Pauline Corbaux

Mévidette El-Madani

Michel Tod

Julien Péron

Denis Maillet

Jonathan Lopez

Gilles Freyer

Prof Benoit You
email: benoit.you@chu-lyon.fr

Clinical efficacy of the optimal biological dose in early-phase trials of anti-cancer targeted therapies

Keywords: Dose-Response Relationship, Drug Antibodies, Monoclonal Antineoplastic Agents Maximum Tolerated Dose

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

Introduction

Since the early 2000s, many molecular targeted therapies have been approved in hematology and solid oncology, including small molecules or monoclonal antibodies (mAbs). New questions were raised about the optimal strategy for defining the best doses and dosing schedules. Indeed, the approach traditionally implemented for conventional chemotherapy agents, relies on dose-escalation trials meant to identify the maximal tolerated doses (MTD) and the recommended phase II trial doses (RP2D). However, this strategy may not be adequate for novel targeted drugs, for which the dose-efficacy and dose-toxicity curves may not be correlated [START_REF] Roda | Are Doses and Schedules of Small-Molecule Targeted Anticancer Drugs Recommended by Phase I Studies Realistic?[END_REF][START_REF] Cannistra | Challenges and pitfalls of combining targeted agents in phase I studies[END_REF][START_REF] Toloi D De | Phase I trials of antitumour agents: fundamental concepts[END_REF], and efficacy may occur at doses that do not induce clinically significant toxicity [START_REF] Postel-Vinay | Clinical benefit in Phase-I trials of novel molecularly targeted agents: does dose matter?[END_REF].

As a consequence, novel drug development strategies meant to identify the best doses and dosing schedules of novel targeted agents have been proposed [START_REF] Rogatko | Translation of innovative designs into phase I trials[END_REF][START_REF] Wong | The changing landscape of phase I trials in oncology[END_REF]. Among them, determination of the "optimal biological dose" (OBD), also called "biologically effective dose" appears promising [START_REF] Sachs | Optimal Dosing for Targeted Therapies in Oncology: Drug Development Cases Leading by Example[END_REF]. Indeed, identification of the minimal dose associated with an optimal predefined biological effect, through measurement of target inhibition and pharmacokinetics analyses, might be a good complementary approach for defining the RP2Ds of targeted therapies [START_REF] Postel-Vinay | Clinical benefit in Phase-I trials of novel molecularly targeted agents: does dose matter?[END_REF][START_REF] Sachs | Optimal Dosing for Targeted Therapies in Oncology: Drug Development Cases Leading by Example[END_REF]. However, there are no data about the actual relevance of this endpoint, in terms of clinical efficacy. The present review study aimed at assessing the clinical relevance of OBDs, defined during early phase trials, for further approvals.

Materials and methods

Study design

This systematic review study was conducted in three consecutive steps. 1) Identification of the FDA approved targeted agents, for which early phase trials defined OBDs:

All the molecular targeted therapies approved before July 2018 by the FDA for hematological or solid malignancies were collected through the National Cancer Institute Database (https://www.cancer.gov/about-cancer/treatment/drugs). The compilation of all early phase trials publications was performed using MEDLINE via PubMed (https://www.ncbi.nlm.nih.gov/pubmed/), and using the American Society of Clinical Oncology (ASCO) website, with the search terms "compound name" or "compound code", "phase I" and "early phase trial". Identifications of OBDs, doses/dosing schedules and biological effects, were tracked in the article bodies. OBD was defined as the lowest dose shown to inhibit a drug target reliably, or to achieve a target plasma concentration [START_REF] Adjei | What is the right dose? The elusive optimal biologic dose in phase I clinical trials[END_REF], and was reported as the biologically effective dose by authors. The following elements were collected for all considered drugs: name of the first author; date of publication; studied tumor types; maximum tolerated dose if any; RP2D if any; reported OBD; OBD-related biological effects; OBD as the primary endpoint of the trial (yes vs no); structure of the drug (small molecules, or mAbs).

Early phase trials investigating more than one investigational molecular targeted agents, or those focusing on specific populations or ethnicities were excluded, as were those involving active immunotherapies, such as vaccines or Chimeric antigen receptor T cell (CAR T cell) therapies, due to their specific and complex mechanisms of action.

2) Identification of the clinically effective doses in subsequent pivotal efficacy trials leading to FDA approvals:

We identified the subsequent efficacy clinical trials leading to the first FDA approvals and the dose/dosing schedules that had been tested, through the "clinical studies" section of the structured product labeling (SPL) document of every compound, using the website https://nctr-crs.fda.gov/fdalabel/ui/search. The corresponding publications were then retrieved using the following keywords on PubMed: "compound name" or "code" and "phase III trial" ("phase II trial" or "phase I trial" when appropriate). The following elements were collected for every selected article: name of the first author; date of publication; studied tumor types; dose and dosing schedule tested; estimated value of the treatment effect on the primary endpoint. Moreover, the following data were collected in the first FDA approvals: approval year; type of approval (accelerated or full), labelled dose/dosing schedules; and tumor indications, using the website https://www.accessdata.fda.gov/scripts/cder/daf/.

3) Comparison of OBDs and clinically effective doses:

The consistency in between the OBDs reported in the early phase trials, the clinically effective doses reported in efficacy trials leading to FDA approval, and the FDA labelled doses/dosing schedules was finally assessed for each selected drug.

The data extraction was performed by two authors (PC and MEM). They were verified by all coauthors.

Statistical analysis

All analyses were descriptive. Qualitative data were described by percentages.

Results

As of July 2018, total 83 molecular targeted therapies were FDA approved for hematological or solid malignancies. Two of them were excluded, because they were active T cell immunotherapies. For the remaining 81 therapies, 87 early phase trials were identified (Figure 1). The concept of OBD was mentioned in the early phase trials of 50/81 of these FDA approved molecules (62%). Actual OBDs were reported in 32 early phase trials, corresponding to 32 molecular targeted therapies (40%) (Appendix). One trial was a pilot biological study of lapatinib, while all others were phase I trials. All assessed trials were published between 1999 and 2017. The median year of publication was 2010. As shown in Table 1, 19/32 were small molecules (59%), while 13/32 were mAbs (41%). OBD identifications were clearly reported as the primary objectives of the early phase trials for 7 drugs (22%). The clinical studies that led to the first FDA approvals of small molecules were phase III efficacy trials for 12/19 of them (63%) and phase II trials for 7/19 of them (37%).

For monoclonal antibodies, these numbers were phase III trials for 10/13 (77%), phase II trials for 2/13 (15%) and phase I trial (cohort expansion) 1/13 (8%).

For 19 of the 32 studied agents (59%), no MTDs were found during the early phase trials despite dose escalations, including 9 small molecules and 10 mAbs, and corresponding to 47% and 77% of all tested small molecules and mAbs, respectively. Among the 13 drugs with reported MTDs, the OBDs were lower than the MTDs for 10 of them (77%), and similar for 3 of them (23%).

