Ayman Tourbah
email: ayman.tourbah@aphp.fr

Caroline Papeix

Patricia Tourniaire

Karin Rerat

Mohamed Meite

Barbara Durand

Fabienne Lamy

Isabelle Chouette

Claude Mekies

Ayman Pr

Tourbah

Reasons for switching to fingolimod in patients relapsing-remitting multiple sclerosis in France: the ESGILE study

Keywords: multiple sclerosis, fingolimod, switching, treatment acceptability, patientreported outcome

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

(2.0 ± 1.3 at inclusion; 2.0 ± 1.5 at M12). With the CGI-C, 67 patients (38.7%) were considered improved and 23 (13.3%) worsened. Although no obvious differences in CGI-C ratings were observed as a function of the reason for switching, when patient preferences entered into the decision, the proportion of patients considered minimally improved was somewhat higher (37.7%) and the proportion considered unchanged somewhat lower (41.5%). With the PGI-C, more patients rated themselves improved than were rated as improved by the physician: of 64 patients rated as 'no change' on the CGI-C, 21 (32.8%) rated themselves as 'improved' and 10 (15.6%) as 'worsened'. The overall level of agreement between the two measures was moderate (κ = 0.48 [95% CI: 0.35 -0.60]). The mean general treatment acceptability score on the ACCEPT® questionnaire was .9] at inclusion (reflecting acceptability of the previous DMT) and ] at M12 (reflecting acceptability of fingolimod). Mean dimension scores ranged from 36.7 for effectiveness to 72.2 for medication inconvenience at inclusion and from 63.4 for effectiveness to 96.8 for medication inconvenience at M12. The frequency and nature of

Introduction

Over the last decade, the options available for the treatment of relapsing remitting sclerosis (RRMS) have increased considerably, in particular with the introduction of disease modifying treatments (DMTs) that can be administered orally, starting with fingolimod in 2011 [START_REF] Druart | Long-term safety and real-world effectiveness of fingolimod in relapsing multiple sclerosis[END_REF], and the availability of a range of therapeutic monoclonal antibodies.

Since none of the current first-line DMTs can be guaranteed to provide an optimal therapeutic response in all patients with RRMS, switching to an alternative therapy in case of inadequate control of disease activity or poor tolerability is a commonly used management strategy [START_REF] Hansen | Adherence to Disease Modifying Drugs among Patients with Multiple Sclerosis in Germany: A Retrospective Cohort Study[END_REF][START_REF] Lebrun-Frenay | Five-year outcome in the copaxone observatory: a nationwide cohort of patients with multiple sclerosis starting treatment with glatiramer acetate in France[END_REF][START_REF] Miller | Switching or Discontinuing Disease-Modifying Therapies for Multiple Sclerosis[END_REF]. The availability of a wider range of DMTs provides more opportunities for switching, and this has led to more frequent and earlier switching. Indeed, current practice guidelines from the European Committee of Treatment and Research in Multiple Sclerosis (ECTRIMS) and the European Academy of Neurology (EAN) recommend that patients starting a DMT should be actively monitored, including with magnetic resonance imaging (MRI) and switched to a more efficacious treatment if clinically or radiologically manifest disease activity persists [START_REF] Montalban | ECTRIMS/EAN Guideline on the pharmacological treatment of people with multiple sclerosis[END_REF]. A recent large (~3000 patients) Italian study reported that 48% of patients switched to a second-line therapy within three years, and that this rate had increased four-fold over the period 2010 -2017 for switches due to inadequate efficacy and six-fold for those due to tolerability issues [START_REF] Sacca | Determinants[END_REF]. In the case of fingolimod, [START_REF] Bergvall | Relapse rates in patients with multiple sclerosis switching from interferon to fingolimod or glatiramer acetate: a US claims database study[END_REF][START_REF] Cohen | Fingolimod versus intramuscular interferon in patient subgroups from TRANSFORMS[END_REF][START_REF] Fox | Outcomes of switching directly to oral fingolimod from injectable therapies: Results of the randomized, open-label, multicenter, Evaluate Patient OutComes (EPOC) study in relapsing multiple sclerosis[END_REF][START_REF] Freedman | The clinical and cost impact of switching to fingolimod versus other first line injectable disease-modifying therapies in patients with relapsing multiple sclerosis[END_REF] a number of studies have shown improvements in disease control in patients who switch to this drug when disease activity persists under therapy with first-line injectable DMTs (interferon-β or glatiramer acetate).

The reasons for switching may include partial efficacy, tolerability issues and patientrelated factors such as lassitude with injections or lifestyle issues. These reasons are likely to differ according to the initial DMT with which the patients were treated and according to the DMT to which they were switched. There is relatively little information on reasons determining switching choices, notably with respect to patient-related factors. For this reason, we have undertaken a study to document the reasons for switching patients from a first-line DMT to fingolimod in everyday clinical practice in France, and to evaluate the extent of improvement from the point of view of the neurologist and of the patient, according to the reasons for choice.

The primary objective of this observational study was to describe the consequences of switching to fingolimod in terms of clinical improvement measured with the Clinical Global Impression (CGI) rating scale after twelve months according to the reasons underlying the switch. Secondary objectives were to evaluate improvement and treatment acceptability from the patient's perspective and to collect further data on the safety profile of fingolimod in routine clinical practice.

Methods

ESGILE was a prospective, multicentre observational study conducted in France. The study was conducted between January 2015 and January 2019 by community or hospitalbased neurologists who treated patients with MS.

Participants

The study was proposed by mail to neurologists identified in a national listing (Onekey; CEGEDIM) covering >95% of all practicing neurologists in France. Each participating physician was expected to invite the next four patients consulting consecutively who fulfilled the eligibility criteria to participate in the study.

Patients diagnosed with relapsing-remitting multiple sclerosis according to the revised (2010) McDonald criteria [START_REF] Polman | Diagnostic criteria for multiple sclerosis: 2010 revisions to the McDonald criteria[END_REF] previously treated with a single DMT for at least six months and for whom a switch to fingolimod had been decided were eligible for the study. The decision to switch to fingolimod should have been made independently of participation in the study and fingolimod should have been initiated at the earliest four weeks before the inclusion visit. Patients were required to be aged between 18 and 65 years inclusive, to correspond to the approved indication for fingolimod as a second-line treatment at the time of the study and to have an Expanded Disability Status Scale (EDSS) [START_REF] Kurtzke | Rating neurologic impairment in multiple sclerosis: an expanded disability status scale (EDSS)[END_REF] no higher than 5.5. Patients who had previously received multiple DMTs for over six months each, patients with a progressive form of MS, patients with contraindications to fingolimod and patients unable to understand or complete the study questionnaire were ineligible.

Study procedures

Since this was an observational study, participation in the study did not affect patient care and there were no protocol-imposed procedures. Physicians were thus free to pursue their treatment and follow-up procedures as they saw fit. Follow-up visits were programmed according to the participating neurologist's normal practice or as needed by the patient's medical state.

During the study, patients were evaluated at an inclusion visit and at two follow-up visits at approximately Month 6 and Month 12. A margin of ±3 months for these follow-up visits was accepted. Fingolimod was to be initiated at, or at most 35 days before, the inclusion visit. The neurologist completed an electronic case report form (e-CRF) for each patient. At each visit, a physical examination was performed and disability rated with the Expanded Disability Status Scale (EDSS) [START_REF] Kurtzke | Rating neurologic impairment in multiple sclerosis: an expanded disability status scale (EDSS)[END_REF]. Magnetic resonance imaging data were documented when available. At inclusion, data were recorded on demographics, medical and treatment history, and on the reasons underlying the switch of treatment. These reasons were identified on a pre-specified checklist which was based on unpublished findings of a previous pilot survey of eighty neurologists in France. At the two follow-up visits, exacerbations since the previous visit were documented, as well as any adverse events occurring in the intervening period, as reported by the patient in response to undirected questions from the physician. The physician completed the Clinical Global Impression -Change questionnaire (CGI-C) [START_REF] Guy | Clinical Global Impressions, ECDEU Assessment Manual for Psychopharmacology-Revised[END_REF] at both follow-up visits.

At each visit, the patients was invited to complete a paper questionnaire which included two patient-reported outcome measures (PRO), the ACCEPT © questionnaire [START_REF] Lambert | Assessing patients' acceptance of their medication to reveal unmet needs: results from a large multi-diseases study using a patient online community[END_REF], which was completed at all visits, and the Clinical Global Impression -Change questionnaire (PGI-C) [START_REF] Guy | Clinical Global Impressions, ECDEU Assessment Manual for Psychopharmacology-Revised[END_REF], which was only completed at the two follow-up visits.

The CGI-C and PGI-C are identically constructed rating scales on which the clinician (CGI-C) or patient (PGI-C) is invited to rate how much the patient's illness has evolved since the previous assessment on a seven-point Likert scale ranging from 1 (very much improved) to 7 (very much worse). A rating of 4 indicates no change. ACCEPT © is a multidimensional 25item questionnaire, composed of six dimensions relating to specific treatment attributes (medication inconvenience, long-term treatment, regimen constraints, numerous medications, side-effects and effectiveness) and a general treatment acceptance dimension.

