaque [1].

Les bêtabloquants ont des effets chronotrope, dromotrope et inotrope négatifs par inhibition des récepteurs β1. Ils entrainent donc de façon dose dépendante, notamment, une diminution de la fréquence cardiaque, une baisse du travail et de la consommation d'oxygène myocardique, avec une balance consommation/transport d'oxygène myocardique plus favorable, ainsi qu'une réduction du débit cardiaque et une baisse de la pression artérielle à plus forte dose, dose-dépendante. Il faut différencier les bétabloquants de l'insuffisant cardiaque, prescrits et titrés à faible dose, qui ont pour objectif de ralentir la fréquence cardiaque sans mauvaise tolérance tensionnelle, des bétabloquants prescrits à plus forte dose dans le cadre de l'hypertension artérielle. L'arrêt d'un traitement par bétabloquant est suivi d'un effet « rebond » avec augmentation de la fréquence cardiaque et de la pression artérielle. Il existe également une diminution de l'effet anti-arythmique, pouvant favoriser les épisodes d'arythmies supra-ventriculaires ou ventriculaires. Au contraire, les bétabloquants diminuent les effets de la stimulation adrénergique liée à l'intubation orotrachéale et aux autres stimuli nociceptifs durant la période per et postopératoire.

Des nombreux essais ont testé l'efficacité des bétabloquants pour la prévention des évènements cardiovasculaires et leur influence sur le pronostic en période peropératoire. Dans plusieurs larges cohortes rétrospectives, une diminution de la mortalité hospitalière en péri-opératoire de chirurgie majeure non cardiaque, chez les patients présentant un risque cardiaque élevé, i.e. un score RCRI (Revised Cardiac Risk Index) de 2 ou plus était observée chez les patients sous traitement bétabloquant [14][15]. Ces résultats ont également été confirmés dans d'autres études prospectives de bonne qualité [16]. Cependant, dans une méta-analyse Cochrane récente, incluant 88 essais randomisés et près de 20 000 opérés, aucun effet n'était retrouvé en chirurgie cardiaque concernant la mortalité, toute cause confondue (RR 0.73 IC 95% 0.35-1.52), l'infarctus du myocarde (RR 1.04 IC 95 % 0.71-1.51) ou les évènements cérébro-vasculaires (RR 1.52, IC 95 % 0.58-4.02) [17]. Cette méta-analyse ne mettait pas en évidence par ailleurs, non plus, d'augmentation du risque d'hypotension artérielle (RR 1.54 IC 95 % 0.67-3.51), de bradycardie (RR 1.61 IC 95 % 0.97-2.66) ou d'insuffisance cardiaque congestive (RR 0.22 IC 95 % 0.04-1.34). En revanche, les bêtabloquants étaient associés à une diminution du risque d'arythmies ventriculaires (RR 0.37 IC 95 % 0.24-0.58) et supra-ventriculaires (RR 0.44 IC 95 % 0.36-0.53) ainsi qu'à une diminution de la durée d'hospitalisation (0.54 jours IC 95 % -0.90 -0.19). En chirurgie non cardiaque, cette méta-analyse sur 35 essais randomisés rapportait une augmentation significative de la mortalité (RR 1.25 IC 95 % 1.00-1.57), des épisodes d'hypotension artérielle (RR 1.50 IC 95 % 1.38-1.64) et des épisodes de bradycardie (RR 2.23 IC 95 % 1.48-3.36) en peropératoire. Deux éléments sont à souligner concernant cette méta-analyse. Le premier est que seules 6 études présentaient des critères méthodologiques de qualité. Le second est que dans la plupart de ces essais, les bétabloquants étaient introduits en préopératoire pour diminuer un risque péri-opératoire et n'étaient pas un traitement pris au long cours.

La recommandation formalisée d'experts de la Société Française d'Anesthésie-Réanimation (RFE SFAR 2009) mais également les recommandations américaines (American College of Cardiology Foundation ACCF/American Heart Association [AHA]) préconisent la poursuite des traitements bétabloquants en période péri-opératoire lorsqu'ils sont prescrits de façon chronique [18][19]. En revanche, il n'y a pas de données suffisantes pour recommander une prescription de bétabloquants, en prophylaxie du risque cardiovasculaire en péri-opératoire, en dehors d'une chirurgie chez un patient présentant un risque cardiovasculaire intermédiaire ou élevé (évalué par un score de Lee clinique ≥ 2), et pour une chirurgie au moins à risque intermédiaire (i.e. en dehors des chirurgies superficielles, ophtalmologiques, ambulatoire et actes endoscopiques) [13][18]. Il est fortement recommandé de ne pas commencer le traitement le jour de la chirurgie mais d'augmenter les posologies progressivement en fonction de la tolérance clinique plusieurs jours avant la chirurgie.