For 18/32 drugs (56%), the OBDs were chosen as the RP2Ds, including 10 small molecules (corresponding to 53% of all small molecules) and 8 mAbs (corresponding to 62% of all mAbs). Of note, no MTD had been found for most of these drugs (11/18, 61%, including 5 small molecules and 6 mAbs). The 14 remaining drugs, for which RP2Ds were not the OBDs (44%), comprised 9 small molecules, and 5 mAbs. These RP2Ds were superior to the reported OBDs for 13/14 of these drugs (93%); cetuximab being the only exception. Among these 14 drugs, MTD had been found for 6 of them, of which 5 were chosen as RP2Ds (36% of drugs for which OBDs were not chosen as RP2Ds). For the 9 remaining drugs (64%), the rationale sustaining the RP2D selection was not clearly reported.

Regarding FDA approvals, the labelled doses were similar to the OBDs for 19 of the 32 drugs with reported OBDs (59%), corresponding to 47% of small molecules and to 77% of mAbs, respectively. Regarding the 18 molecules for which OBDs were chosen as the RP2Ds, the final FDA labelled doses were consistent with the OBDs for 15 of them (83%), including 8/10 small molecules (80%), and 7/8 mAbs (88%). The three remaining molecules approved at doses different from OBDs were lapatinib, midostaurin and siltuximab. Their labelled doses were lower than the reported OBDs, and than their RP2Ds. Two of them (lapatinib and midostaurin) were actually approved in combination with chemotherapy at lower doses. Siltuximab was approved at 11 mg/kg every three weeks, instead of 12 mg/kg every three weeks, that was the OBD and defined as the RP2D.

Interestingly, 4 drugs for which RP2Ds were not similar to the OBDs, were finally approved at the OBDs: abemaciclib, cetuximab, obinutuzumab and necitumumab. Cetuximab was approved at intravenous 400 mg/m 2 loading dose followed by weekly 250 mg/m 2 , although the reported RP2D was intravenous weekly 200 mg/m 2 . Abemaciclib was approved at 150 mg orally twice a day, which is inferior to the MTD RP2D, that was set up at 250 mg orally twice a day. As for obinutuzumab and necitumumab, the approved dosing schedules were adjusted for chemotherapy combinations at doses that were similar to OBDs.

Overall, 19/81 (23%) assessed molecular targeted therapies approved by the FDA were approved at their OBDs.

The impact of the type of approval (accelerated versus full) on the results were explored.

FDA granted accelerated approval to 11/32 (34%) molecules, including 6 small molecules and 5 mAbs. OBDs were selected as the RP2Ds for 4 of them (36%), and the approved doses were similar to the OBDs for 5 of them (45%). When OBDs were chosen as the RP2Ds, the accelerated FDA approved doses were consistent with the OBDs for all of them (100%), against 11/14 (79%) for the drugs that were directly fully approved.

Regarding the biological effects of small molecules, OBDs mainly relied on indirect effects on elements of the involved signaling pathways (11/19, 58%). For example, OBDs of imatinib and ponatinib, two BCR-ABL inhibitors, were based on the inhibitions of CRK-like protein (CRKL) phosphorylation, a substrate of BCR-ABL. Otherwise, the biological effects relied on direct inhibition of the target (6/19, 32%) (i.e. diminution of the formation of poly ADP ribose (PAR) for olaparib, a Poly ADP-Ribose Polymerase (PARP) inhibitor), or receptor occupancy (2/19, 10%). For mAbs, the assessed biological effects involved receptor occupancy (6/13, 46%), followed by direct inhibition of the target activity (4/13, 31%), or indirect effects on components of the signaling pathways (2/13, 15%), and finally on target concentration associated with biological effects based on pharmacokinetic study outcomes (1/13, 8%) (Table 2).

Of note, in the early phase trials of 18 other drugs, representing 22% of all approved targeted molecular therapies, no OBDs were finally reported although the concept of OBD was mentioned in the articles. Most of time, the authors failed to determine the OBDs due to the lacks of correlation between drug doses and the assessed biological effects [START_REF] Orlowski | Phase I trial of the proteasome inhibitor PS-341 in patients with refractory hematologic malignancies[END_REF][START_REF] Rodon | A phase I, multicenter, open-label, first-in-human, dose-escalation study of the oral smoothened inhibitor Sonidegib (LDE225) in patients with advanced solid tumors[END_REF][START_REF] Kelly | Phase I clinical trial of histone deacetylase inhibitor: suberoylanilide hydroxamic acid administered intravenously[END_REF][START_REF] Daud | Phase I study of bosutinib, a src/abl tyrosine kinase inhibitor, administered to patients with advanced solid tumors[END_REF][START_REF] Flaherty | Inhibition of Mutated, Activated BRAF in Metastatic Melanoma[END_REF][START_REF] Demetri | Phase I dose-escalation and pharmacokinetic study of dasatinib in patients with advanced solid tumors[END_REF][START_REF] Stein | Enasidenib in mutant IDH2 relapsed or refractory acute myeloid leukemia[END_REF][START_REF] Herbst | Selective oral epidermal growth factor receptor tyrosine kinase inhibitor ZD1839 is generally well-tolerated and has activity in non-small-cell lung cancer and other solid tumors: results of a phase I trial[END_REF][START_REF] Deangelo | Inotuzumab ozogamicin in adults with relapsed or refractory CD22-positive acute lymphoblastic leukemia: a phase 1/2 study[END_REF][START_REF] Weber | Phase I/II study of ipilimumab for patients with metastatic melanoma[END_REF][START_REF] Yamada | Phase I Dose-Escalation Study and Biomarker Analysis of E7080 in Patients with Advanced Solid Tumors[END_REF][START_REF] Infante | A Phase I Study of the Cyclin-Dependent Kinase 4/6 Inhibitor Ribociclib (LEE011) in Patients with Advanced Solid Tumors and Lymphomas[END_REF][START_REF] Faivre | Safety, Pharmacokinetic, and Antitumor Activity of SU11248, a Novel Oral Multitarget Tyrosine Kinase Inhibitor, in Patients With Cancer[END_REF][START_REF] Younes | Brentuximab Vedotin (SGN-35) for Relapsed CD30-Positive Lymphomas[END_REF][START_REF] Falchook | RAF Inhibitor Dabrafenib (GSK2118436) is Active in Melanoma Brain Metastases, Multiple BRAF Genotypes and Diverse Cancers[END_REF][START_REF] Giles | A Phase I Study of Intravenous LBH589, a Novel Cinnamic Hydroxamic Acid Analogue Histone Deacetylase Inhibitor, in Patients with Refractory Hematologic Malignancies[END_REF][START_REF] Wilson | A phase I study of intravenous and oral rucaparib in combination with chemotherapy in patients with advanced solid tumours[END_REF][START_REF] Verstovsek | Safety and Efficacy of INCB018424, a JAK1 and JAK2 Inhibitor, in Myelofibrosis[END_REF].

The time intervals from the publications of early phase trials reporting OBD to the first FDA approvals were relatively short (median, 1 year, range: less than one year to 16 years for midostaurin), with no differences in between small molecules and mAbs.

