Camille Houron

Marie Danielou

Olivier Mir

Bernard Fromenty

Gabriel Perlemuter
email: gabriel.perlemuter@aphp.fr

Sebastian Cosmin

Voican

Cosmin Sebastian Voican

Gabriel Pr

Perlemuter

Multikinase inhibitor-induced liver injury in patients with cancer: A review for clinicians

Keywords: multikinase inhibitor, drug-induced liver injury, hepatotoxicity

Introduction

Drug induced liver injury (DILI) is the fourth most common cause of liver damage in Western countries and the most frequent reason for market withdrawal of a drug [START_REF] Schuster | Why drugs fail--a study on side effects in new chemical entities[END_REF][START_REF] Temple | Safety of newly approved drugs: implications for prescribing[END_REF]. The incidence of DILI is between 1 per 10,000 and 1 per 100,000 patient-years, but is expected to rise in the context of increasing drug availability and prescription [START_REF] Bjornsson | Incidence, presentation, and outcomes in patients with drug-induced liver injury in the general population of Iceland[END_REF]. DILI is traditionally J o u r n a l P r e -p r o o f classified as intrinsic (or direct) or idiosyncratic. Intrinsic DILI, related to drug accumulation, is dose dependent and therefore predictable, occurring in a high proportion of exposed individuals. Idiosyncratic DILI is generally not dose related and is unpredictable based on dose or the pharmacological properties of the molecule [START_REF] Voican | Antidepressant-induced liver injury: a review for clinicians[END_REF]. DILI caused by most drugs is idiosyncratic. Although the pathogenesis is largely unknown, idiosyncratic DILI is probably the consequence of interactions between host susceptibility, environmental factors, and the pharmacological properties of the drug [START_REF] Kaliyaperumal | Pharmacogenomics of drug-induced liver injury (DILI): Molecular biology to clinical applications[END_REF].

The definition of DILI includes one of the following thresholds: ALT ≥ 5X ULN (upper limit of normal), ALT ≥ 3X ULN associated with bilirubin ≥ 2X ULN, or alkaline phosphatase ≥ 2X ULN in the absence of bone pathology [START_REF] Eaftsot | EASL Clinical Practice Guidelines: Drug-induced liver injury[END_REF]. For patients with abnormal liver function (LF) before initiation of the culprit drug, ULN is replaced by the mean baseline values prior to DILI onset. In the absence of specific tests for DILI, the diagnosis relies on circumstantial evidence and the exclusion of other causes of liver damage [START_REF] Andrade | Diagnostic and prognostic assessment of suspected druginduced liver injury in clinical practice[END_REF].

The emergence of innovative therapies, such as targeted molecular agents, has revolutionized the management of cancer. Among these molecules, multi-kinase inhibitors (MKIs) block phosphorylation cascades involved in tumour proliferation, survival, motility, angiogenesis, and evasion from the immune response [START_REF] Hanahan | Hallmarks of cancer: the next generation[END_REF]. Their targeted action theoretically decreases the risk of adverse events. Nevertheless, MKI use has been associated with serious toxic effects, such as skin toxicity, cardiotoxicity, and hepatotoxicity (idiosyncratic DILI) [START_REF] Hartmann | Tyrosine kinase inhibitors -a review on pharmacology, metabolism and side effects[END_REF][START_REF] Dong | Cardiotoxicity of Anticancer Therapeutics[END_REF][START_REF] Shah | Hepatotoxicity of tyrosine kinase inhibitors: clinical and regulatory perspectives[END_REF]. However, there is a paucity of data concerning MKI-related hepatotoxicity risk and clinical guidelines for the management of liver toxicity in patients receiving MKIs for cancer are scarce.

Early diagnosis of DILI is essential, as the continuation of treatment could cause acute liver failure, associated with a 60 to 80% mortality rate in the absence of liver transplantation [START_REF] Lee | Acute liver failure: Summary of a workshop[END_REF].

On the other hand, an erroneous diagnosis of DILI may lead to unnecessary drug withdrawal, which may affect the prognosis of patients with cancer. We therefore performed an exhaustive J o u r n a l P r e -p r o o f review of the published data on hepatic adverse events and DILI related to MKI use and discuss the issue of hepatotoxicity during MKI treatment for cancer.

J o u r n a l P r e -p r o o f

Methods

We conducted a PubMed search of articles in English published from January 2000 to December 2018 related to hepatic toxicity of the 30 FDA-approved MKI at doses used in clinical practice : alectinib, afatinib, axitinib, bosutinib, cabozantinib, ceritinib, cobimetinib, crizotinib, dabrafenib, dacomitinib, dasatinib, erlotinib, gefitinib, ibrutinib, imatinib, lapatinib, lenvatinib, osimertinib, nilotinib, nintedanib, pazopanib, ponatinib, regorafenib, sorafenib, sunitinib, trametinib, vandetanib, vemurafenib, everolimus, and temsirolimus. The search terms were international non-proprietary name of each agent cross-referenced with «hepatotoxicity», «hepatitis», «hepatic adverse event», or «liver failure», and «phase II clinical trial», «phase III clinical trial», or «case report». The results of the literature search are presented in Figure 1.

Clinical trials reporting hepatic adverse events occurring under treatment and including 20 or more patients (except for bosutinib, for which the threshold was fixed at 10 patients due to a lack of data) were retained. Case reports describing DILI were considered suitable for review by author consensus based on the exclusion of other causes of liver injury and the use of validated scales for the assessment drug imputability. If necessary, drug imputability was reassessed by the authors based on information provided in the manuscripts. Phase I clinical trials were excluded, as we aimed to review hepatotoxicity associated with therapeutic doses.

The potential for causing hepatotoxicity was assessed for each agent on the basis of five criteria: published cases of DILI, published cases of DILI leading to death, ALT or AST > 3X ULN (upper limit of normal) associated with bilirubin ≥ 2X ULN (Hy's law) in clinical trials, published clinical trials describing cases of DILI with acute liver failure, and hepatotoxicity demonstrated by published experimental studies. Based on these criteria, the hepatotoxicity risk of MKI was estimated to be low (absence of Hy's law criteria, absence of DILI with acute liver failure in clinical trials, absence of published cases of DILI), moderate (published cases of DILI without reported death), or high (published cases of DILI with reported death or Hy's law J o u r n a l P r e -p r o o f criteria and DILI with acute liver failure in clinical trials). The isolated elevation of aminotransferases was not considered, as its incidence poorly correlates with the incidence of severe liver injury.

Results

We first present the existing data on the epidemiology of MKI-related hepatotoxicity, starting from that obtained in clinical trials. We then detail the clinical-biological presentation and diagnostic strategy of DILI associated with MKI use. We also present data concerning the physiopathology of liver events.

Epidemiology

In most clinical trials, patients are excluded when baseline aminotransferase levels are > 2.5X ULN in the absence of hepatic metastases or > 5X ULN if hepatic metastases are present or the baseline bilirubin level is > 1.5X ULN [START_REF] Watkins | The clinical liver safety assessment best practices workshop: rationale, goals, accomplishments and the future[END_REF]. However, baseline LF test (LFT) values are often unknown in case-reports or not mentioned in clinical trials. In this context, even grade 3 to 4 elevations in aminotransferase levels reported in clinical trials are difficult to interpret as DILI in the absence of baseline values. Severe hepatic events (grade 3-4), defined as aminotransferase levels > 5X ULN, have been detected in 0 to 29% of patients treated with MKI in clinical trials (Table 1). The heterogeneity between publications on the incidence of increases in aminotransferase levels is high, even for the same agent. For example, severe hepatic events have been described in 4 to 19% of patients treated with bosutinib and in 1 to 11% of those receiving gefitinib (Table 1). In clinical trials, cases of defined DILI have been mentioned for alectinib, afatinib, bosutinib, ceritinib, cobimetinib, crizotinib, gefitinib, imatinib, lenvatinib, osimertinib, pazopanib, ponatinib, regorafenib, sorafenib, sunitinib, and vemurafenib [11, 14-29]. Nevertheless, no description of the clinical features was offered.

J o u r n a l P r e -p r o o f

Based on preregistration clinical trials, we estimate the prevalence of hepatotoxicity with liver failure to be 0.2 to 0.5% of treated patients for molecules such as afatinib, ceritinib, crizotinib, lenvatinib, and regorafenib [START_REF] Shah | Hepatotoxicity of tyrosine kinase inhibitors: clinical and regulatory perspectives[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] Shaw | Crizotinib versus chemotherapy in advanced ALK-positive lung cancer[END_REF][START_REF] Uetake | Clinical Features of Regorafenib-induced Liver Injury in Japanese Patients From Postmarketing Experience[END_REF][START_REF] Schlumberger | Lenvatinib versus placebo in radioiodine-refractory thyroid cancer[END_REF]. No case reports of DILI have been published or reported for clinical trials of axitinib, cabozantinib, dabrafenib, dacomitinib, nilotinib, trametinib, vandetanib, or temsirolimus. The only hepatotoxicity event potentially related to these agents reported in clinical trials is mostly transient increased aminotransferase levels. It should be mentioned that surveillance of patients included in clinical trials is strictly regulated.

