Charanraj Goud Alladi

Ravi Philip Rajkumar

Surendiran Adithan

Cynthia Marie-Claire

Frank Bellivier

Dr Deepak Gopal

Charanraj Goud Alladi

Deepak Gopal Shewade

Shewade Professor

Receptor Gene Polymorphisms do not influence early response to Risperidone in South Indian Patients with Schizophrenia

Keywords: Risperidone, Schizophrenia, Dopamine, Serotonin, Genetic Polymorphism

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INTRODUCTION

Schizophrenia (SCZ) a severe psychotic illness requires long term therapy with antipsychotics.

Atypical antipsychotics (AAP) act by binding to dopamine (DRD2) and serotonin (5HT) receptors [START_REF] Seeman | Atypical antipsychotics: mechanism of action[END_REF]. Clinical efficacy of AAP is highly correlated with potent binding affinity to DRD2 and 5HT receptors [START_REF] Richtand | Role of serotonin and dopamine receptor binding in antipsychotic efficacy[END_REF]. However, a high level of response variability to antipsychotic treatment has been described with regard to acute response, relapse prevention, and side effects. In particular, for the treatment of an acute phase, it is estimated that at least 30 -40% of the patients do not respond to the treatment [START_REF] Simonsen | Early identification of non-remission in first-episode psychosis in a two-year outcome study[END_REF][START_REF] Cui | Early predictors of a clinical response at 8 weeks in patients with first-episode psychosis treated with paliperidone ER[END_REF]. There are no biological markers available to predict therapeutic response and tolerance, thereby leading to lengthy empirical trials with an uncertain outcome. Genetic variation may play a role in AAP response variability and several lines of evidence suggest that polymorphisms within genes coding for proteins implicated in the dopamine and serotonin neurotransmission may influence the AAP efficacy in SCZ [START_REF] Malhotra | Candidate gene studies of antipsychotic drug efficacy and drug-induced weight gain[END_REF][START_REF] Arranz | Toward understanding genetic risk for differential antipsychotic response in individuals with schizophrenia[END_REF]. In this context, several pharmacogenetic studies have been performed to investigate the implication of dopaminergic and serotonergic candidate genes in AAP response [START_REF] Blanc | Pharmacogenetic of response efficacy to antipsychotics in schizophrenia: pharmacodynamic aspects. Review and implications for clinical research[END_REF]. In particular, polymorphisms in DRD2, HTR2A and to a lesser extent in HTR2C genes have been extensively studied in relation with AAP response in SCZ [START_REF] Reynolds | Pharmacogenomics in psychiatry: the relevance of receptor and transporter polymorphisms[END_REF][START_REF] Arranz | Pharmacogenetic studies of drug response in schizophrenia[END_REF]. Several studies have shown that the -141C Ins/Del and the Taq1A polymorphisms of the DRD2 gene may be associated with a reduced receptor density which may alter the therapeutic response [START_REF] Arinami | A functional polymorphism in the promoter region of the dopamine D2 receptor gene is associated with schizophrenia[END_REF][START_REF] Pohjalainen | The A1 allele of the human D2 dopamine receptor gene predicts low D2 receptor availability in healthy volunteers[END_REF]. Previous study reports showed that Ins allele carriers of -141C Ins/Del polymorphism and A1 allele carriers of Taq1A polymorphism of DRD2 gene have a better therapeutic response [START_REF] Wu | Response to chlorpromazine treatment may be associated with polymorphisms of the DRD2 gene in Chinese schizophrenic patients[END_REF][START_REF] Suzuki | Association between TaqI A dopamine D2 receptor polymorphism and therapeutic response to bromperidol: a preliminary report[END_REF]. The association between AAP response and DRD2 Ins/Del polymorphism was also suggested by two meta-analyses [START_REF] Zhang | Dopamine D2 receptor genetic variation and clinical response to antipsychotic drug treatment: A meta-analysis[END_REF][START_REF] Zhang | Pharmacogenetics and Antipsychotics: Therapeutic Efficacy and Side Effects Prediction[END_REF]. However, this association was not replicated in a large study with a prospective evaluation of the response [START_REF] Huang | Preliminary evidence for association of genome-wide significant DRD2 schizophrenia risk variant with clozapine response[END_REF]. With regard to DRD2 Taq1A polymorphism, no association between this polymorphism and AAP response was suggested by a meta-analysis of seven positive studies and five negative studies [START_REF] Zhang | Pharmacogenetics and Antipsychotics: Therapeutic Efficacy and Side Effects Prediction[END_REF]. Two additional negative studies were published following this previously mentioned meta-analysis [START_REF] Huang | Preliminary evidence for association of genome-wide significant DRD2 schizophrenia risk variant with clozapine response[END_REF][START_REF] Kang | DRD2 genotypic and haplotype variation is associated with improvements in negative symptoms after 6 weeks' amisulpride treatment[END_REF]. Overall, the interpretation of these conflicting results remains difficult due to allelic heterogeneity related to ethnic background. In particular, some authors report an association between A1 allele and some A2 alleles with good response [START_REF] Suzuki | The relationship between dopamine D2 receptor polymorphism at the Taq1 A locus and therapeutic response to nemonapride, a selective dopamine antagonist, in schizophrenic patients[END_REF][START_REF] Hwang | Association study of 12 polymorphisms spanning the dopamine D(2) receptor gene and clozapine treatment response in two treatment refractory/intolerant populations[END_REF].