Discussion:

In 2014, Fontes Jardim et al. showed that the RP2Ds had been used as the labelled doses for 97% of cytotoxic chemotherapy agents, against 58% of targeted agents approved in between 1990 and 2011, thereby suggesting the limitations of traditional toxicity-based trials for defining the optimal clinically effective doses of these novel drugs [START_REF] Jardim | Predictive Value of Phase I Trials for Safety in Later Trials and Final Approved Dose: Analysis of 61 Approved Cancer Drugs[END_REF].

The first reason relates to the lack of direct dose-toxicity relationships for many of these drugs. In a review study of 201 phase I trials, DLTs were less frequently identified with targeted therapies (48%) than with cytotoxic drugs (89%) [START_REF] Penel | Nature and subjectivity of dose-limiting toxicities in contemporary phase 1 trials: comparison of cytotoxic versus non-cytotoxic drugs[END_REF]. Consistently, in a review of 10 82 articles about mAb phase I trials, MTDs had been found in 16% of early phase trials only [START_REF] Tosi | Clinical Development Strategies and Outcomes in First-in-Human Trials of Monoclonal Antibodies[END_REF]. In the present study, MTDs were reported for 53% and 23 % of assessed small molecules and mAbs, respectively, corroborating these data.

The second reason is the frequent lack of direct dose-efficacy relationships, meaning that the highest tolerable doses are not necessarily the most effective doses, contrarily to what is accepted with most of cytotoxic chemotherapy agents.

To solve this issue, identification of the optimal biologically effective dose is frequently described as a relevant approach [START_REF] Roda | Are Doses and Schedules of Small-Molecule Targeted Anticancer Drugs Recommended by Phase I Studies Realistic?[END_REF]. Indeed, the knowledge of the mechanisms of actions of these new targeted agents offers the opportunity to assess the biological effect magnitude induced by the novel drugs, and thus to define the lowest dose associated with the expected pharmacodynamic effects. It was reported that OBDs were indeed frequently identified at lower doses compared to MTDs, thereby suggesting that this strategy could potentially reduce the toxicity profiles, and the costs of anti-cancer treatments [START_REF] Sachs | Optimal Dosing for Targeted Therapies in Oncology: Drug Development Cases Leading by Example[END_REF].

The present study first suggests that OBDs are still rarely assessed, reported in less than 40 % of early phase trials of recent FDA approved targeted agents. When reported, they were chosen as the RP2Ds in 56% of cases. Our work suggests that the search for OBDs may be a relevant strategy, since 83% of the final approved doses were consistent with the OBDs, when they were selected as the RP2Ds. This percentage is much higher than the previously reported 58% with MTD [START_REF] Jardim | Predictive Value of Phase I Trials for Safety in Later Trials and Final Approved Dose: Analysis of 61 Approved Cancer Drugs[END_REF]. Moreover, when both MTDs and OBDs had been defined, the OBDs were lower for most of studied agents (77%), hence corroborating the assumption that the OBD identification strategy could improve the safety of anti-cancer treatments.

Niraparib, a recently approved PARP inhibitor, is an illustrative example. The MTD at 300 mg/day was defined as the RP2D, although the lower 80 mg/day dose was identified as the minimum biologically effective dose, based on PARP inhibition effects in peripheral blood mononuclear cells [START_REF] Sandhu | The poly(ADPribose) polymerase inhibitor niraparib (MK4827) in BRCA mutation carriers and patients with sporadic cancer: a phase 1 dose-escalation trial[END_REF]. In 2017, niraparib was approved as a maintenance treatment for recurrent epithelial ovarian cancer at the MTD (300 mg/day), based on the positive NOVA trial outcomes [START_REF] Mirza | Niraparib Maintenance Therapy in Platinum-Sensitive, Recurrent Ovarian Cancer[END_REF]. However, this dose has subsequently been recognized as too high and toxic, and dose reductions based on patient weight and platelet count are now advised for prescriptions of niraparib in the majority of patients, and for on-going clinical trials [START_REF] Berek | Safety and dose modification for patients receiving niraparib[END_REF][START_REF] Gonzalez | 941PDA prospective evaluation of tolerability of niraparib dosing based upon baseline body weight (wt) and platelet (blplt) count: Blinded pooled interim safety data from the PRIMA Study[END_REF].

If these data about the clinical efficacy of OBDs are encouraging, they also raise issues. The selection and measurement of the adequate biological effects required for OBD identification are not easy, especially for mAbs [START_REF] Sachs | Optimal Dosing for Targeted Therapies in Oncology: Drug Development Cases Leading by Example[END_REF]. We noticed that the measured biological effects associated with OBDs were heterogeneous among small molecules and mAbs, but also among some molecules of the same classes. For example, the OBDs of pembrolizumab and nivolumab, two Programmed cell Death protein 1 (PD-1) inhibitors, relied on IL2 stimulation, and on PD-L1 occupancy, respectively [START_REF] Patnaik | Phase I Study of Pembrolizumab (MK-3475; Anti-PD-1 Monoclonal Antibody) in Patients with Advanced Solid Tumors[END_REF][START_REF] Brahmer | Phase I study of single-agent anti-programmed death-1 (MDX-1106) in refractory solid tumors: safety, clinical activity, pharmacodynamics, and immunologic correlates[END_REF]. The lack of validated biomarkers and thresholds for novel study agents contributes to this inconsistency. Such a complexity may explain why OBDs frequently rely on indirect effects on the involved signaling pathway components for small molecules, and on receptor occupancy or saturation for the majority of mAbs, as already reported [START_REF] Tosi | Clinical Development Strategies and Outcomes in First-in-Human Trials of Monoclonal Antibodies[END_REF]. It may also explain why, actual OBDs could not be found for about a quarter of drugs, despite the mention of biological effective dose in the articles. For example, the authors demonstrated some pharmacodynamic effectdose relationships as proofs of concepts, and set up the RP2Ds of bortezomib [START_REF] Orlowski | Phase I trial of the proteasome inhibitor PS-341 in patients with refractory hematologic malignancies[END_REF], sonidegib [START_REF] Rodon | A phase I, multicenter, open-label, first-in-human, dose-escalation study of the oral smoothened inhibitor Sonidegib (LDE225) in patients with advanced solid tumors[END_REF], and vorinostat [START_REF] Kelly | Phase I clinical trial of histone deacetylase inhibitor: suberoylanilide hydroxamic acid administered intravenously[END_REF], based on other criteria, without defining OBDs. This observation is consistent with Sweis et al. who reported that despite their increased uses, the impact of biopsy-derived pharmacodynamic biomarkers in phase I oncology studies remains uncertain on subsequent drug development, as no effects on subsequent dose or schedule were demonstrated [START_REF] Sweis | Analysis of Impact of Post-Treatment Biopsies in Phase I Clinical Trials[END_REF]. Another difficulty relates to the impact of the trial designs on the relevance of identified OBDs. For example, the number of assessed dose levels restricts the search in a dose-window, as illustrated by nilotinib for which only two doses were evaluated [START_REF] Kantarjian | Nilotinib in imatinib-resistant CML and Philadelphia chromosome-positive ALL[END_REF]. Of note, the rationale sustaining the selection of the RP2Ds was not clear in more than 60% of drugs, for which the RP2Ds were not based on OBDs or MTDs.