Therefore, abnormal LFT are picked up early enough such that they do not often progress to serious toxicity that needs to be reported. These data may not translate in real-life settings where hepatotoxicity events are identified later and treated patients can be much sicker at baseline than in clinical trials.

Clinical presentation and laboratory tests

The clinical and biological presentation of DILI related to MKI use has only been described in case reports. MKI-induced liver injury includes various biological and clinical presentations, ranging from isolated abnormalities in LFT values to nonspecific symptoms, such as anorexia, asthenia, nausea, abdominal pain, arthralgia, and rash and more specific symptoms, such as jaundice, progressive liver failure with the loss of hepatocellular function, hepatic encephalopathy, and death. In most cases, patients are completely asymptomatic, and the increased aminotransferase level revealed by routine blood tests is the only element that raises a suspicion of MKI-related hepatotoxicity.

Limited data are available from MKI clinical trials. When provided, the latency of hepatotoxicity varies between the first cycle of treatment for crizotinib [START_REF] Shaw | Crizotinib versus chemotherapy in advanced ALK-positive lung cancer[END_REF][START_REF] Shaw | Ceritinib in ALKrearranged non-small-cell lung cancer[END_REF][START_REF] Rothenstein | Managing treatment-related adverse events associated with Alk inhibitors[END_REF][START_REF] Cao | Efficacy and safety of crizotinib among Chinese EML4-ALK-positive, advanced-stage non-small cell lung cancer patients[END_REF][START_REF] Kwak | Anaplastic lymphoma kinase inhibition in non-small-cell lung cancer[END_REF][START_REF] Sato | Fulminant hepatitis following crizotinib administration for ALK-positive non-small-cell lung carcinoma[END_REF][START_REF] Ripault | Crizotinib-induced acute hepatitis: first case with relapse after reintroduction with reduced dose[END_REF] and several months for molecules such as bosutinib [START_REF] Taylor | Phase 2 study of bosutinib, a Src inhibitor, in adults with recurrent glioblastoma[END_REF][START_REF] Moy | Bosutinib in combination with the aromatase inhibitor letrozole: a phase II trial in postmenopausal women evaluating first-line endocrine therapy in locally advanced or metastatic hormone receptor-positive/HER2negative breast cancer[END_REF][START_REF] Kantarjian | Bosutinib safety and management of toxicity in leukemia patients with resistance or intolerance to imatinib and other tyrosine kinase inhibitors[END_REF][START_REF] Gambacorti-Passerini | Safety of bosutinib versus imatinib in the phase 3 BELA trial in newly diagnosed chronic phase chronic myeloid leukemia[END_REF][START_REF] Gambacorti-Passerini | Bosutinib efficacy and safety in chronic phase chronic myeloid leukemia after imatinib resistance or intolerance: Minimum 24-month follow-up[END_REF][START_REF] Campone | Phase II study of single-agent bosutinib, a Src/Abl tyrosine kinase inhibitor, in patients with locally advanced or metastatic breast cancer pretreated with chemotherapy[END_REF], and imatinib [START_REF] Talpaz | Imatinib induces durable hematologic and cytogenetic responses in patients with accelerated phase chronic myeloid leukemia: results of a phase 2 study[END_REF][START_REF] Gambacorti-Passerini | Safety of bosutinib versus imatinib in the phase 3 BELA trial in newly diagnosed chronic phase chronic myeloid leukemia[END_REF][START_REF] Blay | Nilotinib versus imatinib as first-line therapy for patients with unresectable or metastatic gastrointestinal stromal tumours (ENESTg1): a randomised phase 3 trial[END_REF][START_REF] Matti | Evaluation of the safety of imatinib mesylate in 200 iraqi patients with chronic myeloid leukemia in the chronic phase: single-center study[END_REF], with most cases occurring during the first two months of treatment (Table 2). Significant liver toxicity generally consists of an increase in aminotransferase levels > 5X ULN, but baseline values are not J o u r n a l P r e -p r o o f reported. Nevertheless, associated hyperbilirubinemia (Hy's law) has been described for alectinib, bosutinib, ceritinib, cobimetinib, crizotinib, gefitinib, imatinib, lenvatinib, pazopanib, regorafenib, sorafenib, sunitinib, and the vandetanib-ipilimumab association [14-22, 27, 28, 30, 45-48].

A more detailed description of MKI-related hepatotoxicity is provided by case reports (Table 2). Latency varies between one week for erlotinib [START_REF] Huang | Three cases of severe hepatic impairment caused by erlotinib[END_REF][START_REF] Pellegrinotti | Erlotinib-induced hepatitis complicated by fatal lactic acidosis in an elderly man with lung cancer[END_REF][START_REF] Zenke | Successful treatment with afatinib after grade 3 hepatotoxicity induced by both gefitinib and erlotinib in EGFR mutation-positive non-small cell lung cancer[END_REF], sorafenib [START_REF] Mancuso | Letter: Sorafenib hepatotoxicity may be enhanced during treatment of advanced hepatocellular carcinoma in HIV-infected patients[END_REF], regorafenib [START_REF] Raissouni | Acute liver failure and seizures as a consequence of regorafenib exposure in advanced rectal cancer[END_REF], and more than six months for gefitinib [START_REF] Takeda | Successful treatment with erlotinib after gefitinib-related severe hepatotoxicity[END_REF][START_REF] Yano | Treatment of Non-Small-Cell Lung Cancer with Erlotinib following Gefitinib-Induced Hepatotoxicity: Review of 8 Clinical Cases[END_REF], dasatinib [START_REF] Bonvin | Dasatinibinduced acute hepatitis[END_REF], ibrutinib [START_REF] Kahn | Ibrutinib-induced acute liver failure[END_REF], imatinib [START_REF] Cross | Imatinib mesylate as a cause of acute liver failure[END_REF][START_REF] Perini | Nilotinib post-liver transplantation for acute hepatic failure related to imatinib[END_REF], nindetanib [START_REF] Olin | Delayed Presentation of Hepatocellular Liver Injury After Nintedanib Administration[END_REF], and sunitinib [START_REF] Weise | Fatal liver failure in a patient on acetaminophen treated with sunitinib malate and levothyroxine[END_REF]. DILI related to MKI use is hepatocellular (elevated aminotransferase levels with either no or a small increase in alkaline phosphatase levels), but cases of mixed DILI (elevation of both aminotransferase and alkaline phosphatase levels) have been published for dasatinib [START_REF] Bonvin | Dasatinibinduced acute hepatitis[END_REF], imatinib [START_REF] Ohyashiki | Imatinib mesylate-induced hepato-toxicity in chronic myeloid leukemia demonstrated focal necrosis J o u r n a l P r e -p r o o f resembling acute viral hepatitis[END_REF], pazopanib [START_REF] Klempner | Severe pazopanib-induced hepatotoxicity: clinical and histologic course in two patients[END_REF], sorafenib [START_REF] Mancuso | Letter: Sorafenib hepatotoxicity may be enhanced during treatment of advanced hepatocellular carcinoma in HIV-infected patients[END_REF], everolimus [START_REF] Patel | Drug-Induced Liver Injury Associated with the Use of Everolimus in a Liver Transplant Patient[END_REF], and vemurafenib [START_REF] Spengler | Vemurafenib-induced granulomatous hepatitis[END_REF]. Elevated bilirubin levels meeting Hy's law criteria (hepatocellular type injury seen concurrently with bilirubin > 2X ULN) has been reported for crizotinib (2 cases -death) [START_REF] Sato | Fulminant hepatitis following crizotinib administration for ALK-positive non-small-cell lung carcinoma[END_REF][START_REF] Van Geel | Crizotinib-induced fatal fulminant liver failure[END_REF], erlotinib (3 cases -death) [START_REF] Pellegrinotti | Erlotinib-induced hepatitis complicated by fatal lactic acidosis in an elderly man with lung cancer[END_REF][START_REF] Schacher-Kaufmann | Acute Fatal Liver Toxicity under Erlotinib[END_REF][START_REF] Liu | Fulminant hepatic failure secondary to erlotinib[END_REF], ibrutinib (2 cases -recobery) [START_REF] Kahn | Ibrutinib-induced acute liver failure[END_REF][START_REF] Nandikolla | Ibrutinib-induced severe liver injury[END_REF], imatinib (5 cases -1 death) [START_REF] Suzuki | Imatinibinduced Severe Hepatitis in a 9-Year-old Girl With Philadelphia Chromosome-positive Acute Lymphoblastic Leukemia[END_REF][START_REF] Tonyali | Imatinib mesylate-induced acute liver failure in a patient with gastrointestinal stromal tumors[END_REF][START_REF] Ridruejo | Imatinibinduced fatal acute liver failure[END_REF][START_REF] Fuster | Imatinib-induced toxic hepatitis: description of two cases and review of the literature[END_REF][START_REF] Sobhi | Imatinib-induced immune hepatitis: case report and literature review[END_REF], lapatinib (1 case -recovery) [START_REF] Peroukides | Lapatinib-induced hepatitis: a case report[END_REF], pazopanib (2 casesrecovery) [START_REF] Xu | HLA-B*57:01 Confers Susceptibility to Pazopanib-Associated Liver Injury in Patients with Cancer[END_REF], regorafenib (5 cases -2 deaths) [START_REF] Raissouni | Acute liver failure and seizures as a consequence of regorafenib exposure in advanced rectal cancer[END_REF][START_REF] Bechade | Fatal Acute Liver Failure as a Consequence of Regorafenib Treatment in a Metastatic Colon Cancer[END_REF], sorafenib (3 cases -1 death) [START_REF] Kuroda | Successful treatment for sorafenib-induced liver dysfunction: a report of case with liver biopsy[END_REF][START_REF] Murad | Sorafenib-Induced Grade Four Hepatotoxicity in a Patient with Recurrent Gastrointestinal Stromal Tumor (GIST): A Case Report and Review of Literature[END_REF][START_REF] Fairfax | Fatal case of sorafenib-associated idiosyncratic hepatotoxicity in the adjuvant treatment of a patient with renal cell carcinoma[END_REF], sunitinib (2 cases -death) [START_REF] Weise | Fatal liver failure in a patient on acetaminophen treated with sunitinib malate and levothyroxine[END_REF][START_REF] Mermershtain | Fatal liver failure in a patient treated with sunitinib for renal cell carcinoma[END_REF], and the vemurafenib-ipilimumab association (2 casesrecovery) [START_REF] Ribas | Hepatotoxicity with combination of vemurafenib and ipilimumab[END_REF] (Table 2).