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With regard to the HTR2A gene, several lines of evidences suggest the implication that this gene in AAP response variability in SCZ. Indeed, it has been shown that the -1438G>A polymorphism in HTR2A influences the transcription levels [START_REF] Ruble | Genomic structure and expression of the human serotonin 2A receptor gene (HTR2A) locus: identification of novel HTR2A and antisense (HTR2A-AS1) exons[END_REF]. In addition, previous studies also showed that the T102C polymorphism of this gene plays an important role in HT2RA mediated neurotransmission [START_REF] Parsons | The -1438A/G polymorphism in the 5-hydroxytryptamine type 2A receptor gene affects promoter activity[END_REF][START_REF] Polesskaya | Allele C-specific methylation of the 5-HT2A receptor gene: evidence for correlation with its expression and expression of DNA methylase DNMT1[END_REF]. In connection with therapeutic response, 12 studies analysing the influence of T102C polymorphism of the HTR2A gene on AAP response have been published: one reports an association between the C allele and better response [START_REF] Lane | Association of risperidone treatment response with a polymorphism in the 5-HT(2A) receptor gene[END_REF], another reports an association between T allele and better response [START_REF] Ellingrod | 5HT2A and 5HT2C receptor polymorphisms and predicting clinical response to olanzapine in schizophrenia[END_REF], and ten studies did not show any association [START_REF] Masellis | Genetic variation of 5-HT2A receptor and response to clozapine[END_REF][START_REF] Nöthen | Genetic variation of the 5-HT2A receptor and response to clozapine[END_REF][START_REF] Malhotra | Lack of association between polymorphisms in the 5-HT2A receptor gene and the antipsychotic response to clozapine[END_REF][START_REF] Masellis | Serotonin subtype 2 receptor genes and clinical response to clozapine in schizophrenia patients[END_REF][START_REF] Joober | T102C polymorphism in the 5HT2A gene and schizophrenia: relation to phenotype and drug response variability[END_REF][START_REF] Lin | No evidence for association of serotonin-2A receptor variant (102T/C) with schizophrenia or clozapine response in a Chinese population[END_REF][START_REF] Yamanouchi | Effect of DRD2, 5-HT2A, and COMT genes on antipsychotic response to risperidone[END_REF][START_REF] Vehof | Clinical response to antipsychotic drug treatment: association study of polymorphisms in six candidate genes[END_REF][START_REF] Jönsson | 5HT 2a receptor T102C polymorphism and schizophrenia[END_REF][START_REF] Ikeda | Variants of dopamine and serotonin candidate genes as predictors of response to risperidone treatment in first-episode schizophrenia[END_REF]. Six studies investigated -1438G>A polymorphism, with two reporting an association between G allele and poor treatment response [START_REF] Arranz | Evidence for association between polymorphisms in the promoter and coding regions of the 5-HT2A receptor gene and response to clozapine[END_REF][START_REF] Shen | HTR2A A-1438G/T102C polymorphisms predict negative symptoms performance upon aripiprazole treatment in schizophrenic patients[END_REF], one reporting that the presence of G allele was associated with better response [START_REF] Benmessaoud | Excess of transmission of the G allele of the -1438A/G polymorphism of the 5-HT2A receptor gene in patients with schizophrenia responsive to antipsychotics[END_REF],