The present study has several limitations that are important to notice. First, we chose to restrain our analysis to single agent studies for avoiding the effects of pharmacodynamic interactions. In addition, we assumed that approved doses were clinically effective, but this is debatable. Although clinical efficacy cannot be reduced to drug approvals, the FDA labeled dose appeared as the most objective and assessable endpoint for the present analysis.

In particular, our research did not focus on unpublished early phase trials for feasibility reasons, which did not allow us to assess the number of negative efficacy trials testing the OBDs, thereby leading to drug abandons before approvals.

Conclusions:

While being insufficiently investigated, OBD may be a relevant and complementary endpoint to toxicity endpoints traditionally assessed in oncologic phase I trials. Indeed the reported OBDs were found to be consistent with subsequent doses approved by FDA for the large majority of oncology and hematology targeted therapies, when developed at these doses. The design of future early phase trials should be adjusted to integrate systematic assessment of the biological effective dose, as a way of reducing the high attrition rate and costs in oncology drug development, and improving the quality of life of patients.

Funding:

The work was supported by Lyon University Hospital (Hospices Civils de Lyon).

Role of the Funder: The authors receive salaries from Lyon University Hospital. The funders had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.

Conflict of interest statement

The authors have declared no conflicts of interest.

Tables Table 1: Characteristics of the considered drugs and associated trials MTD reached in early phase trials Yes approved by the FDA before July 2018

drugs (40%)

with optimal biological doses reported in the early phase trial articles (as monotherapy agents)

drugs (56%)

with optimal biological doses selected as the RP2Ds


MD a , Bérengère Papegay

BSc Vincent Nuyens

PhD Adelin Albert

PhD Mustapha Cherkaoui-Malki

PhD Oberdan Leo

PhD Véronique Kruys

MD, PhD Jean G Boogaerts

MD, PhD Joseph Vamecq
email: joseph.vamecq@inserm.fr

Protection in a model of liver injury is parallel to energy mobilization capacity under distinct nutritional status

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

Introduction

The protective properties of dietary and energetic restrictions in health, ageing and disease models assessed through experimental injuries have been previously considered along with the counterbalanced view that a drop of cell energy availability under a critical threshold may prevent protection [START_REF] Papegay | Short fasting does not protect perfused ex vivo rat liver against ischemia-reperfusion. On the importance of a minimal cell energy charge[END_REF]. This latter aspect was inferred from studies on livers isolated from rat donors undergoing 18h-fasting vs feeding, and showing an exacerbation by 18h-fasting of ex vivo liver cytolysis [START_REF] Papegay | Short fasting does not protect perfused ex vivo rat liver against ischemia-reperfusion. On the importance of a minimal cell energy charge[END_REF]. In this respect, necrosis, apoptosis and autophagy represent cell deaths which are accidental, programmed and programmed with optional survival, respectively [2,3]. Extrinsic and intrinsic apoptosis recruit plasma membrane (death-inducing signalling complex DISC) and soluble apoptosomes, respectively [START_REF] Kiraz | Major apoptotic mechanisms and genes involved in apoptosis[END_REF] to trigger auto-activation of initiating caspases which in turn activate executioner caspases for protein cleavage and changes observed during apoptosis [START_REF] Green | Apoptotic pathways: ten minutes to dead[END_REF]. Autophagy involves autophagosomes (encompassing intracytoplasmic components and organelles) which fuse with lysosomes for content degradation; it may lead to cell death or under nutrient deprivation [START_REF] He | Regulation mechanisms and signaling pathways of autophagy[END_REF] supply energy compatible with cell survival [START_REF] Stipanuk | Macroautophagy and its role in nutrient homeostasis[END_REF].

As mentioned above, exacerbation of cytolysis and drop under a critical energy threshold were previously documented in ex vivo perfused livers originating from 18h-fasted vs fed rats [START_REF] Papegay | Short fasting does not protect perfused ex vivo rat liver against ischemia-reperfusion. On the importance of a minimal cell energy charge[END_REF][START_REF] Stadler | Effect of nutritional status on oxidative stress in an ex vivo perfused rat liver[END_REF]. The primary goal of carrying out the present study was to work under energy deprived perfusion and to perform a lengthening of starvation period which precedes liver isolation. This latter was done to provide an additional exacerbation of fasting-driven susceptibility to liver cytolysis previously reported in our liver model [START_REF] Papegay | Short fasting does not protect perfused ex vivo rat liver against ischemia-reperfusion. On the importance of a minimal cell energy charge[END_REF]. The reason of this strategy was to amplify and hence better characterize key determinants responsible for the difference observed in cytolysis between fed and starved states. Unexpectedly, the exacerbation of cytolysis was reduced by increasing starvation period. This has led, secondarily, to consider mechanisms which might protect against fasting-driven cytolysis. In fact, assessing cell death pathways (apoptosis and autophagy) along with energetic stores in various nutritional states here leads to better understand the two issues in our liver model: differences in cytolyses between fed and starved states and mechanisms which might protect against fasting-driven exacerbation of cytolysis. Apoptosis is monitored by changes in activated caspase-3 (extrinsic and intrinsic apoptosis) [START_REF] Hengartner | The biochemistry of apoptosis[END_REF]. Autophagy is assessed by increase in LC3II/actin ratio (and decrease in p62/actin ratio) [10-12], and is modulated by 3-methyladenine (3MA), a specific inhibitor of autophagy [START_REF] Seglen | 3-methyladenine: specific inhibitor of autophagic/lysosomal protein degradation in isolated rat hepatocytes[END_REF].

Materials and methods

Ethics

All animal studies were authorized by the Animal Care Use and Review Committee of the Belgian Free University of Brussels (CEBEA-IBMM-2014-39).

Animals

Female Wistar rats purchased from Charles River Laboratories (Saint Germain Nuelles, France) with a body weight of 200 g were acclimatized for at least five days to the room temperature of 24-26°C with a 12:12-h light-dark cycle. Standard laboratory chow and water were provided ad libidum. Animals were randomly allocated to fed (n=15) and fasting groups (n ≥ 10 in each group), 18h (n=15) and 24h (n=10)-fasted groups keeping free access to tap water, with food withdrawn 18 and 24h before liver isolation, respectively. Challenge of 24h-fasted group by 3-methyladenine (3MA) (n=10) was done by compound inclusion in perfusion of removed livers, avoiding animal exposure to in vivo compound side effects.

Solutions and chemicals for liver perfusions

Albumin-free Hank's Balanced Salt Solution (HBSS) included 0.4 g/L KCl, 0.06 g/L KH2PO4, 0.35 g/L NaHCO3, 0.048 g/L Na2HPO4, and 0.14 g/L CaCl2. HEPES 2.38 g/L were added. All these chemicals were obtained from Sigma (Bornem, Belgium). The solution was saturated with 100% O2 (0.5 L/min), pH adjusted to 7.35±0.05 using NaOH 1.0 M and supplemented with NaCl to achieve 300 mOsm. In part of experiments with livers from 24h-fasted rats, HBSS mixture contained 5 mM 3MA (Sigma-Aldrich, Bornem, Belgium). 24h-Fasting+3MA and 24h-fasting groups were perfused by HBSS mixtures supplemented and not with 5 mM 3MA, respectively.