Histological features

Liver biopsy performed in three published cases of erlotinib or gefitinib-induced hepatotoxicity revealed elements of an immuno-allergic (hypersensitivity) mechanism: active hepatitis with portal inflammation (lymphocyte, plasma cell, and neutrophil infiltration) and bridging necrosis [START_REF] Liu | Fulminant hepatic failure secondary to erlotinib[END_REF][START_REF] Ueda | Successful treatment with afatinib after gefitinib-and erlotinib-induced hepatotoxicity[END_REF][START_REF] Ho | Side effects related to cancer treatment: CASE 1. Hepatitis following treatment with gefitinib[END_REF]. The liver histology for imatinib-induced hepatic injury has been described in 12 case reports [START_REF] Ohyashiki | Imatinib mesylate-induced hepato-toxicity in chronic myeloid leukemia demonstrated focal necrosis J o u r n a l P r e -p r o o f resembling acute viral hepatitis[END_REF][START_REF] Tonyali | Imatinib mesylate-induced acute liver failure in a patient with gastrointestinal stromal tumors[END_REF][START_REF] Sobhi | Imatinib-induced immune hepatitis: case report and literature review[END_REF][START_REF] Rocca | Imatinib mesylate-induced acute cytolytic hepatitis][END_REF][START_REF] Ferrero | Corticosteroids can reverse severe imatinib-induced hepatotoxicity[END_REF][START_REF] Pariente | Imatinib mesylateinduced acute hepatitis in a patient treated for gastrointestinal stromal tumour[END_REF][START_REF] Kikuchi | Severe hepatitis and complete molecular response caused by imatinib mesylate: possible association of its serum concentration with clinical outcomes[END_REF][START_REF] Kong | Early imatinib-mesylateinduced hepatotoxicity in chronic myelogenous leukaemia[END_REF][START_REF] Charier | Glivec induced autoimmune hepatitis[END_REF][START_REF] Ayoub | Imatinib (Gleevec)induced hepatotoxicity[END_REF][START_REF] Mindikoglu | Imatinib mesylate (gleevec) hepatotoxicity[END_REF][START_REF] Harbaum | Treatment with dasatinib for chronic myeloid leukemia following imatinib-induced hepatotoxicity[END_REF]. Bridging necrosis (mainly centrilobular) associated with portal inflammatory infiltrates containing lymphocytes, plasma cells, and many J o u r n a l P r e -p r o o f eosinophils was found, suggesting a hypersensitivity mechanism. In cases of mixed DILI, histological cholestasis was present. Bridging fibrosis and acute viral hepatitis-like histological features (focal necrosis, with lymphocyte infiltration surrounding the necrotic zones and sinusoids) have been described in two cases of imatinib-induced hepatotoxicity [START_REF] Ohyashiki | Imatinib mesylate-induced hepato-toxicity in chronic myeloid leukemia demonstrated focal necrosis J o u r n a l P r e -p r o o f resembling acute viral hepatitis[END_REF][START_REF] Sobhi | Imatinib-induced immune hepatitis: case report and literature review[END_REF][START_REF] Rocca | Imatinib mesylate-induced acute cytolytic hepatitis][END_REF][START_REF] Pariente | Imatinib mesylateinduced acute hepatitis in a patient treated for gastrointestinal stromal tumour[END_REF].

Histological features suggesting hypersensitivity DILI (bridging necrosis, hepatocellular dropout surrounding the centrilobular areas, and portal eosinophil infiltration) have been reported in one case of lapatinib-induced hepatotoxicity [START_REF] Peroukides | Lapatinib-induced hepatitis: a case report[END_REF]. For ibrutinib-and pazopanibinduced liver injury, histology shows cholestatic hepatitis with eosinophil infiltration within the portal tracts [START_REF] Klempner | Severe pazopanib-induced hepatotoxicity: clinical and histologic course in two patients[END_REF][START_REF] Nandikolla | Ibrutinib-induced severe liver injury[END_REF]. Cholestatic hepatitis in association with granulomatous inflammation and eosinophil infiltrate has been described in a case of vemurafenib-related DILI [START_REF] Spengler | Vemurafenib-induced granulomatous hepatitis[END_REF]. Histological lesions compatible with metabolic (centrilobular hepatic necrosis with confluent destruction of acinar zone 3 and scarce inflammation) or immuno-allergic DILI (bridging necrosis with portal lymphocyte and plasma cell infiltration) have been described in eight cases of hepatotoxicity to regorafenib, sorafenib, and osimertinib [46, 77-80, 93, 94]. The only case report of sunitinibinduced hepatotoxicity with available liver biopsy showed liver invasion by cancerous cells and thus the histological features may not be related to DILI [START_REF] Weise | Fatal liver failure in a patient on acetaminophen treated with sunitinib malate and levothyroxine[END_REF]. In a recently reported case of alectinib-induced hepatitis, liver biopsy showed bridging necrosis with an inflammatory infiltrate containing lymphocytes, macrophages, and plasma cells [START_REF] Zhu | Severe Acute Hepatitis in a Patient Receiving Alectinib for ALK-Positive Non-Small-Cell Lung Cancer: Histologic Analysis[END_REF].

Imputability assessment

MKI-associated liver injury can mimic almost all known causes of hepatic disease (viral hepatitis, non-alcoholic fatty liver disease, alcoholic liver disease, autoimmune hepatitis, metabolic diseases, biliary tract obstruction, hepatic ischemia, vascular obstruction, and sepsis) and is consequently diagnosed by ruling out all other possible causes [START_REF] Chalasani | ACG Clinical Guideline: the diagnosis and management of idiosyncratic drug-induced liver[END_REF]. Imputability assessment of MKI-related DILI is not detailed in clinical trials. In case reports, the RUCAM The RUCAM score is the most widely used method for assessing causality in hepatotoxicity induced by various drugs and includes several criteria, such as [97] event chronology, description of the first clinical signs, results of LFTs after drug withdrawal (a 50% decrease of liver enzyme levels following withdrawal of the suspected culprit drug is highly suggestive of DILI) or drug re-challenge (recurrence of LFT abnormalities), concomitant medication (including self-medication, phytotherapy products, and illicit drugs), other potential causes, previous case reports of DILI, nature of the drug, drug dosage, and alcohol consumption. Deliberate or inadvertent drug re-challenge resulting in a deterioration of LF provides the strongest evidence for drug imputability. Positive drug re-challenge has been described for crizotinib (4 cases) [START_REF] Ripault | Crizotinib-induced acute hepatitis: first case with relapse after reintroduction with reduced dose[END_REF]98], erlotinib (2 cases) [START_REF] Liu | Fulminant hepatic failure secondary to erlotinib[END_REF]99], gefitinib (6 cases) [START_REF] Ueda | Successful treatment with afatinib after gefitinib-and erlotinib-induced hepatotoxicity[END_REF][START_REF] Ho | Side effects related to cancer treatment: CASE 1. Hepatitis following treatment with gefitinib[END_REF][100][101][102], imatinib (10 cases), osimertinib (1 case), pazopanib (2 cases) [103], and regorafenib (1 case) [104] (table 2).