It is easily and rapidly completed by the patient and covers relevant and important aspects of the effectiveness and constraints of DMT therapy. The attribute-specific items are presented as multiple-choice questions with three response modalities ('Yes and I don't find this easy to accept'; 'Yes and I find this easy to accept' or 'No'). The general dimension consists of three questions ('Do you agree with the statement that your treatment has more advantages than disadvantages', 'Taking into account its advantages and disadvantages, does you treatments seem to be an acceptable solution for you?' and 'In the long-term, are you convinced that your treatment is worth taking?), each scored on a five-point Likert scale. Scores are linearly transformed to range from 0 to 100 for all multi-item dimensions with a higher score indicating greater acceptance. All questionnaires were used in their validated French translations.

Data analysis

Different study populations were defined. The Included Population included all patients enrolled, the Safety Population included all enrolled patients who had taken at least one dose of fingolimod, the Analysis Population consisted of those members of the Safety Population who fulfilled the eligibility criteria and who attended at least one follow up visit (at M6 or M12) and the Completer population consisted of those members of the Analysis Population for whom the CGI-C had been completed at the inclusion and at both follow-up study visits. The principal analyses were performed on the Analysis Population. The analysis of adverse events was performed in the Safety Population. Adverse events were classified by System-Organ Class and by Preferred Term using the coding specified in the Medical Dictionary for Regulatory Activities Version 20.0.

The presentation of the data is purely descriptive and no a priori hypotheses were tested. Continuous data are presented as mean values ± standard deviations (SD) or median values. Categorical variables are presented as frequency counts with percentages.

Categorical and continuous variables were compared between groups using the χ² test and Student's t-test respectively. Missing data were not replaced and were included in the denominator when calculating percentages.

All statistical analyses were performed using SAS software Version 9.4 (SAS Institute, Cary, United States).

Ethical considerations

The survey was conducted in accordance with relevant current French and international legislation and Good Epidemiological Practice guidelines. Since the patients' identity did not appear on the study questionnaires and patient management was not affected by participation in the study and all patient data were anonymised prior to entry into the study database, submission to an Institutional Review Board for ethical approval was not required. The physicians were required to give a letter of information to potentially eligible patients, who were given the opportunity to agree or refuse to participate in the survey. Refusal to participate had no impact on the patient's future care. The letter of information included a summary of the recommendations of the French Data Protection Authority (Commission Nationale de l'Informatique et des Libertés; CNIL) on the protection of personal data and the processing of the data from the survey.

Participating physicians

Of the 2564 neurologists invited to participate in the study, 192 agree to participate.

For the 322 neurologists who replied to the invitation but declined to participate, the principal reasons for non-participation were that they did not see patients with MS (31.1%), lack of time (30.2%) and that they do not participate in studies (11.2%). One hundred and thirty-two participating centres were opened of whom 71 recruited at least one patient.

Patients

Participating neurologists included 232 patients (Included Population), of whom 201 (86.6%) attended visit M12 and thus completed the study. The Safety Population included 225 patients and the Analysis Population 190 patients. The distribution of patients across the different study populations is illustrated in Fig. 1. Seven of the enrolled patients were not prescribed fingolimod at the inclusion visit and are thus excluded from the Safety and Analysis Populations. A further 30 patients presented at least one violation of the eligibility criteria (certain patients presented more than one violation). These principally involved receipt of more than one DMT for longer than six months before the switch to fingolimod (N = 18), a treatment duration for the previous DMT of <6 months (N = 12), switching to fingolimod >4 weeks prior to the inclusion visit (N = 8), a missing EDSS score at inclusion (N = 5) and no previous DMT treatment (N = 4). During the twelve-month follow-up period, thirty patients (13.7%) interrupted treatment with fingolimod. This was principally due to the occurrence of adverse events (nine patients), or to inadequate efficacy (ten patients). The mean duration of fingolimod treatment was 10.9 months (SD: 3.0; median: 11.8 months; range: 0.5 -17.9]. The characteristics of the Analysis Population are presented in Table 1. The mean time since diagnosis was six years. The majority of patients (89.6%) presented clinical or radiological signs of disease activity in the previous twelve months in spite of their DMT therapy. The DMTs that were most frequently switched were β-interferons (42.6%), glatiramer acetate (16.8%), dimethylfumarate (16.8%) and teriflunomide (13.2%).

Table 1

Sociodemographic and clinical characteristics of the study population.

Reasons for stopping previous treatment

The reasons given by the neurologist for switching treatment are presented in Table 2. Multiple reasons could be provided on the e-CRF and the response modalities were thus not mutually exclusive. The most frequent reason for switching observed in 84.7% of patients was clinical or radiological activity, or progression of clinical disability. Twenty patients had no clinical or radiological signs of disease activity in the year preceding inclusion. The second most frequent class of reasons, accounting for 30.5% of switches were related to the patients' preference, notably fatigue and a desire to change or lassitude with the previous treatment. Adverse events were cited for 18.4% of switches and the mode of administration for 12.1%.

Table 2

Reasons for switching treatment. Multiple reasons for switching were frequently given and no attempt was made to classify these as more or less important. This was most often the case when patient preferences were cited. For these 58 patients, disease worsening was also cited in 43 cases (74.1%) and adverse events in 19 cases (32.8%). On the other hand, for the 161 patients switching for disease worsening, multiple reasons were less frequently cited, with adverse events also being cited in 24 cases (14.9%), patient preferences being cited in 43 cases (26.7%).

Clinical evolution over the study period

During the follow-up period, 38 patients (20.0%) experienced at least one exacerbation, six patients experienced two relapses, four patients experienced three and two patients experienced four. The mean EDSS score did not change over the twelve-month study period (mean EDSS score at inclusion: 2.0 ± 1.3 versus 2.0 ± 1.5 at M12). For the 137 patients for whom the EDSS was evaluated at both inclusion and M12, the mean change in EDSS score was -0.1 ± 0.9, the score was identical on both occasions for 68 patients (49.6%), whereas it had increased in 29 patients (21.2%) and decreased in 40 patients (29.2%). For the 173 patients who attended both the inclusion and the M12 visit, 99 patients underwent an MRI examination and ongoing disease activity was observed in 27 cases (three with Gdenhancing T1 lesions only, fourteen with new T2 lesions only and ten with both types of lesion).

Clinical Global Impression -Change at M12

A completed CGI-C was available at M12 for 173 patients (91.1%). The modal response on the CGI-C was 'No change' (Response 4), accounting for 48.0% of patients overall (N = 83). Response 3 ('Minimally improved') accounted for 27.7% of patients and Response 5 ('Minimally Worse') for 11.6% of patients (Table 3). The other response modalities accounted for <10% of patients each. Overall, 67 patients (38.7%) were considered to be improved and 23 (13.3%) to have worsened. No obvious differences were observed in CGI-C ratings as a function of the reason for switching (Fig. 2). Nonetheless, compared to the other two groups, when patient preferences entered into the decision, the proportion of patients who were considered minimally improved was somewhat higher (37.7% versus 27.6% and 25.8%) and the proportion considered unchanged was somewhat lower (41.5% versus 49.7% and 51.6%).

Table 3

CGI-C and PGI-C ratings at 12 months, overall and by reason for switch

Patient Global Impression -Change at M12

The PGI-C was completed at M12 and returned by 145 patients (83.8% of the Completer Population. As with the CGI-C, 'No change' was the most frequent response modality (Table 4). However, more patients rated themselves improved on the PGI-C than were rated as improved by the physician. For the 132 patients (69.5%) for whom both the CGI-C and the PGI-C were available at M12, ratings were compared between the two measures, grouped into three categories (improved, no change and worsened). For the majority of these patients (N = 91; 62.8%), the two ratings were identical. However, of the 64 patients rated as 'no change' by the physicians on the CGI-C, 21 (32.8%) rated themselves as 'improved' and 10 (15.6%) as 'worsened'. The overall level of agreement was moderate (κ = 0.48 [95% CI: 0.35 -0.60]).

Table 4

Correlation between ratings on the CGI-C and PGI-C at M12. Data are presented for the 132 patients for whom both questionnaires were available.

ACCEPT © questionnaire

The mean dimension scores on the ACCEPT © scale at the inclusion visit, reflecting acceptability of the previous DMT ranged from 36.7 for the effectiveness dimension to 72.2 for medication inconvenience (Table 5). The mean general acceptability score was 42.7 [95%CI: 34.5 -50.9]. At the M12 visit, mean dimension scores had risen for all dimensions with the exception of long-term treatment and numerous medications; they now ranged from 63.4 for effectiveness to 96.8 for medication inconvenience. The mean general acceptability score at M12 was 64.6 [95%CI: 57.6 -71.6]. *This single-item dimension is scored on a scale of 0 -3, rather than 0 -100 for the remaining dimensions.

Safety

During the 12-month follow-up period, 238 adverse events were reported for 130 patients in the Safety Population (57.8%). The most frequently-reported adverse events were lymphopenia (75 events in 66 patients; 29.3%), increased γ-glutamyl-transferase (8 events in 8 patients; 3.6%), hepatocellular injury (8 events in 8 patients; 3.6%) and headaches (7 events in 7 patients; 3.1%). No other individual adverse event was reported in >3% of patients. Twenty patients (8.9%) presented a total of 32 serious adverse events, of which 23 events in fourteen patients were considered treatment-related. These are listed in Table 6. The most frequent of these treatment-related serious adverse events were lymphopenia, bradycardia and hepatocellular injury (three patients each) and increased γglutamyl-transferase (2 patients).