Hepatectomy and ex vivo liver perfusion

Procedure for liver removal and perfusion was extensively described elsewhere [START_REF] Stadler | Effect of nutritional status on oxidative stress in an ex vivo perfused rat liver[END_REF][START_REF] Stadler | Intralipid minimizes hepatocytes injury after anoxiareoxygenation in an ex vivo rat liver model[END_REF]. Briefly, animals were anesthetized with pentobarbital sodium (NEMBUTAL®, Ceva, Libourne, France) intraperitoneally (50 mg/kg), the abdomen was opened and heparin (5000 IU/kg) administered via the inferior vena cava. The portal vein was cannulated with a 22-gauge catheter, secured in place and immediately perfused with the adequate solution. The liver was removed under continuous perfusion and transferred to the closed system ex vivo arrangement. The system (circuit volume of 125 mL) was maintained at 37°C. The perfusate, i.e., enriched HBSS solution supplemented or not with 5 mM 3MA, passed sequentially through a peristaltic pump (ISMATEC REGLO, Fisher Bioblock Scientific, Tournai, Belgium) at a flow rate of 5 mL/min to obtain a perfusion pressure around 12 cm H2O through the portal vein.

General protocol for liver biopsies and perfusate sampling

Protocol included 135 min perfusion of livers with respective solutions. Perfusate samples were collected every 15 min and thin liver biopsies at 0 and 135 min with instantaneous immersion in either liquid nitrogen with storage at -80°C before determination of LC3II and p62 autophagy marker or in 10% formalin before postponed embedding in paraffin before immunohistochemistry of caspase-3 (apoptosis) and periodic acid Schiff staining (glycogen). For apoptosis, livers embedded in paraffin were cut by a microtome with deposition of slices on optic microscopic glasses. These latter were kept at room temperature for 24 hours before deparaffinisation and processing for caspase 3 immunostaining mentioned below in the experimental subsection titled "Apoptosis and Caspase-3". For PAS staining, liver slice deparaffinisations and staining with periodic acid were performed by a Tissue Tek Prisma® apparatus (Sakura, Europe). PAS stained slices were processed for determination of glycogen contents in hepatocytes at two time-points, i.e. 0 and 135 min, using the Image J software (National Institutes of Health, Bethesda, MD, USA) [START_REF] Stadler | Intralipid minimizes hepatocytes injury after anoxiareoxygenation in an ex vivo rat liver model[END_REF]. Five fields per PAS stained slide were analyzed by a video-microscope (LEITZ DIALUX 20ES, Leitz, Wetzlar, Germany) equipped with a x40 objective and combined with a personal computer [START_REF] Stadler | Effect of nutritional status on oxidative stress in an ex vivo perfused rat liver[END_REF]. Generated pictures were captured onto the hard drive of the workstation computer. Thereafter, captured images were opened in Image J program (NIH, Bethesda, Maryland, USA) for evaluating indices of positivity on PAS slides. We selected the threshold of glycogen content of all cells (X value) and we referred it to the surface of all cells (Y value). From these area data, the glycogen index for the image was calculated, and expressed as a percentage (X/Y).

Perfusate markers and liver biopsy glycogen contents

Aspartate aminotransferase (AST, IU/L), alanine aminotransferase (ALT, IU/L), lactate dehydrogenase (LDH, IU/L), glucose (mg/dl), lactate (mmol/L), and potassium (mEq/L) were assayed in perfusate samples on a Architect plus CI4100 (Abbott Diagnostics, IL, USA). Glycogen contents were determined as mentioned above.

Apoptosis and caspase-3

Apoptosis was evaluated in a blinded fashion using light microscopy after in situ immunostaining. Deparaffinised liver sections were pre-treated: hot citrate buffer pH 6, inhibition of endoperoxydases with methanol/H2O2 blocking cross-reaction (Immunologic, Duiven, The Netherlands) and inhibition of endo-biotin (Vector, Burlingame, CA, USA). Slides were incubated at 4°C overnight using a specific primary antibody for activated cleaved Caspase-3 (CASPASE-3 ASP 175, Cell Signalling Technology, Danvers, MA, USA) at a 1:100 dilution. Signal amplification was performed with incubation of the complex Ultrasens Streptavidin Peroxidase RTU (Immunologic, Duiven, The Netherlands). Revelation was done with diaminobenzidine (Biogenex, Fremont, CA, USA). Slides were counterstained with Toluidine Blue. Apoptosis activity was expressed as the percentage of apoptotic cells (reported on number of hepatocytes) in selected regions. Five fields of each stained slide were analysed. Apoptosis counting was performed with the image J software using a selection of cells immunolabelled for activated caspase-3, a determination of total and labelled cells by the image J cell counter and a subsequent calculation of labelled to total liver cell ratios.

Autophagy, LC3II and p62 to actin ratios LC3II and actin were assayed by western blot. After defrosting from -80°C, liver biopsies were weighed and protein concentrations determined to load western blot gels with similar amounts of proteins. Biopsy was grinded in a micro-vial containing a protease inhibitor (mini EDTA free pellets, Roche, Basel, Switzerland), PBS and Lysing Matrix D Bulk on a Fast Prep Instrument (MP Biomedicals, Santa Ana, CA, USA). It was diluted in a Laemmli buffer (Tris 60 mM pH 6.8, 10% glycerol, 0.01% bromophenol blue, 5% β-mercaptoethanol, 2% SDS) to disrupt disulfide bonds and negatively charge proteins for size-based separation. One µl Benzonase (Novagen, EMD Millipore Corporation, Temecula, CA, USA) was added. Samples were heated to denature proteins, loaded in wells in 10% polyacrylamide/bisacrylamide gel (PAGEr TM EX Gels, Lonza , Rockland, ME USA) in a ProSieve TM Ex Running Buffer (Lonza , Rockland , ME, USA) and submitted to 100 volts for 10 minutes, and 150 volts until complete migration of the markers. Gel was transferred onto membrane using a transfer buffer (25mM Tris pH 7.6, 192 mM Glycine, 0.03% SDS, 20% methanol) under 100 Volts during 2h at 4°C, checking complete transfer of proteins with Red Ponceau. Membrane was incubated for 1 h in TBST + 5% milk at room temperature before incubation with a primary antibody LC3B (# 2775, rabbit 1: 1000, Cell Signalling Technology, Danvers, MA, USA) at 4° C overnight. For LC3II detection, a HRPcoupled secondary antibody (Promega, Madison, WI USA) was added 40 minutes at room temperature and stirred for chemiluminescence with reading after disclosure with substrate of enzyme Western lighting plus ECL (Perkin-Elmer, Waltham, MA, USA). For p62 detection, donkey anti-rabbit secondary antibody coupled to IRdye 680 (LI-COR, Lincoln, NE, USA , 1:15000) was added for 1 hour at room temperature with stirring for reading on the LI-COR (ODYSSEY®FC , Lincoln , NE, USA) on the channel 700. Actin levels were measured by chemiluminescence using a mouse antiactin (1:50,000, monoclonal Anti-β-Actin-Peroxidase antibody, Sigma-Aldrich®, St Louis, MO, USA). LC3II/actin and p62/actin ratios were calculated from respective protein determinations.