Management and outcome

In case reports, MKI-related toxicity has typically consisted of an increase in aminotransferase levels with either no or a slight increase in alkaline phosphatase levels that spontaneously normalize within 10 days to five months from drug withdrawal in three quarters of cases (Table 2). Glucocorticoids (20-40 mg/day) were necessary in addition to drug discontinuation in case reports of DILI related to imatinib [START_REF] Sobhi | Imatinib-induced immune hepatitis: case report and literature review[END_REF], pazopanib [103], and the vemurafenib-ipilimumab combination [START_REF] Ribas | Hepatotoxicity with combination of vemurafenib and ipilimumab[END_REF]. Nevertheless, a fatal evolution despite drug withdrawal was reported for crizotinib [START_REF] Sato | Fulminant hepatitis following crizotinib administration for ALK-positive non-small-cell lung carcinoma[END_REF][START_REF] Van Geel | Crizotinib-induced fatal fulminant liver failure[END_REF], erlotinib [START_REF] Huang | Three cases of severe hepatic impairment caused by erlotinib[END_REF][START_REF] Pellegrinotti | Erlotinib-induced hepatitis complicated by fatal lactic acidosis in an elderly man with lung cancer[END_REF][START_REF] Schacher-Kaufmann | Acute Fatal Liver Toxicity under Erlotinib[END_REF][START_REF] Liu | Fulminant hepatic failure secondary to erlotinib[END_REF], imatinib [START_REF] Cross | Imatinib mesylate as a cause of acute liver failure[END_REF][START_REF] Ridruejo | Imatinibinduced fatal acute liver failure[END_REF], pazopanib [START_REF] Klempner | Severe pazopanib-induced hepatotoxicity: clinical and histologic course in two patients[END_REF], regorafenib [START_REF] Sacre | Regorafenib induced severe toxic hepatitis: characterization and discussion[END_REF][START_REF] Bechade | Fatal Acute Liver Failure as a Consequence of Regorafenib Treatment in a Metastatic Colon Cancer[END_REF], sorafenib [START_REF] Fairfax | Fatal case of sorafenib-associated idiosyncratic hepatotoxicity in the adjuvant treatment of a patient with renal cell carcinoma[END_REF], and sunitinib [START_REF] Weise | Fatal liver failure in a patient on acetaminophen treated with sunitinib malate and levothyroxine[END_REF][START_REF] Mermershtain | Fatal liver failure in a patient treated with sunitinib for renal cell carcinoma[END_REF]105]. Two cases of cirrhosis were reported after 18 and 24 months of imatinib treatment [START_REF] Sobhi | Imatinib-induced immune hepatitis: case report and literature review[END_REF]106]. No other cause of liver disease was found in these patients.

J o u r n a l P r e -p r o o f

In case reports of crizotinib-related DILI, LFT abnormalities returned despite drug reintroduction at a lower dose (250 mg daily versus 250 mg twice daily) but completely disappeared following drug withdrawal [START_REF] Ripault | Crizotinib-induced acute hepatitis: first case with relapse after reintroduction with reduced dose[END_REF]. Successful reintroduction at a reduced dose has been reported in clinical trials [START_REF] Shaw | Crizotinib versus chemotherapy in advanced ALK-positive lung cancer[END_REF][START_REF] Cao | Efficacy and safety of crizotinib among Chinese EML4-ALK-positive, advanced-stage non-small cell lung cancer patients[END_REF]107]. LFT abnormalities recurred upon restarting the drug in two case reports of erlotinib-related DILI, with a fatal evolution in one case [START_REF] Liu | Fulminant hepatic failure secondary to erlotinib[END_REF]99].

DILI also recurred after gefitinib or regorafenib re-challenge in case reports with concurrent corticoid administration [START_REF] Ho | Side effects related to cancer treatment: CASE 1. Hepatitis following treatment with gefitinib[END_REF] or dose reduction [START_REF] Ueda | Successful treatment with afatinib after gefitinib-and erlotinib-induced hepatotoxicity[END_REF]104]. Nevertheless, the reintroduction of gefitinib was possible for one patient one year later [102]. For imatinib-and pazopanib-related DILI, LFT abnormalities reappeared upon restarting the drug, but successful reintroduction was reported when combined with prednisone in seven [START_REF] Ferrero | Corticosteroids can reverse severe imatinib-induced hepatotoxicity[END_REF]108,109] and two case reports [103],

respectively. In the only case report describing osimertinib-related DILI with a positive rechallenge, the drug could be restarted following oral desensitization, without the recurrence of LFT abnormalities [110].

Cross-toxicity has not been described for MKIs and the switch to erlotinib [START_REF] Takeda | Successful treatment with erlotinib after gefitinib-related severe hepatotoxicity[END_REF]101] or afatinib [START_REF] Zenke | Successful treatment with afatinib after grade 3 hepatotoxicity induced by both gefitinib and erlotinib in EGFR mutation-positive non-small cell lung cancer[END_REF][START_REF] Ueda | Successful treatment with afatinib after gefitinib-and erlotinib-induced hepatotoxicity[END_REF]111] appears to be a safe option for patients developing gefitinib-associated DILI (Table 2). Furthermore, patients developing erlotinib-related DILI were switched to gefitinib without DILI recurrence [99,112]. The dabrafenib/trametinib combination was also successfully introduced to a patient developing vemurafenib-induced severe liver injury [113].

In clinical trials, liver toxicity related to MKI agents is often transient and non-severe.

For bosutinib, LFT values returned to normal for half of patients without drug withdrawal [START_REF] Campone | Phase II study of single-agent bosutinib, a Src/Abl tyrosine kinase inhibitor, in patients with locally advanced or metastatic breast cancer pretreated with chemotherapy[END_REF].

Nevertheless, fatal cases of MKI-related hepatotoxicity have been reported for crizotinib (1 case) [START_REF] Shaw | Crizotinib versus chemotherapy in advanced ALK-positive lung cancer[END_REF], gefitinib (1 case) [START_REF] Park | Afatinib versus gefitinib as first-line treatment of patients with EGFR mutation-positive non-small-cell lung cancer (LUX-Lung 7): a phase 2B, open-label, randomised controlled trial[END_REF], imatinib (1 case) [START_REF] Talpaz | Imatinib induces durable hematologic and cytogenetic responses in patients with accelerated phase chronic myeloid leukemia: results of a phase 2 study[END_REF], lenvatinib (3 cases) [114, 115], pazopanib (2 cases) [116], sorafenib (7 cases) [117-120], and sunitinib (9 cases) [121, 122].

J o u r n a l P r e -p r o o f Furthermore, the presence of the human leukocyte antigen (HLA)-DQA1*02:01 allele was shown to be associated with a higher risk of lapatinib-induced liver injury in woman with advanced breast cancer [126]. It was also shown that HLA-B*57:01 carriage confers a risk of aminotransferase elevation in patients receiving pazopanib [START_REF] Xu | HLA-B*57:01 Confers Susceptibility to Pazopanib-Associated Liver Injury in Patients with Cancer[END_REF]. The hepatotoxicity of lapatinib and pazopanib may therefore be attributed to the interaction between CYP450 polymorphisms and host immune status. Despite a low risk of liver toxicity risk when given as a monotherapy, the vemurafenib-ipilimumab combination was shown to be associated with hepatotoxicity in 60% of patients in a phase 1 clinical trial [START_REF] Ribas | Hepatotoxicity with combination of vemurafenib and ipilimumab[END_REF]. Ipilimumab is an immune checkpoint inhibitor that swings the balance towards immune stimulation and may therefore favour an immunoallergic DILI to vemurafenib. The results for individual MKI agents are summarized in Table 2 [11, 14, 18, 23, 33-36, 42, 46, 47, 49-56, 58, 59, 61-64, 66-68, 70-92, 94, 98, 99, 101, 102, 105, 106, 109, 111, 112, 114, 115, 124, 132, 136-165].

J o u r n a l P r e -p r o o f 4 Discussion

Data on DILI from MKI drugs are scarce and there are no recent reviews addressing MKI-related hepatic events. Nevertheless, clinical trials suggest that all MKI agents are associated with a risk of hepatotoxicity, defined as an increase in aminotransferase levels, with or without an increase in bilirubin levels. For certain MKI agents, such as bosutinib, the resolution of LFT abnormalities is observed without drug discontinuation or dose reduction.

Such hepatic tolerance to drug toxicity has already been described for isoniazide [166], tacrine [167], and agomelatine [168]: these drugs can initially induce an increase in aminotransferase levels that is thereafter reversible, despite the continuation of treatment. Such an "adaptive phenomenon", which is not synonymous with DILI, is probably underestimated, as druginduced elevation of aminotransferase levels generally results in treatment withdrawal. Overall, the molecules for which the frequency of severe aminotransferase abnormalities (> 5X ULN), Hy's low criteria, and DILI appears to be the highest are crizotinib, erlotinib, gefitinib, imatinib, lenvatinib, pazopanib, regorafenib, sorafenib, sunitinib and vemurafenib-ipilimumab association (table 1 and2). Those with apparently lower risks are axitinib, cabozantinib, dabrafenib, dacomitinib, dasatinib, nilotinib, ponatinib, trametinib, vandetanib, everolimus, and temsirolimus (Tables 1 and2) [14, 18, 31-45, 47, 48, 57, 69, 93, 95, 110, 117-122, 136-142, 155, 169-254] [255-257].

The detection of DILI during pre-marketing clinical trials is a difficult challenge due to the lack of good predictive biomarkers and the small number of patients treated relative to the

J o u r n a l P r e -p r o o f

Drug-drug interactions should be considered. Thus, it would be advisable to avoid coprescriptions that may target or inhibit the same CYP450 pathway in patients treated with MKI.

Immuno-allergic mechanisms may play a role, as suggested by the association between lapatinib and pazopanib-induced liver injury and HLA-DQA1*02:01 and HLA-B*57:01 carriage, respectively. Nevertheless, the clinical relevance of CYP450 pathway and HLA typing of MKI-treated patients has not been proven. DILI related to MKI use is idiosyncratic and generally considered to be dose independent. However, crizotinib and nintedanib were successfully reintroduced at lower doses in most patients who developed hepatotoxicity (Table 2).