another reporting an association between the AA genotype and negative symptoms improvement [START_REF] Ellingrod | 5-HT2A receptor promoter polymorphism, -1438G/A and negative symptom response to olanzapine in schizophrenia[END_REF], and two studies not showing any association with response to AAP [START_REF] Yamanouchi | Effect of DRD2, 5-HT2A, and COMT genes on antipsychotic response to risperidone[END_REF][START_REF] Hamdani | Negative symptoms of schizophrenia could explain discrepant data on the association between the 5-HT2A receptor gene and response to antipsychotics[END_REF]. Of particular interest for the present work, two independent studies among Indian populations, which reported an association between the HTR2A gene (-1438G>A; T102C polymorphisms) and SCZ [START_REF] Araga | Association between the 102T/C polymorphism of serotonin-2A receptor gene and schizophrenia among south Indians[END_REF][START_REF] Sujitha | 5-Hydroxytryptamine (serotonin) 2A receptor gene polymorphism is associated with schizophrenia[END_REF]. Among the eight studies that have investigated the role of HTR2C gene polymorphism on AAP response, only two have shown an association with AAP treatment response [START_REF] Ikeda | Variants of dopamine and serotonin candidate genes as predictors of response to risperidone treatment in first-episode schizophrenia[END_REF][START_REF] Sodhi | Association between clozapine response and allelic variation in the 5-HT2C receptor gene[END_REF][START_REF] Rietschel | Efficacy and sideeffects of clozapine not associated with variation in the 5-HT2C receptor[END_REF][START_REF] Masellis | Serotonin subtype 2 receptor genes and clinical response to clozapine in schizophrenia patients[END_REF][START_REF] Schumacher | Pharmacogenetics of clozapine response[END_REF][START_REF] Ellingrod | 5HT2A and 5HT2C receptor polymorphisms and predicting clinical response to olanzapine in schizophrenia[END_REF][START_REF] Reynolds | Pharmacogenetics of treatment in firstepisode schizophrenia: D3 and 5-HT2C receptor polymorphisms separately associate with positive and negative symptom response[END_REF][START_REF] Malhotra | Clozapine response and the 5HT2C Cys23Ser polymorphism[END_REF]. HTR2C -759C>T is an important genetic polymorphism and reported to affect the its receptor expression [START_REF] Polesskaya | Differential expression of the "C" and "T" alleles of the 5-HT2A receptor gene in the temporal cortex of normal individuals and schizophrenics[END_REF]. Though two studies have been conducted to evaluate the role of HTR2C-759C>T in AAP response, they contradict each other, with one study showing no association with response and another study showed an association with improvement in negative symptoms [START_REF] Ikeda | Variants of dopamine and serotonin candidate genes as predictors of response to risperidone treatment in first-episode schizophrenia[END_REF][START_REF] Reynolds | Pharmacogenetics of treatment in firstepisode schizophrenia: D3 and 5-HT2C receptor polymorphisms separately associate with positive and negative symptom response[END_REF].

There are several possible reasons for these conflicting results including AAP treatment heterogeneity, ethnic background disparities, treatment duration, response assessment tools, power of detection (sample size) and outcome criteria as well as the study design (prospective or retrospective assessment of the response). So far, the influence of these polymorphisms on AAP response has not been investigated in South Indian populations, except for one study that analyzed the influence of DRD2 Taq1A polymorphism on AAP response and another study that investigated the DRD2 Taq1A

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This article is protected by copyright. All rights reserved. and HTR2A (-1438A>G;T102C) polymorphisms with risperidone response among the North-Indian population [START_REF] Vijayan | Association of dopamine receptor polymorphisms with schizophrenia and antipsychotic response in a South Indian population[END_REF][START_REF] Kaur | Identification of genetic correlates of response to Risperidone: Findings of a multicentric schizophrenia study from India[END_REF].

To further explore the association between DRD2, HTR2A and HTR2C genes polymorphisms and AAP response to antipsychotic medication, we conducted an association study between these candidate genes and treatment response in South Indian populations of patients with schizophrenia treated with risperidone (4-8 mg/day).

METHODS

Patients with schizophrenia (n=320) recruited for the study as per Diagnostic and Statistical Manual for Mental Disorders-V, criteria were recruited for the study from the Department of Psychiatry, Jawaharlal Institute of Post graduate Medical Education and Research (JIPMER), Puducherry, India.

All the patients were recruited between December 2013 and August 2015. The study protocol was approved by Institute Ethics Committee (Project No. JIP/IEC/4/2013/189) and after explaining the study procedure, written informed consent was obtained from all the patients or patients' legally acceptable representatives. A total of 320 patients with schizophrenia were recruited for the study.

Among the total patients n=211 patients were recruited cross-sectionally, by assessing the clinical global impression improvement (CGI-I) score for antipsychotic response after receiving the risperidone (4-8mg/day) for minimum of 4 weeks and n=109 patients were recruited with prospective assessment. In the prospective study group, patients who have not received any antipsychotic medication for past 30 days and have been prescribed risperidone (4-8mg/day) as their antipsychotic therapy for a minimum of four weeks were recruited for the study. In the prospective group, baseline characteristics, positive, negative and general psychopathology symptoms were assessed using Positive and Negative Syndrome Scale (PANSS) score before starting the therapy and after receiving the risperidone therapy for a minimum of four weeks. Patients who had history of other medical illness, substance abuse, pregnant women, nursing women and patients with age of <18 years were excluded from the study.

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Response Assessment

In the cross-sectional study group, clinical global impression-improvement (CGI-I) score of sevenpoint rating scale was assessed. After adequate trial of risperidone treatment patients with CGI-I score of 1-3 considered as responders and 4-7 considered as non-responders [START_REF] Busner | The Clinical Global Impressions Scale[END_REF].