Statistical Analysis

Results of liver solutes were expressed as mean and standard deviation and similarly for enzymes after a log-transform to normalize their distribution and homogenize their variances. The impact of experimental conditions on their evolution during the 135 min perfusion was tested by Zerbe's method for growth and response curves, which allows comparing groups at each time point and over any time interval. For glycogen content and ratios, data were displayed as boxplots (median and interquartile range). Changes between T0 and T135 were assessed by the Wilcoxon signed rank test while the Kruskal-Wallis test was used for between group comparisons. Calculations and graphs were done using SAS (version 9.4) and R (version 3.2.2) statistical packages. Significance was considered at the 5% critical level (p < 0.05).

Results

Perfusate marker liver solutes

Fig. 1A illustrates time-dependent changes in perfusate solutes in the four experimental groups. Perfusate glucose concentrations in fed group were systematically higher (p<0.0001) than in 18h, 24h and 24h+3MA fasting groups and likewise for lactate values. Glucose and lactate concentrations were also significantly lower (p<0.0001) in 18h-fasting group than in the two 24h-fasting groups. Potassium concentrations in fed group were significantly lower than in 18h-fasting (p<0.05) and 24h-fasting+3MA groups (p<0.001) after 90 min and 60 min of perfusion, respectively. They were also lower in 24h-fasting group than in 24h-fasting+3MA group after 75 min (p<0.01).

Perfusate marker liver enzymes

Liver enzyme releases in perfusate samples are illustrated in Fig. 1B. Values statistically significant are depicted for each group at 135 minutes. However, the kinetics of the considered enzyme release showed also significant differences from 60 to 120 minutes over the perfusion period. Basically, enzyme levels in 24h-fasted group were observed between those of fed and 18h-starved groups but highest enzyme activities were recorded in 24h-fasting+3MA group. AST values were significantly lower in fed group than in the other three groups (p<0.001). AST in 24h-fasting group was significantly lower than in 24h-fasting+3MA (p<0.001) and 18h-fasting (p<0.05) groups. After 90 min of perfusion, AST curves were highest in 24h-fasting+3MA group and lowest in fed group; intermediate with values, however, higher in 18h-vs 24h-fasting groups (p=0.025). Similar findings were obtained for ALT. At the end of perfusion, ALT activities in 18h-fasting and 24h-fasting+3MA groups were not significantly different, these two groups having higher perfusate ALT levels than 24h-fasting group. LDH values differed between the four groups after 45 min of perfusion. They turned out to be systematically lower in fed group than in 18h-(p=0.014) and 24h-fasting+3MA (p=0.0001) groups but not 24h-fasting group. LDH values in 18h-fasting and 24h-fasting+3MA groups were similar and significantly higher than in 24h-fasting group.

Liver glycogen

At baseline, liver glycogen contents differed significantly between each group except between 24hfasting and 24h-fasting+3MA (Fig. 2 and Fig. 3A). During perfusion, it significantly decreased in all groups. At 135 min, glycogen was still in fed group higher than in 18h-fasted group (p<0.0001), the latter group exhibiting glycogen content higher than in the two 24h-fasted groups (p<0.0001). Glycogen content was lower in 24-fasting group than in 24h-fasting+3MA (p=0.014). Fig. 2 -Photomicrographs of PAS-stained liver slices at 0 min (T0) and 135 min(T135) time-points in fed, 18-h starved, 24 h starved and 24h-starved + 3MA groups. The figure compares and illustrates for one animal in each nutritional group PAS staining at the start and end of liver perfusions. The abundance of glycogen is proportional to the red component of the PAS staining. As mentioned in the Materials and methods section, PAS stained slices were processed for determination of glycogen contents in hepatocytes at two time-points, i.e. 0 and 135 min, using the Image J software (National Institutes of Health, Bethesda, MD, USA). The electronically determined staining intensity values determined on the whole experimental groups are further accounted for by the boxplot representations appearing on Fig. 3 panel A.

Liver apoptosis

Photomicrographs of activated caspase-3 liver immunohistochemistry at 0 min (T0) and 135 min (T135) time-points in fed, 18-h starved, 24 h starved and 24h-starved + 3MA groups are shown in Fig. 4. Apoptosis activity as measured by the percentage of activated caspase-3 was the same in all groups at baseline (Fig. 3B). At the end of perfusion, it differed between all groups except when comparing 24h-fasting+3MA with 18h-and 24h-fasting groups (Fig. 3B). Fig. 4 -Photomicrographs of activated caspase-3 liver immunohistochemistry at 0 min (T0) and 135 min(T135) time-points in fed, 18-h starved, 24 h starved and 24h-starved + 3MA groups. The figure compares and illustrates for one animal in each nutritional group the immunolabelling at the start and end of liver perfusions. The red arrow appearing on 2 photomicrographs, namely those at 135 min for both fed and 18h-starved liver conditions, points to one of the immunolabelled cells seen on these liver photomicrographs. The immunolabelling is indicated by the visible brown coloration on photomicrographs (counterstaining being achieved by toluidine blue). As mentioned in the Materials and methods section, apoptosis counting was performed with the image J software using selection of cells immunolabelled for activated caspase-3, a determination of total and labelled cells by the image J cell counter and subsequent calculation of labelled to total liver cell ratios. These counting values determined on the whole experimental groups are accounted for by the boxplot representations appearing on Fig. 3 panel B.

Liver autophagy LC3 II/actin ratio

The LC3II/actin ratio was similar in all groups at baseline but after 135 min of perfusion it was found significantly higher in 24h-fasting group than in 18h-fasting and 24h-fasting+3MA groups, respectively. During perfusion, LC3II/actin values significantly increased in 24h-fasting group but remained unchanged in other groups. The rise in autophagy was essentially lost under 3MA (Fig. 3C). Fig. 5 visualizes western blotting bands of autophagy LC3II and actin in livers after 135 min ex vivo perfusion in fed, 18-h starved, 24 h starved and 24h-starved + 3MA groups. p62/actin ratio Fig. 5 also visualizes western blotting band of p62 (along with actin) in the various groups. As accounted for by boxplot representations appearing in Fig 3D panel, the p62/actin ratio differed significantly between groups at baseline, being higher in fed group than in 24h-fasting and 24h-fasting+3MA groups and lower in 24h-fasting than in 18h-fasting and 24h-fasting+3MA groups. During perfusion, it decreased in 18h-fasting and in 24h-fasting groups but not in fed and 24h-fasting+3MA groups (Fig. 3D). At 135 min, p62/actin ratio in fed group was still higher than in other groups. Interestingly, it was also higher in 18h-fasting group than in 24h-fasting and 24h-fasting+3MA groups.