En l'absence de contre-indication, la présence de signes cliniques ou électrocardiographiques d'ischémie myocardique aigue est une indication à l'introduction d'un traitement bétabloquant en urgence en période périopératoire. La SFAR recommande l'utilisation d'un agent cardio-sélectif sans activité sympathique intrinsèque si un traitement bétabloquant est introduit en péri-opératoire.

La surveillance hémodynamique devra être particulièrement attentive chez le patient opéré sous bétabloquant, avec un choix de monitorage adapté aux risques liés au patient d'une part et à la chirurgie d'autre part (monitorage de la pression artérielle invasive et/ou monitorage du volume d'éjection systolique).

Gestion des inhibiteurs du Système Rénine-Angiotensine-Aldostérone (SRAA)

Les antagonistes du SRAA, inhibiteurs de IEC et ARA2, sont prescrits en première ligne de façon large dans l'hypertension artérielle, et ce d'autant plus qu'il existe d'autres comorbidités (souvent associées) comme l'insuffisance cardiaque chronique, l'infarctus du myocarde ou l'insuffisance rénale chronique.

Le SRAA est activé en peropératoire par la baisse de pression artérielle secondaire à la baisse du tonus vasculaire et par la baisse du retour veineux secondaire à la baisse de tonus du système capacitif veineux. Les effets de la baisse de pré-charge ventriculaire sont particulièrement marqués en cas d'hypertrophie ventriculaire gauche liée à une cardiopathie hypertrophique chez le patient hypertendu. La stimulation du SRAA potentialise également celle du système nerveux autonome. Par ailleurs, les antagonistes du SRAA entrainent une altération de l'autorégulation rénale et majorent la baisse du débit de filtration glomérulaire lors d'une hypovolémie et/ou d'une hypotension artérielle.

En revanche, la baisse de pré-charge et de post-charge, induite par les inhibiteurs du SRAA, exerce des effets potentiellement bénéfiques chez les patients insuffisants cardiaques.

Les données des études ayant testé l'introduction d'IEC ou d'ARA2 en chirurgie cardiaque et vasculaire, pour la prévention des évènements cardiovasculaires, ne semblent pas retrouver une incidence plus élevée d'épisodes d'hypotension artérielle ou de recours aux vasopresseurs. Une métaanalyse de 7 essais randomisés contrôlés ne retrouvait pas d'effet de l'utilisation d'IEC ou d'ARA2 sur la mortalité ou les complications cérébro-vasculaires (essais de petits effectifs) [20].

Peu d'études ont évalué l'impact de la poursuite ou de l'arrêt de ces médicaments en péri-opératoire chez les patients traités de façon chronique.

Une large étude prospective observationnelle retrouvait un risque cardiovasculaire diminué sur un critère composite associant mortalité, infarctus du myocarde ou accident vasculaire cérébral (12.0 % vs 12.9 % ; RR ajusté 0.82 ; IC 95 % 0.70 to 0.96), ainsi qu'un risque accru d'hypotension artérielle peropératoire (RR 0.80 ; IC 95 % 0.72 to 0.93) [21].

Une méta-analyse récente incluant 5 essais randomisés contrôlés et 4 études de cohorte [22] ne retrouve pas de surmortalité après arrêt des IEC/ARA2 (OR 0.97 ; 95 % CI 0.62-1.52), ni d'augmentation des évènements cardiaques majeurs (OR 1.12 95 % CI 0.82-1.52) en chirurgie non cardiaque, chez les patients traités de façon chronique. Un sur-risque d'hypotension artérielle était en revanche observé (OR 0.63 95 % CI 0.47-0.85) lorsque ceux-ci étaient continués. Il faut noter que les essais randomisés inclus dans cette méta-analyse sont des essais comprenant de petits effectifs (5 essais incluant au total 774 patients) et le lien de causalité ne pouvait être retenu. Un large essai randomisé multicentrique est en cours pour évaluer l'impact de l'arrêt ou de la poursuite des IEC/ARA2 en préopératoire de chirurgie non cardiaque (essai STOP or NOT, NCT03374449).

Les recommandations françaises et européennes actuelles concernant la gestion des IEC et ARA 2 en péri-opératoire sont d'arrêter le traitement en cas d'hypertension artérielle en l'absence d'insuffisance cardiaque [19]. Au contraire, l'AHA recommande le maintien des IEC et ARA2 (niveau d'évidence B) [18]. La RFE commune SFAR-SRLF concernant l'insuffisance rénale aigue en péri-opératoire et en réanimation recommande également (avis d'experts, accord fort) de ne pas utiliser les IEC ou ARA 2 chez les patients à risque d'insuffisance rénale aigue. Parmi les facteurs de risque d'insuffisance rénale aigue, se trouvent justement les indications des IEC/ARA2 : hypertension artérielle, insuffisance cardiaque, diabète, insuffisance rénale chronique. La période péri-opératoire, en particulier de chirurgie majeure, et l'instabilité hémodynamique sont également des facteurs de risque importants d'insuffisance rénale aigue, avec l'âge. En résumé, dans l'état actuel des connaissances et dans l'attente des résultats de l'essai STOP or NOT, le maintien des IEC/ARA2 ne semble pas associé à un risque accru de complication postopératoire mais pourrait être associé à un risque augmenté d'hypotension artérielle peropératoire. Une attention toute particulière devra donc être portée à la détection et au contrôle de la pression artérielle peropératoire.