In the prospective study group, two response assessment criteria were used. The antipsychotic response to risperidone was assessed using PANSS global score to evaluate the early treatment response. A continuous variable "Δ PANSS" defined difference between baseline PANSS score and follow-up PANSS score was used for assessment of patients. Patients were also categorized as responders (who showed more than 20% reduction in PANNS score after four weeks of treatment) and non-responders patients who did not show more than 20% reduction in PANSS score after four weeks of treatment [START_REF] Haas | A 6-week, randomized, double-blind, placebo-controlled study of the efficacy and safety of risperidone in adolescents with schizophrenia[END_REF].

DNA extraction & genotyping

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Statistical analysis

Demographic characteristics of patient details were expressed as mean ± standard deviation. Hardy-Weinberg equilibrium was tested on genotype frequencies. Chi-square test and Fisher's exact test were used to study the association between genotypes and categories of response to risperidone. Kruskal-Wallis test was used to study the association between the genotypes and Δ PANSS. For bimodal categorical variable, Mann-Whitney U test was used to analyse reduction in PANSS scores between genotypes. All statistical analyses were done with Graph Pad Instat version 3.06.

RESULTS

A total of n= 320 patients with schizophrenia were included for the genotype-phenotype analysis.

Among the 320 patients, 109 patients were recruited with prospective assessment and 211 patients were recruited in cross-sectional study group. The PANSS score of prospective study group participants was 91.47±17.71 (mean ±SD) at baseline and after four weeks of risperidone treatment, the PANSS score was 57.96 ± 20.49 (mean ±SD). Categorically, 84 patients were responders (20% reduction on PANSS score) and others 25 patients were non responders. Among the 211 crosssectional study group participants, 154 were responders and 57 were non-responders to risperidone therapy on CGI-I score. All the responders and non-responder's demographic details were given in Table . I. Genotypic distributions were in Hardy-Weinberg equilibrium for the DRD2 Taq1A (n=320, χ 2 =0.32, DF=1, p=0.5), the HTR2A rs6311 (n=320, χ2=1.3, DF=1, p=0.2), HTR2A rs6313 (n=320, χ 2 =1.34, DF=1, p=0.2) and the HTR2C rs3813929 in female subjects (n=132, χ 2 =1.4, DF=1, p=0.2) since this gene is located on X chromosome. During the genotyping of DRD2 -141 C Ins/Del polymorphism, ten samples were not amplified and were excluded. Due to the low frequency of Del allele, the Del/Del subjects (n=5) were combined with Ins/Del genotype to study the association with antipsychotic response.

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No significant association was detected between risperidone response (categorical definition) and DRD2, HTR2A and HTR2C genes polymorphisms (Table . II, IV and V). Similarly, no significant association was detected between the level of response based on the level of reduction of the PANSS score and DRD2, HTR2A and HTR2C genes polymorphisms (Table. III). In post-hoc exploratory analyses, no association was detected between the improvement of positive and negative symptoms and general psychopathology sub scores of PANSS score in relation with candidate genes polymorphisms. (Supplementary Table. I).

DISCUSSION

In the present study, using a prospective assessment of symptom evaluation and using two definitions of response, as well as using CGI-I score response assessment in cross-sectional study group, we have examined the role of the associations of DRD2 (rs1799732, rs1800497), HTR2A (rs6311, rs6313) and HTR2C (rs3813929) gene polymorphisms with antipsychotic response among South Indian patients with SCZ. The present study did not find significant influence of the studied polymorphisms on treatment response to risperidone.

Allele frequencies of the DRD2 and HTR2A polymorphisms in the present study were consistent with those reported in other studies of Indian populations. Indeed, with regard to DRD2 Taq1A polymorphism, in our sample, G allele frequency (0.69) was consistent with those reported by Vijayan et al (0.69) in South Indian populations and another study by Kaur et al (0.68) in North Indian populations [START_REF] Vijayan | Association of dopamine receptor polymorphisms with schizophrenia and antipsychotic response in a South Indian population[END_REF][START_REF] Kaur | Identification of genetic correlates of response to Risperidone: Findings of a multicentric schizophrenia study from India[END_REF]. For DRD2 -141 C Ins/Del polymorphism the Ins allele frequency (0.77) could not be compared to other South Indian population with SCZ but consistent with allele frequency of South Indian alcohol dependant patients (0.77) and controls (0.65) [START_REF] Prasad | Case-control association analysis of Dopamine receptor polymorphisms in alcohol dependence: a pilot study in Indian males[END_REF]. Similarly, another study by Sujitha et al reported the allele frequencies of HTR2A gene polymorphisms -1438A<G (G-0.56; A-0.44), and T102C (C-0.58; T-0.42) in patients with SCZ from South Indian populations [START_REF] Sujitha | 5-Hydroxytryptamine (serotonin) 2A receptor gene polymorphism is associated with schizophrenia[END_REF] which is consistent with the present study and also consistent with another recent study by Kaur et

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North Indian populations [START_REF] Kaur | Identification of genetic correlates of response to Risperidone: Findings of a multicentric schizophrenia study from India[END_REF]. To the best of our knowledge no data have been reported on HTR2C -759 C>T polymorphism frequency in Indian populations.