Fig. 5 -Western blotting bands of autophagy LC3II and p62 proteins and actin in livers after 135 min ex vivo perfusion in fed, 18-h starved, 24 h starved and 24h-starved + 3MA groups. The figure compares and illustrates for one animal in each nutritional group that two separate runs were performed for assessing autophagy markers: one run for LC3II and a separate run for p62, each of these runs being also evaluated for actin content. The LC3II and p62, along with actin associated with each of these runs were quantified by LI-COR software on selected areas. Data generated by the LI-COR are highly reproducible and are not dependent on the operator and light exposure of blots. LI-COR data were used for boxplot representations appearing in Fig. 3 panels C and D.

Discussion

In perfused ex vivo livers, protective mechanisms were previously suggested to require energy availability being above a critical threshold [START_REF] Papegay | Short fasting does not protect perfused ex vivo rat liver against ischemia-reperfusion. On the importance of a minimal cell energy charge[END_REF]. In the present work, induction of autophagy was attested by a rise in LC3II/actin ratio and a drop in p62/actin ratio, which represent highly specific [START_REF] Papegay | Short fasting does not protect perfused ex vivo rat liver against ischemia-reperfusion. On the importance of a minimal cell energy charge[END_REF] and less specific markers of autophagy, respectively [10-12]. It was observed on transition from 18h to 24h-fasting and protected against fasting-driven exacerbation of liver vulnerability. In fasted groups, the link between protection against cytolysis and enhanced autophagy is stronger and more consistent than the link between protection and reduced apoptosis. After 135 min energy-free perfusion, cytolysis was significantly lower and autophagy higher in 24h-fasting group compared to 24h-fasting+3MA and 18h-fasting groups. Moreover, an inverse relationship between cytolysis and available energy sources (glycogen + autophagy) clearly emerges when comparing the four experimental groups. As illustrated by Fig. 6, the inverse link between autophagy and cytolysis is adequately highlighted when considering autophagy as taking part, along with glycogen stores, to cellular energy mobilization capacities.

Because ex vivo liver is disconnected from circulating energetic substrates and infused with energyfree perfusion, glycogen represents a major energetic source. This latter is by far lower in fasted than in fed livers (Fig. 3A). Enhanced autophagy, known to providing cells with energetic substrates [15], would compensate for limited glycogen stores and protect against reduced energy stores. The higher energy mobilization capacity, the lower is cytolysis (Fig. 6). Accordingly, inclusion of 3MA in perfusion reduced autophagic activity (Fig. 3 C andD); it led to increased cytolysis (Fig. 1B) and hence loss of benefit given by 24h-vs 18-fasting of rats, emphasizing that autophagy is well protective.

A key determinant in interpreting changes in levels of released metabolites, in particular lactate, is disconnection of ex vivo livers from circulation, opposing ex vivo and in situ livers. For in situ liver, lactate release in circulation is primarily fuelled from extra-hepatic anaerobic glycolysis to contribute to Cori's cycle [START_REF] Cori | The glucose-lactic acid cycle and gluconeogenesis[END_REF]. For perfused ex vivo livers, lactate release in perfusate, though virtually issued from anaerobic glycolysis by extra-hepatocyte cells (Kupffer, vascular, duct cells, etc.) primarily accounts for, like glucose, hepatocyte gluconeogenesis. Indeed, due to the absence of inclusion of glucose in perfusion and disconnection from circulation, the major glucose source from which lactate may derive is therefore hepatocyte gluconeogenesis, shading that, in return, lactate production depends on extra-hepatocyte glycolysis rates and may aliment hepatocyte gluconeogenesis. Though perhaps paradoxical, higher glucose production in fed vs fasted conditions is indicative of higher glycogen stores [START_REF] Hems | Control of hepatic glycogenolysis[END_REF]. Accordingly, lactate release is higher in fed vs fasted livers (Fig. 1A). As regards to fasted livers, higher lactate levels at 24h-vs 18h-fasting might be explained by a higher background hepatocyte gluconeogenesis, perhaps as a result of a longer in vivo exposure to glucagon which interestingly, in contrast to the autophagy inhibitor insulin [18], promotes autophagy [19]. This higher lactate levels at 24h-vs 18h-fasting is not affected by inclusion of 3MA in perfusion; suggesting little or no compound effect on gluconeogenesis. Higher gluconeogenesis rates in the two 24h vs the one 18h-fasting groups might take place by consuming glycogen explaining why glycogen contents were significantly lowered in former vs latter groups. The significant drop however observed between hepatic glycogen contents of former groups (24h-fasting vs 24h-fasting+3MA) is only very moderate and might be accounted for by glycogen autophagy, a special form of autophagy referred to as glycophagy [START_REF] Jiang | Starch-binding domain-containing protein 1 (Stbd1) and glycogen metabolism: Identification of the Atg8 family interacting motif (AIM) in Stbd1 required for interaction with GABARAPL1[END_REF] and most likely to be eliminated under autophagy inhibition by 3MA.

Taken as a whole, present protective mechanisms are well accounted for by Fig. 6. Due to the present use of female animals, their potentiating by estrogen signalling cannot be ruled out. Since the pioneer work of de Duve, a growing-up attention has been dedicated to the role of autophagy in programmed cell death and optional survival through well designed experimental works [21-23]. In parallel and meanwhile, the clinical relevance of autophagy has also emerged in medical fields including surgery and oncology. Recently, the prognostic value of the autophagic marker LC3 was highlighted in two cohorts of patients undergoing liver resection as a surgical cure of hepatocellular carcinoma [START_REF] Lin | The autophagy marker LC3 strongly predicts immediate mortality after surgical resection for hepatocellular carcinoma[END_REF]. In this respect, liver regeneration after extended resections following neoadjuvant therapies represents a main issue where ischemic tolerance and remnant volume are of major interest. In the light of the present work, these experimental and medical developments might also stress the interest in promoting cellular pathways involved in energy mobilization to trigger protective and survival effects. Fig. 6. Energy mobilization capacities vs cytolysis in ex vivo livers after exposure to a 135 min energyfree perfusion. Energy mobilization capacity in perfused ex vivo liver is essentially accounted for by the sum of glycogen stores and autophagic activity (assessed by LC3II/actin ratio) in liver biopsies (Fig. 3A and 3C, respectively). Cytolysis is accounted for by releases of AST, ALT, and LDH in perfusates (Fig. 1B). Relative mean levels of energy mobilization capacity and proteins released in perfusates are expressed as percentage values, the 100% mean value referring to the highest level throughout experimental groups. Accordingly, a 100% mean value has been ascribed to glycogen from fed livers, to autophagy marker LC3II/actin ratio from 24h-starved livers, and to AST, ALT and LDH releases from 24h-starved+3MA livers. For protein releases in perfusate, the three calculated percentages are individually represented (red and red-like histograms). For energy mobilization capacity (green histograms), calculated percentages of glycogen and LC3II/actin ratio have been summed (so percentage may exceed 100). The inverse relationship between energy mobilization capacity and cytolysis essentially accounts for protective roles of glycogen stores in fed conditions and of autophagic activity in starved conditions.

Fig. 1 .