3 3 2 1 1 1 1 1 1 1 1 3 (1.3%) 3 (1.3%) 3 (1.3%) 2 (0.9%) 1 (0.4%) 1 (0.4%) 1 (0.4%) 1 (0.4%) 1 (0.4%) 1 (0.4%) 1 (0.4%) 1 (0.4%)

Discussion

The goal of this study was to document patient outcome following switching from a first DMT to fingolimod according to the reason for switching. Although multiple reasons were given for switching, lack of efficacy of the first-line treatment contributed to the decision in 85% of cases. This is not particularly surprising since it corresponds to the specific approved indication for fingolimod, and is a treatment strategy for which there is strong evidence for benefit [START_REF] Hansen | Adherence to Disease Modifying Drugs among Patients with Multiple Sclerosis in Germany: A Retrospective Cohort Study[END_REF][START_REF] Lebrun-Frenay | Five-year outcome in the copaxone observatory: a nationwide cohort of patients with multiple sclerosis starting treatment with glatiramer acetate in France[END_REF][START_REF] Miller | Switching or Discontinuing Disease-Modifying Therapies for Multiple Sclerosis[END_REF]. At inclusion, 72% of patients evaluated on MRI had visible disease activity and 67% had experienced at least one exacerbation in the previous year, in spite of taking a first-line DMT.

The data suggest that around twenty percent of patients are being switched treatment on the basis of radiological evidence for disease activity only, in keeping with a treatment goal of 'no evidence for disease activity' (NEDA) [START_REF] Havrdova | Freedom from disease activity in multiple sclerosis[END_REF]. Only twenty patients with NEDA at inclusion were switched to fingolimod, which indicates that fingolimod is not being widely prescribed outside of its approved indication of 'patients with highly active RRMS despite a full and adequate course of at least one DMT'. Since this study only included patients who were switched to fingolimod, it is not possible to know the proportion of patients who had evidence for disease activity that were not switched.

Only a minority of patients were switched due to the occurrence of adverse events, and this was rarely the only reason for the switch. This may reflect the tendency of neurologists to switch a patient whose first-line treatment is poorly tolerated to an alternative first-line DMT, as has been described in the Italian cohort of Saccà et al. [START_REF] Sacca | Determinants[END_REF]. Another recent Italian cohort of patients who switched between DMTs [START_REF] Patti | Clinical and patient determinants of changing therapy in relapsing-remitting multiple sclerosis (SWITCH study)[END_REF] reported that the principal reason for switching was inadequate efficacy and that less than ten percent of patients were switched principally due to the preference of the patient. The reasons for switching identified in the present study are consistent with this hierarchy.

In thirty percent of cases, patient preferences contributed to the decision to switch, although this was not the only reason for most of these. Lassitude with taking the current DMT and a desire to change treatment were frequently cited, together with symptoms of MS such as fatigue. On the other hand, the mode of administration (route or frequency) contributed to the decision to switch in only 12% of cases. This may indicate that neurologists and patients are prepared to continue with injectable therapy as long as disease activity is under control.

Twelve months after the switch to fingolimod, 39% of patients were considered by the physician to be improved and 48% were considered unchanged. It should be noted that the CGI was originally developed for use in psychiatric disorders which present with episodic fluctuating symptoms where the treatment goal is symptom resolution, and not for neurodegenerative diseases such as multiple sclerosis, where the goal is to minimise disease activity and to prevent worsening of the clinical state. This may explain the high proportion of patients rated as unchanged, whereas, objectively, control of disease activity in these highly active patients was improved following the switch, with 80% of patents being exacerbation-free at M12, and 73% of patients who underwent a post-switch MRI having no signs of new disease activity. It is also possible, at least in patients switched for disease worsening, that 'regression to the mean' of disease activity following an active phase at the time of the switch may account for some of the improvement observed. Without complete information of patient history, the extent of such regression to the mean cannot be determined.

There were no major differences in the CGI-C ratings according to the reason of the switch, with the exception of a somewhat higher proportion of patients who improved in the group of patients in whom patient preferences contributed to the reasons for switch. It may be hypothesised that these patients, who were dissatisfied with their previous treatment, may be more adherent to fingolimod than to their first-line DMT, and therefore benefit more from it.

Patients were also asked to rate their impression of change following switch with the PGI-C. Although the overall correlation between physician and patient ratings was respectable, patients less frequently rated themselves as unchanged than did their physicians, with around half of patients considering themselves improved.

The study provided an opportunity to evaluate treatment acceptability using the ACCEPT © questionnaire. Although patients with MS were evaluated during the validation of this questionnaire [START_REF] Lambert | Assessing patients' acceptance of their medication to reveal unmet needs: results from a large multi-diseases study using a patient online community[END_REF], the ACCEPT © has not yet been used in a dedicated study in MS, nor to evaluate changes in treatment acceptability following treatment switches. An increase in around twenty points in acceptability scores was observed following switch for all dimensions with the exception of the 'long-term treatment' and 'numerous medications' items. Low scores prior to the switch are to be anticipated, since the previous treatment was considered for whatever reason inadequate. The creators of the ACCEPT © questionnaire did not provide any guidance on how the scores should be interpreted, nor on what would constitute a clinically-significant change. In their description of the questionnaire, mean general scores in different chronic diseases ranged from 33.7 for fibromyalgia to 61.2 for type 1 diabetes, with a mean score for MS, in an unselected population, of 48.6. In the present study, the mean general score at inclusion was 42.7, somewhat lower than the value observed in the validation study, but increased to 64.6 twelve months after switching to fingolimod, which is higher than the mean scores for all the diseases evaluated in the validation study. This value is close to the highest mean value reported in the validation study. This difference between scores at inclusion and twelve months was of similar magnitude as the difference in treatment acceptability observed in the validation study between, for example, treatments for breast cancer (mean score ~38) and those for hypertension (mean score ~55). Moreover, the mean medication inconvenience score in our patients, most of whom switched from an injectable to an oral treatment, rose to 96.8, which is close to the maximum possible score and to the score in the highest rated disease (hypertension; mean score: 96.3) in the validation study. The increase in medication inconvenience score was around 25 points, which is a larger difference than the difference in mean scores between sixteen of the nineteen diseases evaluated in the validation study. These indirect comparisons would suggest that the changes in ACCEPT scores observed following a switch to fingolimod are clinically relevant.

With respect to safety, the adverse event profile was similar to that described in the Phase III randomised clinical trials [START_REF] Cohen | Oral fingolimod or intramuscular interferon for relapsing multiple sclerosis[END_REF][START_REF] Kappos | A placebo-controlled trial of oral fingolimod in relapsing multiple sclerosis[END_REF]. No unanticipated safety issue or new signal of potential risk was identified.

The study has several limitations, which are principally related to the observational nature of the study. Firstly, inclusion bias cannot be excluded. Neurologists were expected to include consecutively the first four patients fulfilling the eligibility criteria, but there was no requirement to complete a patient registry of all consulting patients, so there is no way of ensuring that this happened nor whether patients who were included were representative of all eligible patients. However, the demographic and clinical features of the included patients are similar to those of a large registry of patients treated with fingolimod in France enrolled over a similar period [START_REF] Castelnovo | Long-term effectiveness, safety and tolerability with fingolimod treatment in patients with multiple sclerosis in real-world treatment settings in France: final results of the VIRGILE study[END_REF]. Likewise, it is not possible to be sure that the decision to switch patients to fingolimod was made independently of study participation. The second limitation is the relatively large amount of missing data, particularly for the questionnaire. At the twelve-month visit, the CGI-C was not completed for 9% of patients, the PGI-C for 27% and the ACCEPT © for 46%.

In conclusion, most patients who switch from a first DMT to fingolimod do so for reasons of persistent disease activity during the initial treatment, although patient preferences enter into the decision in around one third of cases. The switch to fingolimod is followed by a reduction in disease activity, perceived improvement in the clinical state of the patient and improved acceptability of treatment. of therapy switch in multiple sclerosis treatment-naive patients: A real-life study. Multiple sclerosis (Houndmills, Basingstoke, England) 25(9), 1263-1272.

Figure 1

 1 Figure 1 Patient flow diagram.

  Age (mean ± SD; years) 39.5 ± 10.2Patients never treated with fingolimod: N = 7No follow-up visit: N = 5 Eligibility violation: N

  Figure 2Clinical Global Impression -Change at 12 months.

Table 5

 5 ACCEPT © questionnaire scores.

	Dimension	Inclusion visit	Visit M12
	Missing data	N = 65	N = 87
	Medication inconvenience	72.2 [65.0 -79.4]	96.8 [95.1 -98.6]
	Long-term treatment	63.5 [57.8 -69.2]	67.6 [64.8 -70.4]
	Regime Constraints	68.6 [61.9 -75.3]	85.5 [81.7 -89.3]
	Numerous medications*	2.7 [2.55 -2.85]	2.8 [2.7 -2.9]
	Side-effects	67.7 [60.5 -74.9]	85.4 [81.4 -89.4]
	Effectiveness	36.7 [28.0 -45.4]	63.4 [55.8 -71.0]
	General	42.7 [34.5 -50.9]	64.6 [57.6 -71.6]

Table 6

 6 Adverse events

	System-organ class and Preferred Term	Number of events	Number of patients (%)
	Any adverse event	238	130 (57.8%)
	Any serious adverse even	32	20 (8.9%)
	Any adverse event leading to treatment discontinuation	41	34 (15.1%)

Funding

The study was funded by Novartis.

Data availability

Anonymised data will be made available to qualified external researchers, with requests reviewed and approved by an independent review panel on the basis of scientific merit.