Previous studies, two from Japanese, three from Caucasians, one each from Chinese, African American, and Korean populations have found an association between DRD2 Taq1A polymorphism and antipsychotic response [START_REF] Suzuki | The relationship between dopamine D2 receptor polymorphism at the Taq1 A locus and therapeutic response to nemonapride, a selective dopamine antagonist, in schizophrenic patients[END_REF][START_REF] Hwang | Association study of 12 polymorphisms spanning the dopamine D(2) receptor gene and clozapine treatment response in two treatment refractory/intolerant populations[END_REF][START_REF] Yamanouchi | Effect of DRD2, 5-HT2A, and COMT genes on antipsychotic response to risperidone[END_REF][START_REF] Ikeda | Variants of dopamine and serotonin candidate genes as predictors of response to risperidone treatment in first-episode schizophrenia[END_REF][START_REF] Schäfer | Association of short-term response to haloperidol treatment with a polymorphism in the dopamine D(2) receptor gene[END_REF][START_REF] Dahmen | Genetic polymorphisms of the dopamine D2 and D3 receptor and neuroleptic drug effects in schizophrenic patients[END_REF][START_REF] Shen | Effects of DRD2/ANKK1 gene variations and clinical factors on aripiprazole efficacy in schizophrenic patients[END_REF][START_REF] Kwon | Taq1A polymorphism in the dopamine D2 receptor gene as a predictor of clinical response to aripiprazole[END_REF]. However, present study results are consistent with many other studies that did not find an association between these genetic polymorphisms and treatment response to antipsychotics in other ethnic groups [START_REF] Wu | Response to chlorpromazine treatment may be associated with polymorphisms of the DRD2 gene in Chinese schizophrenic patients[END_REF][START_REF] Suzuki | Association between TaqI A dopamine D2 receptor polymorphism and therapeutic response to bromperidol: a preliminary report[END_REF][START_REF] Vehof | Clinical response to antipsychotic drug treatment: association study of polymorphisms in six candidate genes[END_REF][START_REF] Reynolds | Pharmacogenetics of treatment in firstepisode schizophrenia: D3 and 5-HT2C receptor polymorphisms separately associate with positive and negative symptom response[END_REF][START_REF] Xing | The relationship between the therapeutic response to risperidone and the dopamine D2 receptor polymorphism in Chinese schizophrenia patients[END_REF]. In particular, DRD2