The strengths of this review include the exhaustive analysis of published cases of MKIrelated DILI and the evaluation of LFT abnormalities described in clinical trials.

The main limitation of this review is related to publication biases that must be considered in the analysis of the literature. Any analysis involving case reports is subject to an inherent bias toward the publication of more severe MKI-related hepatic toxicity. Furthermore, the number of reported cases of DILI is inevitably higher for the most frequently used MKI agents, which may tend to falsely indicate a higher hepatotoxicity risk. Finally, alternative causes of liver injury are incompletely ruled out in a considerable number of clinical trials and case reports.

Recommendations for clinical practice

Early detection followed by prompt drug withdrawal can prevent hepatic failure and partially explains the low rate of clinically-significant hepatic adverse events in MKI clinical trials. Furthermore, a correct diagnosis of DILI is essential to avoid inappropriate therapy discontinuation. Therefore, LFT assessment at baseline can provide an estimation of the reference values, identify any underling liver injury, and help in the interpretation of LFT abnormalities during MKI treatment. Of note, hepatitis B virus (HBV) reactivation has been J o u r n a l P r e -p r o o f described during treatment with certain MKI molecules, such as everolimus, temsirolimus, imatinib, sunitinib, sorafenib, erlotinib, and afatinib [262, 263]. This adverse event may progress to severe hepatitis and have a mortality rate as high as 11%, despite antiviral treatment [262]. Furthermore, MKI withdrawal is required irrespective of HBV reactivation severity.

Therefore, the European Association for the Study of the Liver recommends that all candidates for chemotherapy should be screened for HBsAg and anti-HBc antibody before initiation of treatment [264]. Once HBsAg detected, prophylactic antiviral therapy may reduce the risk of HBV reactivation and prevent chemotherapy disruption. HBV reactivation during chemotherapy was previously reviewed [262].

The utility of regular LFT monitoring to prevent clinically-significant hepatic adverse events is difficult to evaluate in practice due to the very low incidence of DILI. However, LFT monitoring is a valuable tool for the early detection of drug-related hepatotoxicity until more specific markers of DILI are available [265]. According to clinical trials and case reports, most cases of MKI-related DILI occur during the first two months of exposure. Therefore, weekly monitoring of LFT should be performed for the first two months when using a MKI with a greater risk of hepatotoxicity (Table 2; Figure 2). LFT assessment may then be continued on a monthly basis. For MKIs with an apparently lower liver toxicity risk (Table 2; Figure 2), monthly monitoring of LFT is reasonable. In patients with long-term exposure to imatinib, noninvasive evaluation of hepatic fibrosis by transient elastometry once a year may be useful, due to the risk of developing cirrhosis (table 2) [START_REF] Sobhi | Imatinib-induced immune hepatitis: case report and literature review[END_REF]106]. Nevertheless, there is only a weak level of evidence to support this strategy. The surveillance of LF (ALT, alkaline phosphatase, and bilirubin) is essential until normalization or a return to baseline values. In most cases, LF improves after drug discontinuation, but hepatic injury may sometimes persist for several months. For cases with immuno-allergic characteristics, oral prednisone initiated at a daily dose of 25 to 30 mg and gradually tapered over 2 to 8 months may be an option if LFT abnormalities fail to resolve or worsen following drug discontinuation [START_REF] Ribas | Hepatotoxicity with combination of vemurafenib and ipilimumab[END_REF][START_REF] Sobhi | Imatinib-induced immune hepatitis: case report and literature review[END_REF]103]. Once LFT values return to baseline, patients should be considered for treatment with another MKI molecule due to the lack of crosshepatotoxicity between different MKI drugs (Table 2). In the absence of an alternative anticancer treatment, a re-challenge may be performed under strict surveillance of LF due to the risk of hepatic failure [START_REF] Shaw | Crizotinib versus chemotherapy in advanced ALK-positive lung cancer[END_REF][START_REF] Cao | Efficacy and safety of crizotinib among Chinese EML4-ALK-positive, advanced-stage non-small cell lung cancer patients[END_REF]99,102,107]. Concomitant administration of corticoids at a daily dose of 0.5 mg/kg may be an option for patients for whom imatinib or pazopanib rechallenge is attempted [START_REF] Ferrero | Corticosteroids can reverse severe imatinib-induced hepatotoxicity[END_REF]103,108,109]. Future studies are required to determine whether Nacetylcysteine is an efficient preventive or curative treatment for MKI-induced hepatotoxicity [270]. Figure 2 proposes an algorithm for the management of patients with suspected MKIinduced liver toxicity.

Conclusions

All MKI molecules may potentially cause liver damage, which is sometimes irreversible. As there is still no strategy available to prevent MKI-related hepatotoxicity, early detection remains crucial. Surveillance of LF during treatment may help in the early detection of hepatic adverse events. It is essential that potential causes of hepatic injury be excluded to avoid unnecessary MKI withdrawal.

J o u r n a l P r e -p r o o f injury. Am J Gastroenterol. 2014;109 [START_REF] Andrade | Diagnostic and prognostic assessment of suspected druginduced liver injury in clinical practice[END_REF] J o u r n a l P r e -p r o o f

  J o u r n a l P r e -p r o o f (Roussel Uclaf Causality Assessment Method) causality assessment score has been used and diagnostic tests discussed.

3. 6

 6 Pathophysiology of MKI-induced hepatotoxicityThe risk of MKI-induced liver injury is likely determined by drug properties, including the generation of toxic reactive metabolites, host factors, and interactions between the two [123]. Most MKIs are metabolized by the cytochrome P450 (CYP450) pathway. Thus, variations in CYP450 isoenzyme activity may critically influence the development of DILI by modifying the level of exposure to the reactive metabolites and/or altering the disposition of the MKI molecule. Gefitinib is metabolized in the liver by CYP3A4, CYP2D6, CYP3A5, and CYP1A1. It has been suggested that CYP2D6 is specifically used as an alternative pathway for gefitinib metabolism [124]. CYP2D6 deficiency may therefore favour gefitinib-induced liver injury in patients taking CYP3A4-inhibitory drugs [124]. Lapatinib undergoes extensive metabolism by CYP3A4 and CYP3A5, generating reactive metabolites that may covalently modify cellular proteins, potentially leading to immune-mediated DILI [125, 126].

A

  recent study showed that erlotinib-induced hepatocyte apoptosis in vitro is mediated by mitochondrial damage [127]. Regorafenib can also induce hepatocyte necrosis in vitro at clinically relevant concentrations by uncoupling liver mitochondrial respiration and inducing J o u r n a l P r e -p r o o f autophagy [128]. Furthermore, imatinib, lapatinib, and sunitinib increase the production of reactive oxygen species, impair cellular oxygen consumption, alter glycolysis, and induce apoptosis in human hepatoma HepG2 cells [129]. Overall, these experimental data suggest that mitochondrial injury is a possible mechanism for hepatotoxicity related to MKI use. Consistent with this assumption, various MKIs that induce metabolic-idiosyncratic hepatotoxicity, such as gefitinib, sunitinib, and sorafenib, are able to impair mitochondrial function in isolated rodent liver mitochondria at clinically relevant concentrations [130, 131]. The adenosine triphosphate-binding cassette transporters ABCG2 and ABCB1 are involved in the efflux of xenobiotics from hepatocytes into the bile. It was recently shown that concomitant use of anti-acid-secreting, agents, such H2 antagonists and proton-pump inhibitors, is associated with an increased risk of gefitinib-induced liver toxicity [132]. Gefitinib is known to be a substrate of ABCG2 and ABCB1 expressed in hepatocytes [133]. Thus, inhibition of ABCG2 and ABCB1 by proton-pump inhibitors and H2 antagonists, respectively, may theoretically increase the concentration of gefitinib in the liver, contributing to hepatotoxicity. Sorafenib and regorafenib are metabolized primarily by oxidative metabolism in the liver via the CYP3A4 pathway and glucuronidation mediated by the uridine diphosphateglucuronosyl-transferase 1A (UGT1A) 9 pathway [134]. Both drugs potently inhibit UGT1A 9 and 1, contributing to the hyperbilirubinemia observed in patients treated with sorafenib and regorafenib [135].

  frequency of DILI. The FDA has proposed indicators of the potential to cause severe hepatotoxicity to guide drug development research: elevation of aminotransferase values to > 3X ULN, with reference to a placebo; any marked elevation of aminotransferases to > 5X ULN in the treatment group, without a corresponding increase in the placebo group; or one or more cases of bilirubin titres increasing to > 2X ULN associated with aminotransferase levels of > J o u r n a l P r e -p r o o f 3X ULN (Hy's law), with no other explanation [258]. The presence of at least one of these criteria may indicate a significant risk of hepatic toxicity and cases of severe DILI can be expected in post-marketing settings. Of note, the definition of hepatic event severity in clinical trials involving MKI drugs is based on the Common Terminology Criteria for Adverse Events (CTCAE), developed by the Cancer Therapy Evaluation Program of the National Cancer Institute (NCI) [259]. According to these criteria, the elevation of aminotransferase > 5X ULN is defined as a severe hepatic event. However, it should be mentioned that asymptomatic increases in aminotransferase levels without jaundice cannot be considered as severe hepatotoxicity. A more reliable scale for grading the severity of liver injury related to drug use was proposed by the Drug-Induced Liver Injury Network (DILIN) [260]. In the DILIN scale, the severity of hepatic events is based on the presence of jaundice, signs of hepatic or other organ failure, the necessity of hospitalization, and death. The presence of these severity criteria is not required in the CTCAE definition. Therefore, the use of the CTCAE criteria in MKI clinical trials may overestimate the severity of MKI-induced hepatotoxicity. It should also be mentioned that LFT abnormalities do not necessarily reflect drug-related toxicity in MKI treated patients. Liver metastases are present in up to 50% of patients in clinical trials and may induce LFT abnormalities. Therefore, LFT perturbations should be interpreted with caution to avoid inappropriate MKI withdrawal. Routine LFT (ALT, AST, PAL, bilirubin) are not specific biomarkers of DILI. Nevertheless, ALT and AST specifically indicate hepatocyte damage and are still the gold standard that should be used to monitor patients [261]. Studies of various individual gene polymorphisms may allow the identification of more reliable indicators of the risk of MKI-related DILI for use in the future. Of note, pre-existing chronic liver disease does not appear to favour MKI-induced hepatotoxicity, but once it develops, DILI may have devastating consequences for patients with a reduced baseline hepatic reserve.