Competing interests

AT has received consultancy or speaker's fees from Medday Pharmaceuticals, Biogen, Novartis, Hikma and Roche.

CP has received has received compensation as a consultant, advisory board member, or speaker, for Roche, Novartis, Biogen, Teva, Sanofi Aventis, Genzyme PT has received consulting and lecturing fees and travel grants from Biogen, Genzyme, Novartis, Merck Serono, Teva Pharma, Sanofi Aventis, and Roche.

CM has participated in scientific advisory boards for Novartis, Teva, Biogen, Sanofi, Genzyme, Bayer, Merck, Eisai, and Lilly, and spoken at or chaired scientific meetings for Novartis,Teva, Biogen, Merck, Genzyme, Bayer, BMS, Pfizer and GSK. IC, BD, FL, MM and KR are employee of Novartis, a pharmaceutical company that developed and markets fingolimod.

Author's contributions


Elodie Lamy

Ayoub Mahjoubi

Hélène Salvator

Françoise Lambinet

Philippe Devillier

Stanislas Grassin-Delyle
email: stanislas.grassin-delyle@uvsq.fr

Liquid chromatography-mass spectrometry method for the quantification of corticosteroids, β2-adrenoreceptor agonists and anticholinergics in human hair

Keywords: corticosteroids, β2-adrenoreceptor agonists, anticholinergics, liquid chromatographymass spectrometry, hair, therapeutic drug monitoring

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

INTRODUCTION

Asthma affects 3-10% of the European population and is a heterogeneous disease in its clinical manifestations and also in its response to treatment. Conventional pharmacological treatments are classified as relievers (bronchodilators such as short-acting β2-adrenoreceptor agonists or anticholinergics) or controllers (inhaled corticosteroids, long-acting β2-adrenoreceptor agonists, leukotriene modifiers, oral corticosteroids and anti-IgE treatments). Patients with severe asthma are defined as those requiring treatment with high-dose inhaled (or systemic) corticosteroids in combination with a second controller medication [START_REF] Ray | Current concepts of severe asthma[END_REF]. Approximately 50% of adults and children on long-term therapy for asthma and even less in chronic obstructive pulmonary disease (COPD) patients fail to take medication as directed at least part of the time. In difficult-to-treat asthma, up to 75% patients has insufficient adherence [2][START_REF]Global Initiative for Asthma. Global strategy for asthma managment and prevention[END_REF][START_REF] Cochrane | Inhaled corticosteroids for asthma therapy: patient compliance, devices, and inhalation technique[END_REF][START_REF] Gamble | The prevalence of nonadherence in difficult asthma[END_REF]. In addition, only 46 to 59% of patients have an efficient inhalation technique, resulting in insufficient drug exposure and participating to poor asthma control [START_REF] Cochrane | Inhaled corticosteroids for asthma therapy: patient compliance, devices, and inhalation technique[END_REF][START_REF] Al-Jahdali | Improper inhaler technique is associated with poor asthma control and frequent emergency department visits[END_REF][START_REF] Pessoa | Incorrect use of inhalation devices among patients with bronchial asthma. A hospital-based cross-sectional study in Rio de Janeiro, Brazil[END_REF]. In clinical practice, the effectiveness of inhaled medications on an individual patient can be hopelessly confounded by a lack of objective accurate measure of both intentional and unintentional nonadherence. This lack of information is potentially leading to overprescription or unnecessary additional treatment, particularly the expensive add-on type 2 biologic therapy in severe asthma. Monitoring the adherence/exposure to those therapies therefore remains an unmet need and new techniques are needed to assess and improve treatment long-term adherence and efficacy. Traditional therapeutic drug monitoring with the measurement of plasma drug concentration has no benefits for asthma and COPD drugs since plasma concentration is a snapshot of the body drug load at a single timepoint and does not allow to assess the chronic exposure, in addition to analytical challenges related to the very low plasma concentrations observed after lowdose inhaled administration. Since drugs present in the bloodstream also reach and stay in the forming hair where their concentrations can be measured and interpreted as an overall chronic exposure, hair analysis could become a valuable tool in the monitoring of inhaled asthma and 4 COPD treatments. Recent reports suggest an interest for hair therapeutic drug monitoring in the clinics in other areas: for HIV infection, there is a strong relationship between hair antiviral concentration and virologic suppression and hair drug concentration is correlated to the dose [START_REF] Baxi | Nevirapine Concentration in Hair Samples Is a Strong Predictor of Virologic Suppression in a Prospective Cohort of HIV-Infected Patients[END_REF][START_REF] Liu | Strong relationship between oral dose and tenofovir hair levels in a randomized trial: hair as a potential adherence measure for pre-exposure prophylaxis (PrEP)[END_REF][START_REF] Gandhi | Atazanavir concentration in hair is the strongest predictor of outcomes on antiretroviral therapy[END_REF][START_REF] Van Zyl | Low lopinavir plasma or hair concentrations explain second-line protease inhibitor failures in a resource-limited setting[END_REF]; the hair imatinib concentration in patients with chronic myeloid leukaemia was also lower in patients with therapeutic failure than in those with optimal response [START_REF] Capron | First report of imatinib measurement in hair: Method development and preliminary evaluation of the relation between hair and plasma concentrations with therapeutic response in chronic myeloid leukemia[END_REF] and an interest in hair concentration monitoring was also suggested to assess compliance in epileptic patients [START_REF] Williams | Relation between dosage of carbamazepine and concentration in hair and plasma samples from a compliant inpatient epileptic population[END_REF]. The incorporation of corticosteroid into human hair has already been documented with prednisone and beclometasone in forensic or clinical cases; with endogenous hormones as well as betametasone, methylprednisolone and triamcinolone in doping controls, or with inhaled fluticasone propionate [START_REF] Noppe | LC-MS/MS-based method for long-term steroid profiling in human scalp hair[END_REF][START_REF] Cirimele | Identification of ten corticosteroids in human hair by liquid chromatography-ionspray mass spectrometry[END_REF][START_REF] Cirimele | First identification of prednisone in human hair by liquid chromatography-ionspray mass spectrometry[END_REF][START_REF] Bevalot | Analysis of corticosteroids in hair by liquid chromatography-electrospray ionization mass spectrometry[END_REF][START_REF] Gaillard | Compared interest between hair analysis and urinalysis in doping controls. Results for amphetamines, corticosteroids and anabolic steroids in racing cyclists[END_REF][START_REF] Hassall | Hair analysis to monitor adherence to prescribed chronic inhaler drug therapy in patients with asthma or COPD[END_REF]. For β2-adrenoreceptor agonists, hair concentrations were reported for salbutamol, salmeterol, formoterol and vilanterol in patients with asthma or in asthma-related death [START_REF] Hassall | Hair analysis to monitor adherence to prescribed chronic inhaler drug therapy in patients with asthma or COPD[END_REF][START_REF] Kintz | Doping control for beta-adrenergic compounds through hair analysis[END_REF].

With respect to anticholinergics, there were reports with the detection of tiotropium and umeclidinium, each in one patient, but with concentrations below the lower limit of quantification [START_REF] Hassall | Hair analysis to monitor adherence to prescribed chronic inhaler drug therapy in patients with asthma or COPD[END_REF]. Although there were numerous liquid chromatography -mass spectrometry methods already published for the quantification of asthma or COPD drugs in human serum, plasma, urine or sputum, methods for hair analysis are much scarcer and either concern endogenous compounds or anabolic steroids [START_REF] Noppe | LC-MS/MS-based method for long-term steroid profiling in human scalp hair[END_REF][START_REF] Bevalot | Analysis of corticosteroids in hair by liquid chromatography-electrospray ionization mass spectrometry[END_REF][START_REF] Gaillard | Compared interest between hair analysis and urinalysis in doping controls. Results for amphetamines, corticosteroids and anabolic steroids in racing cyclists[END_REF], oral or intravenous corticosteroids [START_REF] Cirimele | Identification of ten corticosteroids in human hair by liquid chromatography-ionspray mass spectrometry[END_REF][START_REF] Cirimele | First identification of prednisone in human hair by liquid chromatography-ionspray mass spectrometry[END_REF][START_REF] Gaillard | Compared interest between hair analysis and urinalysis in doping controls. Results for amphetamines, corticosteroids and anabolic steroids in racing cyclists[END_REF] or inhaled drugs with no method validation [START_REF] Cirimele | Identification of ten corticosteroids in human hair by liquid chromatography-ionspray mass spectrometry[END_REF][START_REF] Hassall | Hair analysis to monitor adherence to prescribed chronic inhaler drug therapy in patients with asthma or COPD[END_REF]. The objectives of the present study were therefore to develop and validate according to regulatory agencies guidelines a highly sensitive liquid chromatographytandem mass spectrometry (LC-MS/MS) method for the simultaneous quantitation of inhaled or oral corticosteroids, β2-adrenoreceptor agonists and anticholinergics in human hair. Budesonide metabolites were also included in the method since budesonide is transformed to 16αhydroxyprednisolone and 6ß-hydroxybudesonide by liver metabolism and also forms lipophilic intracellular fatty acid esters (oleate, palmitate, linoleate, palmitoleate) in airways [START_REF] Miller-Larsson | Reversible fatty acid conjugation of budesonide. Novel mechanism for prolonged retention of topically applied steroid in airway tissue[END_REF][START_REF] Van Den Brink | Evidence of the in vivo esterification of budesonide in human airways[END_REF], which metabolites may have hair incorporation profiles different from their parent drug. 6 working solution were added. Ten microliters of the appropriate working solution were added for calibration standards and control samples (concentrations of each calibration standard are shown in supplementary Table 1). One millilitre of the extraction solvent (methanol/chloroform -70/30, v/v) was then added; samples were put in ultrasonic bath for 30 minutes then pulverized in a ball mill for 10 min at 50 Hz with a TissueLyser LT (Qiagen, Hilden, Germany). Samples were centrifuged at 13000 rpm for 10 minutes and 800 µL of the supernatant layer were dispensed into conic 1.5 mL tube and evaporated under vacuum at 43°C for 60 minutes. Fifty microliters of mobile phase (methanol/20 mM pH 9.2 ammonium carbonate buffer -5/95, v/v) were finally added. Samples were vortex mixed for 30 seconds and transferred into injection vials. Each vial was centrifuged at 3000 rpm for 10 minutes at 4°C before LC-MS/MS analysis.