Taq1A and HTR2A (-1438A>G;T102C) polymorphisms results are consistent with previous association studies from Indian populations [START_REF] Vijayan | Association of dopamine receptor polymorphisms with schizophrenia and antipsychotic response in a South Indian population[END_REF][START_REF] Kaur | Identification of genetic correlates of response to Risperidone: Findings of a multicentric schizophrenia study from India[END_REF]. With regard to the DRD2 -141 Ins/Del polymorphism, present study results did not find an association with antipsychotic response and results are consistent with many of the previous studies [START_REF] Hwang | Association study of 12 polymorphisms spanning the dopamine D(2) receptor gene and clozapine treatment response in two treatment refractory/intolerant populations[END_REF][START_REF] Yamanouchi | Effect of DRD2, 5-HT2A, and COMT genes on antipsychotic response to risperidone[END_REF][START_REF] Vehof | Clinical response to antipsychotic drug treatment: association study of polymorphisms in six candidate genes[END_REF][START_REF] Ikeda | Variants of dopamine and serotonin candidate genes as predictors of response to risperidone treatment in first-episode schizophrenia[END_REF][START_REF] Arranz | Evidence for association between polymorphisms in the promoter and coding regions of the 5-HT2A receptor gene and response to clozapine[END_REF][START_REF] Xing | The relationship between the therapeutic response to risperidone and the dopamine D2 receptor polymorphism in Chinese schizophrenia patients[END_REF][START_REF] Shen | Effects of DRD2/ANKK1 gene variations and clinical factors on aripiprazole efficacy in schizophrenic patients[END_REF], with exception of few studies in Chinese, Caucasians and African Americans in whom they have reported an association between DRD2 -141 C Ins/Del polymorphism and antipsychotic response [START_REF] Wu | Response to chlorpromazine treatment may be associated with polymorphisms of the DRD2 gene in Chinese schizophrenic patients[END_REF][START_REF] Malhotra | Allelic variation in the promoter region of the dopamine D2 receptor gene influences the antipsychotic response to clozapine[END_REF][START_REF] Lencz | DRD2 promoter region variation as a predictor of sustained response to antipsychotic medication in first-episode schizophrenia patients[END_REF]. In the present study HTR2A gene -1438 A/G polymorphism results are consistent with reported previous studies in Japanese, Dutch, and Caucasian populations [START_REF] Yamanouchi | Effect of DRD2, 5-HT2A, and COMT genes on antipsychotic response to risperidone[END_REF][START_REF] Vehof | Clinical response to antipsychotic drug treatment: association study of polymorphisms in six candidate genes[END_REF][START_REF] Hamdani | Negative symptoms of schizophrenia could explain discrepant data on the association between the 5-HT2A receptor gene and response to antipsychotics[END_REF] and not consistent with a few studies such as studies in Caucasians, Algerian, American and Chinese population in whom they have reported an association [START_REF] Arranz | Evidence for association between polymorphisms in the promoter and coding regions of the 5-HT2A receptor gene and response to clozapine[END_REF][START_REF] Shen | HTR2A A-1438G/T102C polymorphisms predict negative symptoms performance upon aripiprazole treatment in schizophrenic patients[END_REF][START_REF] Ellingrod | 5-HT2A receptor promoter polymorphism, -1438G/A and negative symptom response to olanzapine in schizophrenia[END_REF][START_REF] Benmessaoud | Excess of transmission of the G allele of the -1438A/G polymorphism of the 5-HT2A receptor gene in patients with schizophrenia responsive to antipsychotics[END_REF]. Similarly, HTR2A T102C polymorphism results are consistent with many previous studies [START_REF] Masellis | Genetic variation of 5-HT2A receptor and response to clozapine[END_REF][START_REF] Nöthen | Genetic variation of the 5-HT2A receptor and response to clozapine[END_REF][START_REF] Malhotra | Lack of association between polymorphisms in the 5-HT2A receptor gene and the antipsychotic response to clozapine[END_REF][START_REF] Masellis | Serotonin subtype 2 receptor genes and clinical response to clozapine in schizophrenia patients[END_REF][START_REF] Joober | T102C polymorphism in the 5HT2A gene and schizophrenia: relation to phenotype and drug response variability[END_REF][START_REF] Lin | No evidence for association of serotonin-2A receptor variant (102T/C) with schizophrenia or clozapine response in a Chinese population[END_REF][START_REF] Yamanouchi | Effect of DRD2, 5-HT2A, and COMT genes on antipsychotic response to risperidone[END_REF][START_REF] Jönsson | 5HT 2a receptor T102C polymorphism and schizophrenia[END_REF][START_REF] Ikeda | Variants of dopamine and serotonin candidate genes as predictors of response to risperidone treatment in first-episode schizophrenia[END_REF] and not consistent with few studies from Chinese, American, Caucasians, African American, and Korean populations in whom they have reported an association with antipsychotic response [START_REF] Lane | Association of risperidone treatment response with a polymorphism in the 5-HT(2A) receptor gene[END_REF][START_REF] Shen | HTR2A A-1438G/T102C polymorphisms predict negative symptoms performance upon aripiprazole treatment in schizophrenic patients[END_REF][START_REF] Ellingrod | 5HT2A and 5HT2C receptor polymorphisms and predicting clinical response to olanzapine in schizophrenia[END_REF][START_REF] Nimgaonkar | 5-HT2 receptor gene locus: association with schizophrenia or treatment response not detected[END_REF][START_REF] Kim | Could HTR2A T102C and DRD3 Ser9Gly predict clinical improvement in patients with acutely exacerbated schizophrenia? Results from treatment responses to risperidone in a naturalistic setting[END_REF]. So far very few studies have investigated the role of HTR2C-759 C/T polymorphism in association with antipsychotic response. In the present study, HTR2C-759 C/T polymorphism did not show significant association with antipsychotic response.

Present study findings are consistent with previous study by Ikeda et al in the Japanese population [START_REF] Ikeda | Variants of dopamine and serotonin candidate genes as predictors of response to risperidone treatment in first-episode schizophrenia[END_REF] and inconsistent with Reynolds et al who have reported a negative symptom response in the Chinese population [START_REF] Reynolds | Pharmacogenetics of treatment in firstepisode schizophrenia: D3 and 5-HT2C receptor polymorphisms separately associate with positive and negative symptom response[END_REF].

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To the best of our knowledge our study is the first to assess these genetic associations in a large sample of South Indian population with the strengths of ethnic homogeneity, treatment homogeneity with the same atypical antipsychotic tested (risperidone) and definitive response criteria with prospective and cross-sectional assessment. However, previous association studies were reported with use of typical antipsychotics (chlorpromazine, haloperidol, bromperidol, fluphenazine, flupentixol), atypical antipsychotics (clozapine, olanzapine, aripiprazole, nemonapride, amisulpride, sulpride), few studies with mixed medications (typical and atypical) and only six studies have reported with risperidone therapy.