La survenue d'une insuffisance rénale aigue en postopératoire justifie cependant probablement l'arrêt temporaire des IEC/ARA2. Une reprise de ces traitements après la résolution de l'épisode d'insuffisance rénale aigue est par contre à recommander, deux études récentes suggérant un bénéfice des IEC/ARA2 sur la mortalité après un épisode d'insuffisance rénale aigue.

Gestion des inhibiteurs calciques

Les inhibiteurs calciques sont représentés par les dihydropyridines et des antagonistes calciques anti-arythmiques de la classe IV de Vaughan- Williams (verapamil et diltiazem). Les dihydropyridines induisent une vasodilatation artérielle préférentielle. La baisse des résistances vasculaires périphériques diminue la postcharge cardiaque sans effet inotrope négatif et entraine une tachycardie réflexe par stimulation du baroréflexe. Au contraire, les inhibiteurs calciques anti-arythmiques possèdent des propriétés inotropes négatives modérées.

De même que pour les bétabloquants, l'arrêt du traitement expose au risque de rebond hypertensif, pouvant s'accompagner d'une insuffisance cardiaque aigue diastolique ou systolo-diastolique, et/ou d'une souffrance myocardique.

Au cours de l'anesthésie, l'utilisation d'inhibiteurs calciques pourrait majorer le risque d'hypotension artérielle peropératoire. Cependant, les données sont actuellement largement insuffisantes pour évaluer précisément le risque cardiovasculaire en péri-opératoire induit par ces médicaments. Le diltiazem est associé, dans une méta-analyse, à une diminution significative des épisodes de tachycardies supraventriculaires et d'ischémie myocardique [23]. L'attitude actuelle et les recommandations françaises sont de ne pas les interrompre avant l'anesthésie (recommandation de grade C).

Gestion des diurétiques

Les diurétiques exposent principalement à un risque d'hypovolémie et de troubles électrolytiques.

La gestion des diurétiques est plus consensuelle et leur arrêt, le jour de la chirurgie, est la règle. Un ionogramme récent devra s'assurer de l'absence de complication électrolytique avant l'anesthésie. Il s'agit d'une part de s'assurer de l'absence d'insuffisance rénale aigue préopératoire sous diurétiques et, également, de vérifier l'absence de troubles électrolytiques (dyskaliémie, hypomagnésiémie, hypophosphorémie, dysnatrémie) sévères préopératoires sous diurétiques.

La reprise du traitement pourra être effectuée précocement en postopératoire, en l'absence d'instabilité hémodynamique et d'insuffisance rénale aigue et/ou en cas de surcharge hydrosodée.

Gestion des alpha-2 agonistes

De nombreux essais de petits effectifs ont évalué l'utilisation des alpha-2 agonistes, le plus souvent la clonidine, et le plus souvent en chirurgie cardiaque ou vasculaire, pour la réduction de la morbi-mortalité péri-opératoire. Une métaanalyse retrouvait, en 2003, une diminution des épisodes d'ischémie myocardique en chirurgie vasculaire ou cardiaque et de la mortalité en chirurgie vasculaire. Plus récemment, l'étude POISE-2 ne retrouvait pas ces bénéfices en chirurgie non cardiaque, y compris dans l'analyse en sous-groupe en chirurgie vasculaire. L'utilisation de la clonidine était par contre associée à une incidence plus importante d'hypotension artérielle (PAS < 90 mmHg et nécessitant un traitement) et d'arrêts cardiaques non fatals. Cependant, comme beaucoup d'études concernant les traitements antihypertenseurs, ces essais évaluaient l'introduction de ces molécules dans la prévention des complications périopératoires et non une stratégie de poursuite versus arrêt chez des patients recevant ces traitements de façon chronique.

Les alpha-2 agonistes ne sont pas traités dans les recommandations SFAR concernant la gestion péri-opératoire des traitements chroniques et dispositifs médicaux [19]. L'AHA ne recommande pas leur utilisation en prévention des évènements cardiovasculaires en chirurgie non cardiaque, mais n'émet pas de recommandation sur leur arrêt ou leur maintien chez les patients sous traitement chronique.

Conclusion

Une hypertension artérielle chronique expose au risque de complication postopératoire, notamment rénale. Les évènements cardiaques, rénaux et cérébro-vasculaires péri-opératoires, en lien avec le terrain, et potentiellement aggravés par les traitements, ont également un impact important sur le pronostic à long terme des patients hypertendus. La bonne connaissance des pathologies associées à l'hypertension artérielle et de ses traitements doit permettre une gestion optimale et individualisée par l'anesthésiste-réanimateur pour minimiser la morbi-mortalité péri-opératoire.