LC-MS/MS system and analysis procedure

Method optimization was performed for the analytical conditions such as chromatographic column and mobile phase composition, to setup the most sensitive and fastest method. The final chromatography and mass spectrometry parameters were as follows. Chromatography was performed with an UltiMate 3000 Quaternary Rapid Separation Pump (Thermo Scientific Dionex, Villebon, France), using a 1.7 µm Kinetex evo C18 column (150 x 2.1 mm i.d.) (Phenomenex, California, United States) maintained at 40°C in an UltiMate 3000 Rapid Separation Thermostated Column Compartment (Thermo Scientific Dionex). Ten microliters (10 µL) were injected in the chromatographic system with an UltiMate 3000 Rapid Separation Autosampler (Thermo Scientific Dionex) with a sample stack maintained at +8°C. The following mobile phase was used: methanol and 20 mM pH 9.2 ammonium carbonate buffer. The gradient starting composition was 5% methanol, held for 2 min and reaching 100% at 10 min. Then a clean-up step at 100% methanol was run for 5 minutes. Finally, initial conditions were restored and kept for 6 minutes. The flow rate was ionization source and detected with a triple quadrupole Quantiva mass spectrometer (Thermofisher) using multiple reaction monitoring (MRM). Nitrogen (N2-45 nitrogen generator, VWR International, Fontenay sous bois, France) was employed as sheath and auxiliary gas and argon (Messer, Puteaux, France) was used as a collision gas. The electrospray source was set in the positive ionization mode. Instrument parameters were optimized using a continuous 20 µL/min infusion of each compound (1 mg/L in mobile phase). The source parameters were as follows: ion spray voltage: 4100V; vaporizer temperature: 115°C; sheath and aux gas pressure: 31 and 11 arbitrary units, respectively; ion transfer tube temperature: 350°C; collision gas pressure: 1.5 mTor; Q1 and Q3 resolution: 0.7 FWHM and cycle time: 1s. The ions, transitions and RF lens values selected for the analysis of each compound are detailed in supplementary Table 2. Data acquisition and processing was performed using Chromeleon v 6.80 (Thermo Scientific Dionex) and Xcalibur v3.0.63 (Thermofisher).

Method validation procedure

The developed method was validated according to the European Medicines Agency guideline (EMEA/CHMP/EWP/192217/2009 Rev.1 Corr.2 -https://www.ema.europa.eu/en/bioanalyticalmethod-validation) and Food and Drug Administration guideline (Bioanalytical Method Validation -Guidance for Industry, May 2018 -https://www.fda.gov/regulatory-information/search-fdaguidance-documents/bioanalytical-method-validation-guidance-industry) for the following parameters: selectivity, carry-over, lower limit of quantification, calibration curve, accuracy, precision, matrix effect and stability.

Selectivity and carry over

Blank hair samples from six different donors were used. According to the Fischer-Saller scale, hair colours were light brown (2), dark brown (1) and red [START_REF] Ray | Current concepts of severe asthma[END_REF]. Two donors also had grey hair, which is not included in the Fischer-Saller scale. First, 6 blank samples (without compound nor IS) were processed and compared to samples from the same donors spiked with the internal standards and with each compound at the lower limit of quantification. To evaluate the carry-over, a blank sample was analysed immediately after the highest CS in each run. Assay selectivity and carry-over were defined by a mean area of less than 20% for each compound and less than 5% for the IS in blank samples injected before the lower CS (for selectivity) or just after the highest CS (for carry-over) in comparison to the signal obtained for the LLOQ.

Lower limit of quantification

The LLOQ was defined as the lowest concentration measurable with a signal superior or equal to 5 times the signal of a blank sample, an accuracy between 80% and 120% and a precision with a coefficient of variation of ± 20% or less over six measurements. The LLOQ was used as the lowest calibration standard.

Calibration curve

Calibration curves included a blank sample, a zero sample, and seven CS was made each day.

Quantitation was achieved by plotting the peak area ratios of each compound to the internal standard versus concentration followed by regression analysis, excluding blank and zero samples.

Back-calculated concentrations of at least six CS out of the seven (including the LLOQ) had to be within 85-115% of the nominal concentrations, except for the LLOQ for which the range was 80-120%.

Accuracy and precision

Accuracy (measured value/nominal value) and precision (coefficient of variation (CV)) were determined for the three QC levels and for the LLOQ. Six replicates of each QC level were processed the same day for the intraday assay, whereas each QC level was processed six times three different days over a period of two weeks for the interday assay. The data was analysed using analysis of variance. For QCs, accuracy had to be within 85-115% of the nominal values and precision of ±15%, while values between 80-120% and ±20% were accepted for accuracy and precision of the LLOQ, respectively.

Extraction recovery, matrix effect and overall recovery

Human hair samples from six different donors were used to investigate extraction recovery, matrix effect and overall recovery. Three procedures (A, B and C) were performed on each blank hair sample or matrix-free samples spiked at two QC concentrations (corresponding to the lowest and highest quality control levels) to evaluate these parameters: (A) each compound and IS were spiked in hair samples and the complete sample preparation procedure was carried through; (B) blank matrix samples were prepared using the complete sample preparation procedure, compounds and IS were spiked after the evaporation step and the preparation procedure finished; (C) compounds and IS were spiked in mobile phase and directly injected.

Extraction recovery was defined by the ratio between the peak area obtained in procedure A and the peak area obtained in procedure B. Matrix effect was assessed by calculating the matrix factor, defined as the ratio between the peak area obtained in procedure B and the peak area obtained in procedure C. The IS-normalised matrix factor was calculated by dividing the matrix factor of each compound by the matrix factor of appropriate IS. The CV of this IS-normalised matrix factor had to be less than 15%. Overall recovery was defined as the ratio between the peak area obtained in procedure A and the peak area obtained in procedure C. 10

Stability

The stability of processed samples was studied after storage for 24 hours at +8°C in the autosampler.

Analysis of patient samples

To confirm the suitability of the method for the analysis of clinical samples, it was applied to measure corticosteroids and bronchodilator concentration in hair samples from 6 adult patients participating to a clinical trial (CorticHair, NCT03691961). Those patients had controlled/partly controlled asthma and were receiving a maintenance treatment with budesonide/formoterol at a stable dose (≥ 400 μg/day of budesonide) for at least 2 months. The use of inhaled short acting β2adrenergic receptor agonists was allowed during the study and systemic corticosteroids could be administered if required. Sampling was performed as recommended in the Society of Hair Testing (SoHT) guidelines for drug testing in hair [START_REF] Cooper | Society of Hair Testing guidelines for drug testing in hair[END_REF]: a strand of hair from the vertex posterior region of the scalp about the width of a thin pencil and with a length equal to or greater than 3 cm was fastened with a string to distinguish the proximal and distal extremities. The hair was cut with clean scissors as close to the scalp as possible, aligned with the root end of the sample clearly identified, secured with an aluminium foil and stored until analysis into an envelope in a dry, dark environment at room temperature, away from direct sunlight.

RESULTS AND DISCUSSION

Sample preparation

Washing optimization

Several solvents for hair washing were compared with the objective of maximizing the removal from drugs which may originate from contamination and be deposited at the external surface of the hair and to minimize the extraction of compounds integrated from the bloodstream to the inside of the hair. Therefore, this optimization step was performed with samples from both treated patients and spiked hair samples, using budesonide as a representative compound. The comparison between dichloromethane, MTBE and hot water resulted in the lowest extraction from incorporated drug and the highest recovery of spiked drug when hot water was used. In order to ensure that the use of hot water for hair washing would not affect the stability of the molecules studied, a triplicate analysis of a solution of all the molecules at the concentration corresponding to the 5 th calibration standard was immersed for 2 minutes in hot water before analysis and compared to the same solution kept at room temperature. The area ratio of the chromatographic peaks with and without heating was between 1.02 and 1.08 for all the molecules, indicating that the temperature did not cause any significant degradation.

Extraction method optimization

First, the effect of sample pulverisation was assessed by extracting spiked cut hair samples with or without previous pulverisation. The pulverisation step allowed to increase the peak area of most compounds up to 1.8-fold and was included to the sample extraction procedure.

Then, different proportions of the extraction solvent (methanol/chloroform) were compared on hair spiked with all compounds with the ratios 100/0, 70/30, 50/50, 20/80 and 0/100 and peak areas were compared to mobile phase spiked with the same quantities. At this step, the proportions 100/0 and 12 70/30 provided similar, high and reproducible recovery. Samples from patients treated with budesonide/formoterol were used in a last step to compare the recovery with native samples and allowed to conclude that the greatest recovery was observed for the 70/30 ratio.