Our results deserve several comments with regard to potential weaknesses. First, short term evaluation of response (only four weeks of treatment may be too short to capture the sufficient variance in primary outcome measure. Of note, Kaur et al 2017 also detected no association between the DRD2 Taq1A and HTR2A (-1438A>G; T102 C) polymorphisms in association with treatment response to risperidone after 12 weeks of treatment in North Indian populations. Second, in the present study, categorical outcome criteria may appear arbitrary (20% improvement) and outcome was also studied using a continuous definition for the response. However, previous studies have used various response assessment scales with different cut off values and treatment durations to detect the association between genetic polymorphisms and antipsychotic response.

In conclusion, the results obtained from the present study are in line with recent studies by Kaur et al and Vijayan et al to suggest DRD2 (rs1799732, rs1800497), HTR2A (rs6311, rs6313) and HTR2C (rs3813929) genetic polymorphisms are not associated with antipsychotic response. DRD2, HTR2A and HTR2C are the important genes involved in the pharmacodynamics of risperidone and several studies have showed the importance of these genes in antipsychotic response. However, recent evidences indicate the role of epigenetic mechanisms in antipsychotic response. A study by Shi et al (2017) showed that methylation status of CYP3A4 gene has a significant role in response to risperidone therapy. Few studies have also suggested the association of gene expression levels from peripheral blood with antipsychotic response. However, no studies have been conducted to study the methylation Further studies are required to investigate the methylation pattern of promotor regions or

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gene regulatory regions for DRD2, HTR2A, HTR2C genes and their expression pattern in association with antipsychotic response.

Five

  millilitre of blood was collected from each patient to study the genetic polymorphisms in dopamine DRD2 (-141 C Ins/Del, Taq 1A) and serotonin [HTR2A (-1438 A/G, 102 C/T), HTR2C -759 C/T] receptor genes. DNA extraction was done using standard phenol-chloroform method and quantified by NanoDrop TM (ThermoFisher). Genotyping for genetic polymorphisms were done using real time thermocycler (ABI Prism 7300) with Taqman ® SNP probes for DRD2 genes rs1799732 Assay ID: 0150250174, rs1800497 Assay ID: C__7486676_10 and HTR2A rs6311 Assay ID: C__8695278_10, rs6313 Assay ID: C__3042197_1_ and HTR2C rs3813929 Assay ID: C__27488117_10 from Applied Biosystems (Foster City, CA, USA).

  al in which -1438A<G (G-0.59; A-0.41), and T102C (C-0.56; T-0.44) were reported in patients with SCZ from

Table . II Genotype association tests between DRD2, HTR2A and HTR2C genes polymorphisms and antipsychotic response to risperidone in the prospective and cross-sectional samples pooled (categorical definition of responders and non-responders)

 .

	DRD2 -141 C Ins/Del	Ins/Ins (n=177)	Ins/Del +Del/Del	Odds ratio	p value
	(rs1799732)		(n=133)	(95% CI)
	Responders	127	104	0.70 (0.4 -1.2)	0.2
	Non responders	50	29
		Fisher's exact test p<0.05 considered as significant
	DRD2 Taq1A	AA (n=35)	AG (n=132)	GG (n=153)	p value
	(rs1800497)
	Responders	24	104	110	0.2
	Non responders	11	28	43
		χ 2 =2.4, DF=2, p<0.05 considered as significant
	HTR2A -1438 A>G	AA (n=39)	AG (n=164)	GG (n=117)	p value
	(rs6311)
	Responders	31	126	81	0.2
	Non responders	8	38	36
		χ 2 =2.7, DF=2, p<0.05 considered as significant
	HTR2A T102C	CC (n=115)	CT (n=165)	TT (n=40)	p value
	(rs6313)
	Responders	80	124	34	0.1
	Non responders	35	41	6
		χ 2 =3.8, DF=2, p<0.05 considered as significant
	HTR2C -759 C>T	CC (n=65)	CT +TT (n=67)	Odds ratio	p value
	(rs3813929)			(95% CI)
	Females
	Responders	50	48	1.3 (0.6 -2.8)	0.5
	Non responders	15	19
	Males	C (n=120)	T (n=66)	Odds ratio	p value
				(95% CI)
	Responders	91	47	1.2 (0.6 -2.4)	0.4
	Non responders	29	19

Fisher's exact test p<0.05 considered as significant

ACKNOWLEDGEMENT

The study was supported by Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER) Intramural Research Grant (Grant No. JIP/Res/Intra-PhD/01/2014) and Indo-French Centre for the Promotion of Advanced Research (IFCPAR/ CEFIPRA) for promoting the collaboration (Grant No. IFC/4098/RCF2016/853) between JIPMER and University of Sorbonne universities, Paris, France.

CONFLICT OF INTEREST

Authors declared no conflicts of interest.