  The cut-offs for LFTs for MKI withdrawal are difficult to define, especially for patients with a hepatic extension of disease. MKI treatment should be promptly discontinued if serum aminotransferase levels are > 5X ULN or if the criteria for Hy's law are present. In cases of moderate aminotransferase elevation < 5X ULN, LFT monitoring should be performed twice a J o u r n a l P r e -p r o o f week and MKI treatment may be continued. For patients with high baseline aminotransferase levels, the MKI should be discontinued if serum aminotransferase levels reach > 5 times the baseline value. Of note, MKIs that inhibit UGT1A 1 (sorafenib and regorafenib) impair the elimination of bilirubin, with a resulting increase in unconjugated bilirubin levels, mimicking the criteria for Hy's law. Permanent drug withdrawal may become necessary, but this poses a dilemma if the tumour is responsive to the MKI concerned. Exhaustive investigations should therefore be performed to exclude other potential causes of liver injury and confirm the diagnosis of MKIrelated DILI, including serological tests for hepatotropic viruses (hepatitis A, B, C, and E viruses, Epstein-Barr virus, cytomegalovirus, and herpes simplex virus), autoantibody tests, iron and copper levels, and abdominal ultrasonography. A recent study showed that immunemediated DILI related to immune-checkpoint inhibitor use has specific histologic characteristics such as granulomatous hepatitis [266]. Furthermore, the severity of histological lesions guided patient management (those with severe lesions benefited of corticosteroid administration while those with mild lesions spontaneously improved) supporting the necessity of liver biopsy for this indication. For MKI-related DILI, liver histology does not reveal any specific lesions and is therefore not mandatory for diagnosis. Nevertheless, liver biopsy is useful if autoimmune hepatitis is suspected [265]. Liver biopsy may also be considered if LFT worsens or fails to resolve after drug withdrawal to exclude alternative diagnoses, notably the hepatic extension of cancer. Hepatitis E virus (HEV) infection is not considered in the differential diagnosis of DILI in most published cases of MKI-induced liver damage. However, HEV infection has been shown to be the cause of liver injury in 3 to 7% of cases initially adjudicated as DILI [267, 268]. Of note, patients with cancer are immunocompromised and might not mount a good antibody response. Furthermore, false positive HEV serology can occur in autoimmune hepatitis and Epstein-Barr infection [269]. Both anti-HEV IgM and HEV-RNA J o u r n a l P r e -p r o o f testing should therefore be performed in all cases of suspected MKI-induced hepatotoxicity, particularly if the clinical features are compatible with acute viral hepatitis.

Figure 1 .

 1 Figure 1. Results of the literature search.

Figure 2 .

 2 Figure 2. Management of patients with suspected MKI-induced liver toxicity.

  :950-66; quiz 67. Epub 2014/06/18. doi: 10.1038/ajg.2014.131. PubMed PMID: 24935270. 97. Benichou C, Danan G, Flahault A. Causality assessment of adverse reactions to drugs--II. An original model for validation of drug causality assessment methods: case reports with positive rechallenge. J Clin Epidemiol. 1993;46(11):1331-6. Epub 1993/11/01. PubMed PMID: Mennecier B, Gschwend A, Rein M, Coquerel A, Humbert X, et al. Successful treatment with ceritinib after crizotinib induced hepatitis. Lung Cancer. 2016;95:15-6. Epub 2016/04/05. doi: 10.1016/j.lungcan.2016.02.008. PubMed PMID: 27040846. 99. Kunimasa K, Yoshioka H, Iwasaku M, Nishiyama A, Korogi Y, Masuda G, et al. Successful treatment of non-small cell lung cancer with gefitinib after severe erlotinib-related hepatotoxicity. Intern Med. 2012;51(4):431-4. Epub 2012/02/16. PubMed PMID: 22333382. 100. Seki N, Uematsu K, Shibakuki R, Eguchi K. Promising new treatment schedule for gefitinib responders after severe hepatotoxicity with daily administration. J Clin Oncol. 2006;24(19):3213-4; author reply 4-5. Epub 2006/07/01. doi: 10.1200/JCO.2006.05.7109. PubMed PMID: 16809744. 101. Kijima T, Shimizu T, Nonen S, Furukawa M, Otani Y, Minami T, et al. Safe and successful treatment with erlotinib after gefitinib-induced hepatotoxicity: difference in metabolism as a possible mechanism. J Clin Oncol. 2011;29(19):e588-90. Epub 2011/04/20. doi: 10.1200/JCO.2010.34.3368. PubMed PMID: 21502555. 102. Chen X, Pan Y, Zhang S, Chen D, Yang S, Li X, et al. Rechallenge with gefitinib following severe drug-induced hepatotoxicity in a patient with advanced non-small cell lung cancer: A case report and literature review. Oncol Lett. 2014;7(3):878-80. Epub 2014/02/15.

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	98.	Sassier M,

doi

: 10.3892/ol.2013.1756

. PubMed PMID: 24527096; PubMed Central PMCID: PMCPMC3919904.

Acknowledgements

None declared.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for profit sectors.

Authorship contributions

Camille Houron and Marie Danielou performed research studies, analysed the data and wrote the manuscript. Olivier Mir contributed to the research studies and data analysis. Bernard Fromenty contributed to the data analysis. Gabriel Perlemuter and Cosmin Sebastian Voican contributed to the research design, research studies, data analysis, writing the manuscript and supervision of the study.

Conflict of interest statement

Olivier Mir acted as consultant for Eli-Lilly, Janssen, Lundbeck, Pfizer, Roche, Servier, Vifor Pharma; as a speaker for Eli-Lilly, Roche, Servier, Medscape and PrimeOncology; and owns stock options from Amplitude Surgical, Transgene and Ipsen. Gabriel Perlemuter received travel funds from Janssen and Gilead; consulting fees from Bayer, Biocodex, Roche, Gilead, Pierre Fabre, and Servier; and royalties from Elsevier-Masson, Solar, Flammation/Versilio, and John Libbey Eurotext. Camille Houron, Marie Danielou, Bernard Fromenty and Cosmin Sebastian Voican declare that they have no competing interests. [18, 33-36, 66, 98] [49-51, 67, 68, 82, 99, 111, 112, 156-158] everolimus Mixed Metabolic [54, 55, 83, 100-102, 124, 132, 159-161] [58, 59, 62, 70-74, 84-92, 106, 108, 109, 162-165] [START_REF] Ribas | Hepatotoxicity with combination of vemurafenib and ipilimumab[END_REF] Abbreviations: MKI: multi-kinase inhibitor, LFT: liver function tests


Yves Yeboue

Marion Jean

Gilles Subra

Jean Martinez

Frédéric Lamaty
email: frederic.lamaty@umontpellier.fr

Thomas-Xavier Métro
email: thomas-xavier.metro@umontpellier.fr

Epimerization-free C-term Activation of Peptide Fragments by Ball-Milling

Ball-milling enabled to perform [2+1], [2+2], and [2+3] peptide couplings with high yields and, if any, very low epimerization. Very good results were obtained with peptide fragments containing highly epimerization-prone and/or highly hindered amino acids at C-term such as phenylglycine, cysteine and valine. Ball-milling was clearly identified as the key element to obtain both high yield and purity along with low epimerization. Indeed, the ball-milling conditions proved to be superior to the classical solution synthesis approach on a various array of widely used coupling agents. These results open avenues for the development of highly efficient, convergent and flexible peptide synthesis strategies based on peptide fragment couplings mediated by ballmilling.