LC-MS/MS analysis

During chromatographic method development, methanol or acetonitrile were compared as organic modifiers and 20 mM pH 9.2 ammonium carbonate buffer or 0.1% formic acid as aqueous solvents.

Two different analytical columns, Acquity BEH C18 1.7 µm 100 x 2.1 mm i.d. (Waters, Guyancourt, France) and Kinetex Evo C18 1.7 µm 150 x 2.1 mm i.d. (Phenomenex, Le Pecq, France) were also compared. With respect to the organic modifier, intensities and peak area were greater when methanol was used. For the aqueous solvent, a 2-to 3-fold increase in signal intensity was observed with the ammonium carbonate buffer. The Kinetex Evo C18 column provided the better molecule retention and was therefore retained as the analytical column.

With respect to compound detection, multiple reaction monitoring mass spectrometry was used.

Source parameters (ion spray voltage, vaporizer temperature, sheath and aux gas pressure, ion transfer tube temperature) were first optimized individually for each molecule, then the best compromise was retained and confirmed to be suitable for all compounds. These final analytical conditions were finally validated on the whole set of compounds and representative chromatograms are shown in Supplementary Figures 1 and2 for molecules and their internal standards, respectively.

Since ipratropium and tiotropium are quaternary ammoniums, already carrying a positive charge in the physiological milieu, the precursor ions for the detection of these compounds in the positive ionization mode were therefore [M] + ions, i.e. m/z = 332.4 and 392.1, respectively. For the same reasons, the precursor ion for tiotropium-d3 was m/z = 395.2. On the other hand, all other compounds are bases with secondary amines, hydroxyl groups or both. Consequently, the precursor ions observed in the positive ionization mode were the protonated [M+H] + adducts in each case.

The most intense MRM transitions were used for the quantification of each compound and IS, whereas two other transitions were used for confirmation for all compounds. One or two confirmation transitions were used for internal standards (supplementary Table 2).

For the quantification, budesonide-d8 was used as an internal standard for the measurement of budesonide, budesonide oleate, budesonide palmitate, 6β-hydroxy budesonide, beclomethasone, fluticasone and prednisolone; formoterol-d6 was used for the measurement of formoterol, ipratropium and salmeterol; salbutamol-d9 was used for the measurement of salbutamol and terbutaline; tiotropium-d3 was used for the measurement of tiotropium.

Method validation

Selectivity and carry over

The mean ratio between peak areas in samples spiked at the LLOQ and in blank samples was between 8 and 2600 for all compounds (n = 6) and 4715, 14127, 86242 and 1868 for budesonide-d8, formoterol-d6, tiotropium-d3 and salbutamol-d9, respectively (n = 6), demonstrating acceptable selectivity for all molecules. With respect to carry-over, the peak area in blank samples injected after the ULOQ fulfilled the acceptance criteria for budesonide, budesonide oleate, budesonide palmitate, 6β-hydroxy budesonide, beclomethasone, fluticasone, prednisolone, salbutamol, terbutaline, ipratropium and tiotropium. For formoterol and salmeterol, the peak area in blank samples injected after the ULOQ were up to 2-fold the peak area observed at the LLOQ, suggesting that carry over may occur. When the injection of 2 supplemental blank mobile phase samples was added to the procedure after the injection of the highest calibration standard, the peak area in the last washing sample was in accordance with the validation criteria for all compounds. Therefore, in case of concentrations of formoterol or salmeterol in patient samples in the highest range of the calibration curve, care has to be taken to inject washing samples to avoid carry-over. For this 14 method, our procedure systematically includes the injection of a blank mobile phase sample before each new patient to ensure the absence of carry-over from previous samples.

Limit of quantification

The LLOQ for all compounds ranged from 1 to 25 pg/mg, except for 6β-hydroxy budesonide (150 pg/mg). At the LLOQ, a signal superior to 5 times the signal of a blank sample was obtained for each compound. Accuracy and precision results are available in Table 1 and comply with the validation criteria with intra-and inter-day CV lower than 9 and 20%, respectively, and accuracies in the range 88.8-117.1%. The developed method is also highly sensitive with LLOQ for most compounds in the range 1-5 pg/mg. Previously reported LLOQ values were 100 pg/mg for 10 corticosteroids [START_REF] Cirimele | Identification of ten corticosteroids in human hair by liquid chromatography-ionspray mass spectrometry[END_REF][START_REF] Cirimele | First identification of prednisone in human hair by liquid chromatography-ionspray mass spectrometry[END_REF], 160 pg/mg for fluticasone furoate, 80 pg/mg for fluticasone propionate, 0.8 pg/mg for formoterol, 4 pg/mg for salmeterol and 0.4 pg/mg for tiotropium [START_REF] Hassall | Hair analysis to monitor adherence to prescribed chronic inhaler drug therapy in patients with asthma or COPD[END_REF]but the LLOQ definition was different from established guidelines or validation not always reported [START_REF] Cirimele | Identification of ten corticosteroids in human hair by liquid chromatography-ionspray mass spectrometry[END_REF][START_REF] Cirimele | First identification of prednisone in human hair by liquid chromatography-ionspray mass spectrometry[END_REF][START_REF] Hassall | Hair analysis to monitor adherence to prescribed chronic inhaler drug therapy in patients with asthma or COPD[END_REF].

Calibration curve

The calibration range was defined by a seven-point calibration curve. The visual inspection of the graphs plotting the peak area ratios of each compounds to the internal standard versus concentration suggested the linearity of the response, justifying the choice of an ordinary least square model to fit the data. Since the pattern of residuals plotted as a function of the concentration was heterogeneous, demonstrating heteroscedasticity, a weighted least square regression model was applied with a 1/x weighing factor [START_REF] Hubert | The SFSTP guide on the validation of chromatographic methods for drug bioanalysis: from the Washington Conference to the laboratory[END_REF]. Mean fitted equation obtained from six curves and mean least squares linear regression correlation coefficient (r²) are shown in supplementary Table 3. Back-calculated concentrations were in the range of ± 20% for the LLOQ and ± 15% for all other calibration standards.

Accuracy and precision

Intraday and interday precision and accuracies of the QC samples at the 3 concentration levels are summarized in Table 2 and are all within the range of accepted criteria. The developed method is thus highly precise and accurate in a wide range of concentrations. However, since certified reference materials do not exist for hair drug analysis with these classes of compounds, the accuracy measured from authentic hair samples may be different. This method may be useful to compare drug concentrations in different hair segments from the same individual, allowing to assess the month-by-month drug exposure.

Extraction recovery, matrix effect and overall recovery

The results are summarized in supplementary Table 4. Extraction recovery was in the range 30.4-111.7% for the two QC levels, allowing accurate and precise quantification in a large range of concentrations, as described above. The lowest extraction recoveries were observed for salbutamol and terbutaline, which can be explained by their hydrophilic properties with XLog P of 0.3 and 0.9, respectively, while other compounds have XLogP between 1.6 and 3.9. This highest value is for salmeterol, which also had the highest extraction recovery (101.7-111.7%), showing a direct association between the compound hydrophilicity and the extraction recovery. Tiotropium and ipratropium are also quaternary ammoniums with a positive charge, which may also explain their extraction recovery. The matrix factor was in the range 86.0-107.7% for formoterol, beclomethasone, prednisolone, 6β-hydroxy budesonide and ipratropium, suggesting that matrix components do not affect the signal for those compounds. For budesonide, fluticasone, salmeterol, budesonide oleate and budesonide palmitate, the matrix factor was in the interval 29.3-59.9%, meaning that a decrease in signal intensity related to matrix components may occur. On the other hand, the signal was modestly increased for salbutamol and terbutaline, with matrix factors between 131.8 and 152.6% whereas a huge and unexpected increase in signal intensity was observed for tiotropium, with matrix factors between 4456 and 79410% for all six donors for the 2 QC levels.

For all compounds except tiotropium and tiotropium-d3, the coefficients of variation of the matrix factor was between 9.6 and 35%. Interestingly, the internal standard-normalized matrix factor was between 96.5 and 127.2% for budesonide, budesonide oleate, budesonide palmitate, formoterol, salbutamol, terbutaline, ipratropium and tiotropium, demonstrating that the internal standards appropriately corrected for matrix effects, including for tiotropium. An increase in the internal standard-normalized matrix factor was observed when compared to the non-normalized matrix factor for beclomethasone, prednisolone and 6β-hydroxy budesonide, suggesting that the use of internal standards others than budesonide-d8 could be considered. However, all internal standards allowed to reduce the variability since all coefficients of variation of the internal standardnormalized matrix factors were between 1.5 and 14.7% for the two QC levels, meeting all requirements for validation according to the EMA and FDA guidelines. Observations of matrix effect with tiotropium warranted further investigations. There were only few reports of mass spectrometry methods for the measurement of tiotropium in human samples [START_REF] Chi | Ultrasensitive sub-pg/ml determination of tiotropium bromide in human plasma by 2D-UHPLC-MS/MS: challenges and solutions[END_REF][START_REF] Ding | Sensitive HPLC-ESI-MS method for the determination of tiotropium in human plasma[END_REF][START_REF] Wang | Highly sensitive assay for tiotropium, a quaternary ammonium, in human plasma by high-performance liquid chromatography/tandem mass spectrometry[END_REF]. Two methods in human plasma included a method validation according to the US FDA guidelines with the investigation of matrix effect, but either the value of the matrix effect was not reported [START_REF] Wang | Highly sensitive assay for tiotropium, a quaternary ammonium, in human plasma by high-performance liquid chromatography/tandem mass spectrometry[END_REF] or only the values of the internal standard-normalized matrix factors [START_REF] Chi | Ultrasensitive sub-pg/ml determination of tiotropium bromide in human plasma by 2D-UHPLC-MS/MS: challenges and solutions[END_REF] were reported. As shown in Supplementary Figure 3, when compared to compounds directly spiked in mobile phase (procedure C), the peak area in samples with matrix spiked at the same concentration before or after extraction increased greatly for tiotropium and tiotropium-d3 but not for the other molecules. We hypothesized that this matrix effect for tiotropium could be related either to hair components or to solvents used for hair extraction. We thus spiked the compounds in mobile phase and performed the full extraction procedure, which didn't reveal any signal enhancement and allowed to conclude that some hair components may increase the signal of tiotropium. As exposed before, the same phenomenon was observed for tiotropium-d3 and this deuterated internal standard perfectly corrected the matrix effect, allowing accurate and precise quantification of tiotropium in hair samples.