 1 Fig.1. Perfusate markers of ex vivo rat livers in fed, 18h-starved, 24h-starved and 24h-starved+3MA groups. Perfusate concentrations of solutes (A) and enzyme activity (B) over time are expressed in normal and logarithmic scaling, respectively with significant statistical differences between liver groups being given for values at the 135 min time point. Abbreviations: 3MA, 3-methyladenine; AST, aspartate transaminase; ALT, alanine transaminase; LDH, lactate dehydrogenase. Note that in contrast to all other ordinate scales which start from the zero, the ordinate scale of perfusate kalium starts at a 5 mEq/L value.

Fig. 3 .

 3 Fig. 3. Liver markers of ex vivo rat livers in fed, 18h-starved, 24h-starved and 24h-starved+3MA groups. Box plots account for glycogen content (A), apoptosis activated caspase-3 (B), autophagy specific LC3 II/actin (C) p62/actin (D) ratios. Significant statistical differences between liver groups are given for values at 0 min and at 135 min time points. Abbreviations: 3MA, 3-methyladenine


MD Patrice Guiraudet

MD Sébastien Morel

MD Firas Dhouib

MD, PhD Yurdagül Uzunhan

MD Robin Dhote

PhD Emmanuel Martinod
email: emmanuel.martinod@aphp.fr

Pr Emmanuel

Subglottic tracheal stenosis associated with Ehlers-Danlos Syndrome, Hypermobilty Type Short running title: Subglottic stenosis in Ehlers-Danlos Syndrome

Keywords: Subglottic, trachea, stenosis, Ehlers-Danlos syndrome

Text length: 743 words

Introduction: Subglottic tracheal stenoses are most frequently related to postintubation injuries following prolonged mechanical ventilator support [START_REF] Grillo | Postintubation stenosis[END_REF] . It could be also idiopathic or associated with trauma, inhalation burns, irradiation, infections (bacterial, tuberculosis, histoplasmosis and diphtheria), collagen vascular diseases including granulomatosis with polyangiitis, relapsing polychondritis, polyarteritis and scleroderma [START_REF] Grillo | Infectious, inflammatory, infiltrative, idiopathic and miscellaneous tracheal lesions[END_REF] . Subglottic tracheal stenoses are usually treated by endoscopic techniques and/or surgical resection with end-toend anastomosis [START_REF] Feinstein | Endoscopic Management of Subglottic Stenosis[END_REF][START_REF] Lewis | Systematic review for surgical treatment of adult and adolescent laryngotracheal stenosis[END_REF] . More rarely, airway transplantation has been proposed for end-stage tracheal stenoses [START_REF] Martinod | Feasibility of bioengineered tracheal and bronchial reconstruction using stented aortic matrices[END_REF] . We report here a subglottic tracheal stenosis associated with Ehlers-Danlos Syndrome (EDS), Hypermobility Type (HT).

Case reports:

A 51-year old male patient was referred to our center (H24 "SOS Airway") for a symptomatic subglottic tracheal stenosis. His family history found an EDS in his mother and brother. His past medical history showed an EDS-HT, a bilateral knee ligamentoplasty (1983, 1993), five episodes of ankle sprain, a L4-L5 lumbar disc herniation, a surgical management of urethral stricture followed by six repeated dilation (1991), a bilateral pyelonephritis, a repeated hemorrhagic cystitis, a surgical cure of hemorrhoids (1994), a primary hypogonadism treated by dehydroepiandrosterone, respiratory allergies (pollen, shellfish), a Quincke's edema following a bumblebee sting (1980), a gastric reflux, an obstructive sleep apnea syndrome requiring continuous airway pressure, a dyslipidemia and finally a dyspnea at exercise related to a 30% subglottic tracheal stenosis (2011). In 2016, physical examination showed polyarticular joint pain, a joint hypermobility (score=6 on Beighton scale), a soft skin and an inspiratory dyspnea at rest. Computed-scan [ Discussion: To our knowledge, this is the first case reporting a subglottic stenosis in a patient with EDS-HT. According to the Ehlers-Danlos Society, EDS are currently classified in a system of thirteen subtypes. The diagnosis of EDS-HT is based on clinical evaluation and family history [START_REF] Beighton | Ehlers-Danlos syndromes: revised nosology, Villefranche, 1997. Ehlers-Danlos National Foundation (USA) and Ehlers-Danlos Support Group (UK)[END_REF] . Major diagnostic criteria are joint hypermobility; soft skin with normal or only slightly increased extensibility and the absence of skin or soft tissue fragility. Minor diagnostic criteria are positive family history of EDS-HT (or family history of joint laxity) without skin or soft tissue fragility in a pattern consistent with autosomal dominant inheritance; recurrent dislocation or subluxation of multiple joints; chronic joint, limb, and/or back pain; easy bruising; functional bowel disorders; neutrally mediated hypotension or postural orthostatic tachycardia; high, narrow palate and dental crowding. Upper airway obstruction due to laryngeal hypermobility and tracheobronchomegaly have been reported in patients with EDS [START_REF] Chatzoudis | Upper airway obstruction in a patient with Ehlers-Danlos syndrome[END_REF][START_REF] Girit | Ehlers-Danlos Syndrome Type IVB and Tracheobronchomegaly[END_REF] . In the present case, the patient did not have prolonged mechanical ventilation but multiple intubations which could have played a role in the development of the subglottic tracheal stenosis. We hypothesize that EDS was the main mechanism of subglottic tracheal stenosis even though this association has never been clearly described. Indeed, complications related to tracheal intubation have already been reported in patients with EDS. Sood et al. described a difficult intubation during rapid sequence induction in a parturient with EDS-HT [START_REF] Sood | Difficult intubation during rapid sequence induction in a parturient with Ehlers-Danlos syndrome, hypermobility type[END_REF] . This event was probably due to an unknown subglottic stenosis. Furthermore, a postintubation tracheal rupture was observed by Besselink-Lobanova and colleagues in a patient with tenascin-X-deficient type EDS [START_REF] Besselink-Lobanova | Trachea rupture in tenascin-X-deficient type Ehlers-Danlos syndrome[END_REF] .

Conclusion:

We think that the possibility of subglottic tracheal stenosis in patients with EDS must be known. We can also suggest to perform a flexible fiberoptic bronchoscopy in patients with EDS-HT requiring non-urgent tracheal intubation.

  Fig.1] and flexible bronchoscopy revealed a 80% subglottic tracheal stenosis. According to the Myer-Cotton classification, it was a grade III stenosis. Pulmonary function tests showed a significant flattening of the inspiratory flow volume loop [Fig.2a]. Under general anesthesia, mechanical dilation was performed using esophageal bougies [Fig.3]. Tracheal biopsies showed a cicatricial tissue without any specific lesions. Postoperative course was uneventful and the patient was discharged at day 1. There was no recurrence of the stenosis at 32 months of follow-up with significant improvement in postoperative pulmonary function tests [Fig.2b].

Figure Legends:Figure1:Figure2:

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Figure3:

  Figure3: Bronchoscopic management. a) under general anesthesia, a rigid bronchoscopy