Peptides can be found in many and diverse applications as nutriments, cosmetics and pharmaceuticals. In the field of medicinal chemistry, they can act as therapeutics and diagnostic agents, including for the fight against the Covid-19 disease. [START_REF] Xia | Inhibition of SARS-CoV-2 (previously 2019-nCoV) infection by a highly potent pan-coronavirus fusion inhibitor targeting its spike protein that harbors a high capacity to mediate membrane fusion[END_REF][START_REF] Liu | Research and Development on Therapeutic Agents and Vaccines for COVID-19 and Related Human Coronavirus Diseases[END_REF] Over the years, interest in this class of compounds has increased at a steady rate, [START_REF] Raines | Peptides on the Rise[END_REF][START_REF] Lau | Therapeutic peptides: Historical perspectives, current development trends, and future directions[END_REF][START_REF] Henninot | The Current State of Peptide Drug Discovery: Back to the Future?[END_REF][START_REF]Impact Story: Developing the Tools to Evaluate Complex Drug Products: Peptides[END_REF][START_REF] Shaer | FDA TIDES (Peptides and Oligonucleotides) Harvest[END_REF] due to their good biological activities at very low doses, high selectivity, and high probability of success at clinical trial stages. [START_REF] Lau | Therapeutic peptides: Historical perspectives, current development trends, and future directions[END_REF][START_REF] Vlieghe | Synthetic therapeutic peptides: science and market[END_REF] In 2019, the FDA has estimated that approximately 100 peptide drugs were marketed in the U.S., Europe, and Japan, accounting for annual global sales ranging between $15 billion to $20 billion. [START_REF]Impact Story: Developing the Tools to Evaluate Complex Drug Products: Peptides[END_REF] Among these peptide-based drugs, we can highlight Macrilen, which was discovered in our institute (IBMM) in 1999 and that was recently approved by both FDA and EMA to be used in the diagnosis of patients with adult growth hormone deficiency. [START_REF] Martinez | From JMV-1843 to Macrilen[END_REF] When the stepwise synthesis of low molecular weight peptides do not generally present any difficulty, the chemical synthesis of longer peptides and proteins is a much more challenging task. Indeed, the efficiency of coupling and deprotection reactions decreases with the length of the peptide chain, leading to amino acid deletions and uncompleted sequences. These difficulties have certainly slowed down, if not prevented, commercialization of some long peptide chains, and may also explain why recombinant protein strategy is generally preferred when sequences of more than 60 amino acid residues are targeted. [START_REF] Lax | In Peptide Therapeutics: Strategy and Tactics for Chemistry, Manufacturing, and Controls[END_REF] Significant efforts have been deployed by peptide scientists to tackle this challenge, mainly by using convergent fragment coupling approaches, in particular using native chemical ligation (NCL) of peptides fragments. [START_REF] Raibaut | Sequential native peptide ligation strategies for total chemical protein synthesis[END_REF][START_REF] Conibear | Native chemical ligation in protein synthesis and semi-synthesis[END_REF][START_REF] Agouridas | Native Chemical Ligation and Extended Methods: Mechanisms, Catalysis, Scope, and Limitations[END_REF] Yet, these approaches suffer from a limited scope: peptide bond disconnections cannot be performed at any point of the peptide chain. For instance, a N-terminal cysteine or an amino acid modified by a thiol auxiliary are required to perform NCL. Limitations are even more constraining when coupling a C-term activated peptide fragment with a free N-term fragment; the risk of epimerization can be extremely high, leading to the production of highly undesirable diastereomers. [START_REF] Yang | In Side Reactions in Peptide Synthesis[END_REF] This hurdle can be overcome: some examples are reporting peptide fragment couplings with low to null epimerization levels. [START_REF] Hiebl | Large-scale synthesis of hematoregulatory nonapeptide SK&F 107647 by fragment coupling[END_REF][START_REF] Spengler | A Novel Protecting/Activating Strategy for β-Hydroxy Acids and Its Use in Convergent Peptide Synthesis[END_REF][START_REF] Popovic | Epimerization-Free C-Terminal Peptide Activation[END_REF][START_REF] Zhang | Kilogram-Scale Synthesis of Osteogenic Growth Peptide (10-14) Using a Fragment Coupling Approach[END_REF][START_REF] Zhang | Full Solution-Phase Synthesis of Acetyl Hexapeptide-3 by Fragments Coupling Strategy[END_REF][START_REF] Gurjar | Process for preparation of octreotide acetate via an improved 4+4 soln. phase synthesis of octreotide[END_REF] However, these examples remain relatively scarce. In this context, and based on our knowledge of peptide synthesis under solvent-less/solvent-free conditions by mechanochemical means, [START_REF] Declerck | Solvent-Free Synthesis of Peptides[END_REF][START_REF] Bonnamour | Environmentally benign peptide synthesis using liquid-assisted ball-milling: application to the synthesis of Leu-enkephalin[END_REF][START_REF] Porte | Peptide Mechanosynthesis by Direct Coupling of N-Protected α-Amino Acids with Amino Esters[END_REF][START_REF] Maurin | Peptide synthesis: ball-milling, in solution, or on solid support, what is the best strategy?[END_REF][START_REF] Pétry | A mechanochemical approach to access the proline-proline diketopiperazine framework[END_REF][START_REF] Yeboue | Peptide couplings by reactive extrusion: solid-tolerant and free from carcinogenic, mutagenic and reprotoxic chemicals[END_REF] we considered evaluating the capacity of ball-mills to reduce or eliminate epimerization during C-term activation of peptide fragments, and to compare these results with the classical synthesis in solution.

The [2+1] coupling between Z-Ala-Phg-OH and HCl•H-Ile-OMe was considered as a probe of choice. [START_REF]For sake of clarity, absolute configuration indications of L amino acids have been omitted throughout the manuscript. Only D configurations have been indicated[END_REF] Indeed, phenylglycine (Phg) is known as being one of the most epimerization-prone amino acid, [START_REF] Yang | In Side Reactions in Peptide Synthesis[END_REF][START_REF] Popovic | Epimerization-Free C-Terminal Peptide Activation[END_REF][28][START_REF] Elsawy | Racemisation of N-Fmoc phenylglycine under mild microwave-SPPS and conventional stepwise SPPS conditions: attempts to develop strategies for overcoming this[END_REF][START_REF] Fuse | Total synthesis of feglymycin based on a linear/convergent hybrid approach using micro-flow amide bond formation[END_REF][START_REF] Liang | Phenylglycine racemization in Fmoc-based solid-phase peptide synthesis: Stereochemical stability is achieved by choice of reaction conditions[END_REF] while isoleucine is a hindered amino acid exhibiting low coupling speeds. [START_REF] Ostresh | Peptide libraries: Determination of relative reaction rates of protected amino acids in competitive couplings[END_REF] For this study, the target dipeptide Z-Ala-Phg-OH was prepared using Z/tBu orthogonal strategy (see ESI for details). Then, Z-Ala-Phg-OH was reacted with HCl•H-Ile-OMe (1.5 equiv.), in the presence of EDC•HCl (1.2 equiv.), Oxyma (1.2 equiv.) and a small amount of DMF (η = 0.45 µL/mg) in a 5 mL PTFE ball-mill reactor containing three stainless steel balls (5 mm diameter), for 10 min at 25 Hz (see ESI for details). Here, the amount of DMF was not sufficient to solubilize the reaction mixture but improved the homogeneity of the reaction mixture. [START_REF] Bowmaker | Adding a liquid additive to a mechanochemical reaction, known as Liquid Assisted Grinding (LAG), is known to have beneficial effects during peptide couplings under mechanochemical conditions[END_REF] Although being a highly problematic solvent, [START_REF]DMF) is classified as presenting reproductive toxicity according to Regulation[END_REF] DMF is considered as the most widely used solvent for peptide synthesis. [START_REF] Macmillan | Evaluation of alternative solvents in common amide coupling reactions: replacement of dichloromethane and N,N-dimethylformamide[END_REF][START_REF] Jad | Peptide synthesis beyond DMF: THF and ACN as excellent and friendlier alternatives[END_REF][START_REF] Pawlas | SPPS: enabling circular chemistry in environmentally sensible solid-phase peptide synthesis[END_REF] It appeared as the liquid additive of choice to ensure a rigorous comparison between the ball-milling and the classical solution-phase approach. Following the same idea, the η parameter was calculated to facilitate the comparison of the solvent quantities that were used. [START_REF]The η parameter is defined by the ratio of the volume of liquid (in µl) to the total mass of solids[END_REF] After reaction mixture recovery from the milling reactor and work-up, the corresponding tripeptide Z-Ala-Phg-Ile-OMe was isolated in 93% yield, with excellent purity and no detectable traces of the Z-Ala-D-Phg-Ile-OMe epimer as indicated by HPLC (Entry 1, Table 1; tR(LLL) = 5.08 min and tR(LDL) = 5.20 min, see ESI for details).

Entry Reagents

Temp (°C) t (min) Yield (%) Purity (%) a,b LDL (%) Ball-milling is known to induce a slight temperature raise of the milled materials. [START_REF] Kulla | Warming up for mechanosynthesis -Temperature development in ball mills during synthesis[END_REF][START_REF] Užarević | Enthalpy vs. friction: Heat flow modelling of unexpected temperature profiles in mechanochemistry of metal-organic frameworks[END_REF][START_REF] Ardila-Fierro | Direct Visualization of a Mechanochemically Induced Molecular Rearrangement[END_REF] As the temperature inside the reactor could have an influence on the reaction course, the temperature of the milled material was measured directly after milling. During the coupling under ball-milling conditions, the temperature reached 33 °C. The same temperature was applied to the synthesis of Z-Ala-Phg-Ile-OMe under classical magnetic stirring conditions, while using the minimal amount of DMF enabling proper mixing (η = 20 µL/mg). In these experimental conditions, Z-Ala-Phg-Ile-OMe was produced in 88% yield, 32% purity and 9% of the LDL epimer (Entry 1, figures in parentheses, Table 1). These results clearly showed that solvent-less ball-milling could minimize epimerization of a highly epimerization-prone C-term activated peptide fragment. Additionally, the coupling was complete after 10 min of ball-milling, while 30 min were required under classical solution conditions. This latter observation could be explained by the higher concentration of the reaction mixture in the ball-mill compared to that in solution. Other coupling reagents and additives were screened to establish the generality of our observations.