Stability

The stability of processed QC samples was evaluated by reinjection (with a new calibration curve) of the samples kept at +8°C in the autosampler after 24 hrs and for the 3 QC levels (n = 6 for each level). All the differences between the measured and nominal concentrations were between -15% and +15%, showing the stability of processed samples up to 24 hours in the autosampler for each compound.

Analysis of patient samples

A representative chromatogram for patient analysis is shown in Figure 1 and quantitative values in the samples from the 6 patients are shown in Figure 2A. Budesonide was found in the hair from 5 of the 6 patients at concentrations between 6.1 and 8.5 pg/mg; formoterol was quantified between 1.0 and 13.1 pg/mg in the hair from 4 patients, with another patient below the LLOQ (0.9 pg/mg) and salbutamol was detected in the samples from 5 patients also, with only one value above the LLOQ, at 6.4 pg/mg. Observed formoterol concentrations are in line with previous findings for patients receiving ≥12 µg formoterol daily [START_REF] Hassall | Hair analysis to monitor adherence to prescribed chronic inhaler drug therapy in patients with asthma or COPD[END_REF]. Salbutamol concentrations in the range 27-192 pg/mg were also reported in asthma patients [START_REF] Kintz | Doping control for beta-adrenergic compounds through hair analysis[END_REF], while this is the first report of budesonide concentrations in human hair. Interestingly, the concentrations of budesonide and formoterol in four consecutive hair segments corresponding to a 4-months treatment period at constant dose in one patient were stable over time (Figure 2B). Values are expressed as percentages; n = 6 for both intraday and interday results. Representative LC-MS/MS chromatograms of salbutamol, budesonide and internal standard (budesonide-d8, formterol-d6 and salbutamol-d9) obtained after the analysis of an asthmatic patient treated with budesonide, formoterol and salbutamol.

TABLES

FIGURE LEGENDS

Figure 1 :

 1 Figure 1: Chromatograms obtained during the analysis of a patient hair sample.

Figure 2 :

 2 Figure 2: Patient samples analysis. Hair drug concentrations in single hair segments from 6

Table 1 :

 1 Accuracy and precision data for corticosteroids, β2-agonists and anticholinergics in hair LLOQ samples.CV and accuracy values are expressed as percentages. *: One (tiotropium) and two (budesonide palmitate) of 18 values were considered as outliers according to Grubbs test and were excluded from the analysis.

				LLOQ
		Concentration (pg/mg)	Intraday (n = 6) CV (%) Accuracy (%)	Interday (n = 6) Accuracy CV (%) (%)
	Budesonide	5	5.3	106.1-112.2	6.8	109.1
	Formoterol	1	8.7	104.4-115.3	12.4	109.2
	Beclomethasone	10	7.1	102.9-112.5	12.3	106.4
	Prednisolone	25	4.3	99.8-106.3	8.3	102.3
	Fluticasone	25	4.3	90.9-102.4	15.9	95.3
	Salmeterol	5	5.8	95.5-102.2	8.7	98.3
	Salbutamol	5	6.6	99.2-110.8	15.7	103.2
	Terbutaline	5	4.9	93.7-108.9	18.4	101.8
	Ipratropium	5	2.6	107.0-116.4	11.1	112.8
	Tiotropium*	5	8.3	92.7-96.3	4.8	95.0
	Budesonide oleate	5	6.7	101.3-117.1	19.3	107.4
	Budesonide palmitate*	5	4.6	98.8-116.0	18.5	106.4
	6β-hydroxy budesonide	150	6.7	88.8-101.6	16.6	95.1

Table 2: Accuracy and precision for corticosteroids, β2-agonists and anticholinergics in spiked hair QC samples.

CONCLUSION

We describe a method for the simultaneous quantification of corticosteroids, β2-agonists and anticholinergics in human hair samples. This method was validated according to the EMA and FDA guidelines and was shown suitable for the analysis of clinical hair samples which revealed the presence of budesonide, formoterol or salbutamol, part of the treatments used during the clinical trial, demonstrating that this method may be used for hair therapeutic drug monitoring of inhaled medicines in asthma or COPD patients in future studies. This method may be particularly suitable for assessing month-by-month exposure by applying segmental analysis since one segment is compared to another segment from the same patient.

ACKNOWLEDGEMENTS

None

AUTHOR CONTRIBUTIONS

All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.

RESEARCH FUNDING

This research was conducted with support from Astra Zeneca (ESR-16-11931).

DISCLOSURE OF CONFLICTS OF INTEREST

The authors declare no conflict of interest.


S D Ramarao

Amit Pawbake

Ashutosh Kumar Singh

M Núñez-Regueiro

M.-A Méasson

Sebastian C Peter

M Nunez-Regueiro

Marie-Aude Measson

Electrical transport properties of half-heusler ScPdBi single crystals under extreme conditions

Keywords: Single crystals, Flux-growth, Electrical transport, High-Pressure

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

Introduction

Half-Heusler compounds with general formula XYZ (X and Y = transition or rare-earth elements and Z = post-transition metal) attract much attention in the field of topological quantum materials because of their narrow band gap [1][2][3], tunability of spin-orbit coupling (SOC) to induce the band inversion and possibility to close the band gap and form nearly linear dispersive bands resembling the Dirac cone by introducing heavier elements [4][5][6]. This tunability of band gap makes these materials particularly suitable for various applications based on spintronic, thermoelectric, and magneto-caloric effects [7][8][9]. Among all the ternary half-Heusler compounds, special attention has been given to REPdBi and REPtBi (RE: rare-earth) compounds because of their potential topological superconductivity [10,11]. A classification (topologically nontrivial and topologically trivial) has been proposed from NMR studies on a range of these half-Heusler systems RETBi (RE = Sc, Y, and Lu; T = Ni, Pd, and Pt) [12,13].

Notably, ScPtBi and ScPdBi compounds were found to be topologically nontrivial and trivial, respectively [12][13][14]. Among these two compounds, the non-trivial insulating nature of ScPtBi was well studied with help of first principle calculations which reveal that the ScPtBi has an inverted band gap of 0.7 -0.9 eV [3,[13][14][15][16][17]. Apart from these, ScPtBi was reported for its poor thermoelectric properties with a negative Seebeck coefficient and n-type carrier behavior [18,19]. Apart from numerous theoretical studies, experimental studies on ScPtBi was least explored. Hou et al. reported the synthesis of ScPtBi single crystal by self-flux growth technique, and the non-magnetic semi-metallic behavior was observed from the low field magneto-transport studies [20]. Interestingly, ScPdBi substituted by Pt is claimed to be a superconductor below 1.2 K and paving a path to reach the topological non-trivial superconductivity [21]. ScPdBi was reported to have a trivial band topology with a semiconducting band gap in the range of 0.3 -0.7 eV [3,6,14]. It has been reported that the band inversion strength (∆ = Γ6-Γ8) of ScPdBi was close to the trivial to non-trivial insulating phase transition region [3,6,14]. Nowak et al. confirmed the trivial band topology of ScPdBi with help of 209 Bi NMR [17]. It was reported that the uniaxial pressure induces the trivial to non-trivial insulating phase transition in half-Heusler compounds [3,15]. To understand the effect of pressure on the band order, Narimani et al. explored the pressure-dependent electronic band structure of ScPdBi and could not observe any band inversion. Apart from this, the energy difference between Γ6 and Γ8 was enhanced with pressure [22]. Narimani studied the band topology of ScPdBi by incorporating the heavy elements (Gd, Np, and Cm) at the Sc site and could not observe any band inversion [23]. These studies motivated us to grow the single crystals of ScPdBi and study their electrical transport properties at ambient and extreme conditions such as magnetic field up to 7 T and pressure up to 19 GPa assuming that these can cause a change in the electronic band structure, which may lead into interesting topological properties.

Experimental sDetails

Single crystals of ScPdBi were prepared by self-flux growth technique in which stoichiometric amount of Sc (31.2 mg, Alfa Aesar, 99.9% pure chunks), Pd (73.8 mg, Alfa Aesar, 99.99% pure wire) and Bi (145 mg, Sigma Aldrich, 99.9% pure shots) metals were taken in a tantalum ampule. Extra Bi (2.5 gm) was added to these reagents which act as a self-flux.