When couplings were performed in solution, the quantity of DMF was kept as low as possible to ensure maximal concentration of the reactants while enabling proper mixing under classical magnetic stirring ( 11 < η < 26 µL/mg). When Oxyma was replaced with HOBt•H2O, ball-milling conditions led to 25% of the LDL epimer and 70% purity, while 35% of the same epimer and 48% purity were obtained for the synthesis in solution (Entry 2, Table 1). The use of HOAt gave better results than HOBt•H2O; only 1% of the LDL epimer was produced in the ball-milling conditions while 26% LDL epimer were obtained after synthesis in solution (Entry 3, Table 1). These results were in agreement with previous publications comparing HOBt•H2O and HOAt. [START_REF] El-Faham | Peptide Coupling Reagents, More than a Letter Soup[END_REF][START_REF] Subirós-Funosas | An Efficient Additive for Peptide Synthesis to Replace the Benzotriazole-Based HOBt and HOAt with a Lower Risk of Explosion[END_REF] Using DIC with HOAt under ball-milling conditions furnished the tripeptide with 67% purity along with 17% of the LDL epimer, while synthesis in solution led to lower purity (39%) and higher epimerization (33%) (Entry 4, Table 1). These results obtained with DIC were not improved when replacing HOAt with Oxyma (Entry 5, Table 1). Concerning the stand-alone coupling reagents HBTU and HATU, their use induced low epimerization levels both by ball-milling and in solution (Entries 6 & 7, Table 1). Yet, the HPLC purity profiles of the products obtained by ballmilling were much better than when synthesized in solution. The comparison between ball-milling and solution-phase clearly showed the superiority of the mechanochemical approach over the classical synthesis in solution, since lower epimerization rates and better purity profiles were obtained with almost all tested coupling agents and additives, while yields remained comparable.

We next applied the best ball-milling experimental conditions (EDC•HCl/Oxyma) to the synthesis of other challenging peptides. Here, EtOAc was used instead of DMF to improve the overall environmental impact of our ball-milling conditions. Thus, ball-milling Z-Ala-Phg-OH with HCl•H-Phe-OMe produced the tripeptide Z-Ala-Phg-Phe-OMe in 89% yield with full conservation of the stereogenic centers (Entry 1, Table 2).

Entry Peptides Time (min) Isolated yield (%) de (%) When Z-Ala-D-Phg-OH was used instead of Z-Ala-L-Phg-OH, the tripeptide Z-Ala-D-Phg-Phe-OMe was isolated with a similar yield of 92% and excellent purity (Entry 2, Table 2). Cysteine, which is another epimerization-prone amino acid, [START_REF] Yang | In Side Reactions in Peptide Synthesis[END_REF][START_REF] Han | Occurrence and Minimization of Cysteine Racemization during Stepwise Solid-Phase Peptide Synthesis[END_REF] was next tested under the same experimental conditions. Thus, Z-Ala-Cys(Bn)-Ala-OMe and Z-Ala-Cys(Bn)-Phe-OMe were synthesized from Z-Ala-Cys(Bn)-OH. In both cases, tripeptides were obtained in excellent yields (> 94%) without any detectable epimerization (Entries 3 & 4, Table 2). Like isoleucine, valine is a β-branched amino acid known to be more difficult for coupling than most of other amino acids. [START_REF] Ostresh | Peptide libraries: Determination of relative reaction rates of protected amino acids in competitive couplings[END_REF] Yet, the reaction between Z-Phe-Val-OH and HCl•H-Cys(Bn)-OMe led to the desired tripeptide, Z-Phe-Val-Cys(Bn)-OMe, in almost quantitative yield after 30 minutes ball-milling (Entry 5, Table 2). Importantly, no trace of the LDL epimer could be detected by HPLC analysis. Similar results were obtained starting with the Z-Phe-D-Val-OH (Entry 6, Table 2). When HCl•H-Cys(Bn)-OMe was replaced with HCl•H-Ser(tBu)-OtBu, excellent yields were obtained, again with no epimerization (Entries 7 & 8, Table 2). Of note, the t-butyl ester was stable in these experimental conditions. In classical peptide synthesis in solution, Z and Boc N-protecting groups are commonly used. In order to evaluate the coupling of Boc-protected fragments, Boc-Trp-Phe-Glu(OBn)-OBn, Boc-Trp-Phe-Gly-OBn, and Boc-Trp-D-Phe-Gly-OBn were synthesized and successfully isolated in good to excellent yields (84-95%), without any detectable epimerization (Entries 9-11, Table 2). These results supported that ball-milling conditions were compatible with Boc N-protecting groups, without affecting the isolated yields and diastereomeric excesses of the expected products. The presence of benzyl groups was not an obstacle to the success of these syntheses.

After clearly demonstrating the efficiency of our method in epimerization-free [2+1] peptide couplings, we aimed at demonstrating that peptide fragment couplings mediated by ball-milling could be applied to longer peptide fragments. For this, [2+2] and [2+3] couplings involving peptide fragments bearing the hindered valine at C-term were performed. When the optimal experimental reaction conditions were applied to the coupling of Z-Phe-Val-OH with HCl•H-Leu2-OBn, the expected tetrapeptide was obtained in 95% yield without epimerization (Entry 12, Table 2). The same conditions applied to the synthesis of the diastereomer Z-Phe-D-Val-Leu2-OBn yielded the desired tetrapeptide in 91%, yet containing 2% of the epimer (Entry 13, Table 2). 2). So far, the longest peptide synthesized by ball-milling was the pentapeptide Leu-enkephalin. [START_REF] Bonnamour | Environmentally benign peptide synthesis using liquid-assisted ball-milling: application to the synthesis of Leu-enkephalin[END_REF] The successful synthesis of these two pentapeptides by a fragment coupling approach consolidates the potential of mechanochemistry to produce oligopeptides with high efficiency. Overall, the lower levels of epimerization observed under ball-milling conditions could find explanations in the concentrations of the reaction mixtures. Indeed, epimerization during peptide couplings occurs either through direct Habstraction in basic medium and/or through the formation of an oxazolone ring during activation. [START_REF] Yang | In Side Reactions in Peptide Synthesis[END_REF] Of note, the latter mechanism is an intramolecular process favored in diluted conditions, whereas "direct" peptide coupling (in other words, acylation of the free amine) is an intermolecular process, comparatively favored in concentrated conditions such as those enabled by ball-milling. Consequently, our results suggest that the absence of epimerization could be explained by the highly concentrated reaction conditions enabled by ball-milling, that would prevent the transient formation of an oxazolone ring.

To conclude, using the combination of EDC•HCl, Oxyma and small amounts of a liquid additive (DMF or EtOAc) under ball-milling conditions allowed to perform [2+1], [2+2], and

[2+3] peptide couplings with high yields and, if any, very low epimerization. Noteworthy, very good results were obtained with peptide fragments containing highly epimerizationprone and/or highly hindered amino acids at C-term such as phenylglycine, cysteine and valine. Ball-milling was clearly identified as the key element to obtain both high yield and purity along with low epimerization. Indeed, the ball-milling conditions proved to be superior to the classical solution synthesis approach on a various array of widely used coupling agents. These results open avenues for the development of highly efficient, convergent and flexible peptide synthesis strategies based on peptide fragment couplings. Further investigations aiming at i) deciphering the mechanism enabling epimerization suppression during ball-milling when compared to synthesis in solution ii) applying this strategy to longer peptide fragments are currently being explored in our laboratory and will be reported in due course.

  . of HCl•H-AAn-OR were used.b 1.2 equiv. of EDC•HCl were used. c 1.0 equiv. of HCl•H-AAn-OR were used. d 2.2 equiv. of EDC•HCl and η (EtOAc) = 0.9 µL/mg were used.

  To our delight, when the tripeptide HCl•H-Leu3-OBn was reacted with Z-Phe-Val-OH and Z-Phe-D-Val-OH, the corresponding pentapeptides Z-Phe-Val-Leu3-OBn and Z-Phe-D-Val-Leu3-OBn were successfully isolated with high yields (> 91%) and no to negligible level of epimerization (Entries 14 & 15, Table

Table 1 :

 1

Comparative study between vibrating ball-milling (main figures) and solution synthesis (in parentheses) of Z-Ala-Phg-Ile-OMe

Table 2 :

 2 Scope of the peptide couplings by ball-milling

Acknowledgments

Université de Montpellier, Centre National de la Recherche Scientifique (CNRS), and LabEx CheMISyst (through ANR programme ANR-10-LABX-05-01) are acknowledged for financial support. Monique Calmès is aknowleged for assistance in chiral HPLC analysis.