The tantalum ampule was placed in a 13 mm quartz tube and was flame sealed under a vacuum of 10 -5 torr for preventing oxidation during the reaction. The reactant mixture was heated to 1173 K in 8 h and maintained at that temperature for 48 h. Finally, the sample was cooled down to room temperature with a rate of 3 o C/h. Excess flux present in the sample was removed by heating the sample at 773 K and subsequently centrifuged through a coarse frit. To remove the flux present over the crystals, these crystals were soaked in dilute HNO3 solution for a period of 16 h. Finally, these crystals were rinsed with acetone and sonicated for several times. The obtained ScPdBi crystal was having a cube shape with an average size of 200 µm.

Single crystal X-ray diffraction measurements were done on the as cleaned ScPdBi single crystals with the help of Bruker Smart Apex 2-CCD diffractometer having a normal focus, 2.4 kW sealed tube X-ray source with graphite monochromatic Mo Kα radiation (λ = 0.71073 Å). SAINT was used for the integration of diffraction profiles and SADABS was used for the absorption correction. The structure was solved by direct and Fourier methods and refined by full-matrix least-squares, using standard crystallographic software: SHELXT-2014, SHELXL-2014 [24,25]. Olex2 software package was used for these refinements. The crystal structure of ScPdBi was built with help of DIAMOND software.

Temperature-dependent resistivity measurements down to 2 K were done on ScPdBi single crystals at ambient and different magnetic fields with help of physical property measurement system (PPMS, Quantum Design). The dependence of electrical transport on temperature and the magnetic field is systematically investigated between 2 K and 300 K up to 9

T. Electrical transport measurements at ambient and different pressure values (2-19 GPa) were performed using a linear four-probe technique in which we use Keithley 220 and Keithley 2182 as a source and nano voltmeter respectively. For this pressure-dependent measurements, we used a sintered diamond Bridgman anvil apparatus in which a pyrophyllite gasket and two steatite disks act as a pressure transmitting medium [26].

Results and Discussion

The X-ray diffraction pattern of ScPdBi is depicted in Fig. 1a. It has been seen that the compound is phase pure and forming in the MgAgAs structure type having F 3m space group.

The inset of Fig. 1a shows the unit cell of half Heusler ScPdBi compound within which Sc, Pd, and Bi atoms occupy 4b, 4c, and 4a Wyckoff sites, respectively. The lattice parameters (a = b = c = 6.449 Å; and α = β = γ = 90 o ) obtained from the single crystal refinement are in good agreement with reported values [13]. The cubic morphology of the as-synthesized ScPdBi single crystals was confirmed using a scanning electron micrograph (inset of Fig. 1a). The dimensions of these single crystals are in the range of 150 µm as shown in the inset of Fig. 1a. EDAX and elemental color mapping studies were done on ScPdBi single crystals and are shown in Fig. 1b and Fig. 1c respectively. It has been seen that all the Sc, Pd, and Bi elements are uniformly distributed throughout these single crystals. Apart from this, a slight deficiency in Sc was observed from the composition analysis.

Fig. 2a shows the temperature-dependent resistivity of ScPdBi single crystals at ambient pressure. Resistivity decreases with the decreasing temperature down to 2 K, confirming the metallic nature of the system. However, it contradicts the expected semiconducting behavior as predicted from the previous theoretical reports [3,6,14]. Results of the temperature-dependent resistivity under constant magnetic field ranging from 1 T to 9 T are depicted in Fig. 2b. We could not observe any appreciable effect on the resistivity with the application of an external magnetic field which is expected for a trivial band system. Moreover, Sc, Pd, and Bi atoms probably do not contribute any magnetic moment to the system which is in agreement with the reported theoretical prediction [23].

Apart from the metallic nature, we could observe an upturn in the resistivity below 30 K (Fig. 2a). In general, this kind of behavior in resistivity is expected in the case of f-electronbased systems because of the hybridization of conduction bands with the localized f-electrons at low temperature and giving rise to the Kondo effect [27]. It was reported that Kondo behavior can also be observed in 3d-electron-based systems [28][29][30][31]. To understand the origin of the upturn in the resistivity of ScPdBi, we have performed the temperature-dependent specific heat measurements down to 2 K. Fig. 3 shows the C/T vs T 2 and this variation is fitted with the usual quadratic equation C/T=γ+β*T 2 . From this, we extract a Sommerfeld coefficient (γ ≈ 2.59 ± 0.87 mJ mol -1 K -2 ) and a Debye term (β ≈ 0.87 ± 0.01 mJ mol -1 K -4 ). These results ruled out the possibility of Kondo behavior in the ScPdBi compound.

To further understand the electrical transport properties under extreme conditions, we have carried out the temperature-dependent resistivity at different hydrostatic pressures ranging from 2 -19 GPa (see Fig. 4).

These measurements were performed with a four-probe technique and observed a ground of 0.55 kΩ above 10 GPa in the electronic set-up which affects the absolute value of the resistivity without changing the general trends. For completeness, we present all the temperature sweeps at constant pressure where the results above 10 GPa have been normalized by multiplying the factor (0.97+0.00000381) (Fig. 4a). These results indicate that the metallic nature of ScPdBi persists even up to 19 GPa. Interestingly, we also observe an upturn in the resistivity at low temperature in all the measured pressure range. As we rule out the presence of Kondo state in ScPdBi by specific heat measurements, we ascribe this upturn in resistivity to the conduction in strongly disordered systems.

Considering the very low RRR of the sample that suggests defects and impurities, we turn to a model that describes conduction in strongly disordered systems with localized charge carrier states, i.e. the Mott variable-range hopping model. Analyzing a model in which charge is transported via phonon-induced tunneling of electrons between localized states which are randomly distributed in energy and position, Mott [32] has obtained an electrical conductivity of the form

ρ = ρ0 exp[2(T0/T) 1/4 ]
In general, this type of temperature dependence has been observed in a large number of systems [32], including high-temperature superconductors [33]. show a decreasing variation of , i.e. increasing for N(EF), as a function of pressure.

According to band structure calculations [22], the valence Γ8 band is almost on the Fermi level, at all pressures. Thus, the increase of N(EF) that we observe, would indicate an increase of hole charge transfer from defects and impurities to this band. This analysis also supports an explanation for the metallicity of the samples: doping of the valence band by impurities. The low-temperature deviation from 40 K can be attributed due to percolative transport via interlayer coupling or electronic disorders [34,35].

Conclusions

Single crystals of ScPdBi were successfully grown by the self-flux growth technique and observed to crystallize in MgAgAs structure type with F 3m space group. The temperature dependences of the resistivity of ScPdBi was measured at ambient pressure at different magnetic field up to 9 T and under pressure up to 19 GPa. ScPdBi observed to have metallic nature down to 2 K and persists its behavior even under extreme conditions (up to 9 T and 19

GPa). ScPdBi did not show a noticeable magneto-transport effect. The Sommerfeld coefficient (γ) and Debye term (β) were extracted from the specific heat measurements and found to be 2.59 ±0.87 mJ mol -1 K -2 and 0.87 ±0.01 mJ mol -1 K -4 , respectively. The low γ value and smooth temperature dependence of the specific heat indicate the absence of the Kondo effect in ScPdBi.

The upturn in resistivity observed in the low-temperature region at all pressure probably due to either electron-hole scattering process or carrier imbalance.

Graphical abstract

Graphical abstract legend

Resistivity measurements at extreme conditions on ScPdBi single crystals reveal the metallic nature, which further confirmed by band structure calculations.

Fig. 4 (

 4 c) presents the resistivity versus temperature as ln(ρ) versus . The linear t of the low-temperature part (i.e. T < 25 K) determines T0=α 3 /kBN(EF), where α is the localization distance, which can be assumed to be constant with pressure, and N(EF) is the density of states at the Fermi level. On Fig. 4(b) we

FiguresFigure 1 .

 1 Figures

  Figure 1. (a) Powder X-ray diffraction pattern of ScPdBi, insets show the crystal structure and scanning electron microscopic image of ScPdBi single crystal (b) Compositional analysis on ScPdBi single crystals (c) Elemental colour mapping on ScPdBi single crystal, showing uniform distribution of Sc, Pd and Bi elements.

Figure 2 .

 2 Figure 2. (a) Temperature dependent resistivity of ScPdBi showing the metallic nature down to 2 K (b) Temperature dependent resistivity of ScPdBi at different magnetic fields, indicating there is no appreciable change in the resistivity with external magnetic field.

Figure 3 .

 3 Figure 3. Temperature dependent specific heat of ScPdBi. A linear fit to the low temperature region resulted in a low Sommerfeld coefficient (γ ≈ 2.59 ± 0.87 mJ mol -1 K -2 ) and Debye term (β ≈ 0.87 ± 0.01 mJ mol -1 K -4 ).

Figure 4 .

 4 Figure 4. (a) Temperature dependence of the resistivity of ScPdBi at different pressure (2 -19 GPa), (b) Variation of with pressure, and (c) 3D Mott variable-range hopping (VRH) model.

Acknowledgment

We thank Jawaharlal Nehru Centre for Advanced Scientific Research, Sheikh Saqr Laboratory and Department of Science and Technology (DST), India for financial support. The authors thank Indo-French Centre for the Promotion of Advanced Research (CEFIPRA, grant no. 5608-2) for financial support. SCP thanks DST for SwarnaJayanti Fellowship (DST/SJF/CSA-02/2017-18).