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Baptiste Brun-Cottan

Pauline Assemat

Vincent Doyeux

Franck Accadbled

Jérôme Sales De Gauzy

Roxane Compagnon

Pascal Swider

An energy approach describes spine equilibrium in adolescent idiopathie scoliosis

Keywords: Global spine, Scoliosis, Mechanical model, Functional minimization, Inverse problem, Medical imaging

The adolescent idiopathie scoliosis (AIS) is a 3D deformity of the spine whose origin is unknown and clinical evolution unpredictable. In this work, a mixed theoretical and numerical approach based on energetic considerations is proposed to study the global spine deformations. The introduced mechanical model aims at overcoming the limitations of computational cost and high variability in physical parameters. The model is constituted of rigid vertebral bodies associated with 3D effective stiffness tensors. The spine equilibrium is found using minimization methods of the mechanical total energy which circumvents forces and loading calculation. The values of the model parameters exhibited in the stiffness tensor are retrieved using a combination of clinical images post-processing and inverse algorithms implementation. Energy distribution patterns can then be evaluated at the global spine scale to investigate given time patient-specific features. To verify the reliability of the numerical methods, a simplified model of spine was implemented. The methodology was then applied to a clinical case of AIS (13-year-old girl, Lenke lA). Comparisons of the numerical spine geometry with clinical data equilibria showed numerical calculations were performed with great accuracy. The patient follow-up allowed us to highlight the energetic role of the apical and junctional zones of the deformed spine, the repercussion of sagittal bending in sacro-illiac junctions and the significant role of torsion with scoliosis aggravation. Tangible comparisons of output measures with clinical pathology knowledge provided a reliable basis for further use of those numerical developments in AIS classification, scoliosis evolution prediction and potentially surgical planning.

Introduction

The adolescent idiopathie scoliosis (AIS) is a multifacto rial disease potentially involving genetic, metabolic and mechanical aspects, and which affects up to 3% of the 10--16 years population with a significant female prevalence. AIS is characterized by the deviation of anatomical spine cur vature toward coupled 3D deformation involving abnormal bending and torsion. It generally shows a rapid evolution during the growth spurt (Riseborough and Wynne-Davies 1973) which can lead to respiratory and cardiac disorders. If orthotic treatment fails to stop progression which happens in 10% of AIS, the surgery is indicated. Scoliosis clinical management might improve the early diagnosis, the predic tion of its unstable evolution and deterministic information for the surgical planning.

The proposed methodology concemed a mechanical approach to explore the equilibrium of adolescent scoliotic spines. The causality of the disease is not studied. In the literature, several modeling strategies have been proposed to predict the mechanical response of a scoliotic spine. In van der [START_REF] Van Der Plaats | Numerical simulation of asymmetrically altered growth as initiation mechanism of scoliosis[END_REF], the authors developed a numerical finite element model to test a buckling hypothesis as initia tion of the AIS. The results were descriptive and comparison with clinical data remained to be performed. [START_REF] Lafage | 3d finite element simulation of Cotrel-Dubousset correction[END_REF] investigated an all spine model using adapted struc tural elements, but as pointed out by the authors, no physical model was developed to adjust the mechanical parameters used. [START_REF] Drevelle | Analysis of idiopathic scoliosis progression by using numerical simulation[END_REF] proposed a 3D FEM model to mimick AIS evolution, a study that was limited by the lack of follow-up to assess the clinical evolution of the spine. Despite significant progress in the anatomy characterization of AIS [START_REF] Fábio | A shared biomechanical environment for bone and posture development in children[END_REF]) and previously cited modeling efforts, most of studies resulted in qualitative propositions for scoliosis description.

In particular, the 3D generic approach of FEM finds its limitation in the size of the problem and the lack of knowledge in tissue properties and boundary conditions in vivo [START_REF] Stephen | Fluid flow and convective transport of solutes within the intervertebral disc[END_REF]Jebaseelan et al. 2012). Alternative discrete mechanical models of children spines involving soft tissues and muscles have been proposed (Schmid et al. 2019). To examine the soft tissues and, especially the intervertebral disk (IVD) role into the AIS time course, studies at reduced scale have been proposed. For instance, [START_REF] Stokes Ian | Analysis and simulation of progressive adolescent scoliosis by biomechanical growth modulation[END_REF] focused on the biomechanical interaction between the IVD and the vertebral body in scoliosis evolution. This hypothesis is supported by recent clinical studies which highlight the altered response of disk in AIS population [START_REF] Abelin-Genevois | Spino-pelvic alignment influences disc hydration properties after AIS surgery: a prospective MRI-based study[END_REF]Langlais et al. 2018). The poromechanical response of vertebral segments was numerically predicted using a dedicated substructuring technique to reduce computation costs [START_REF] Swider | Substructuring and poroelastic modelling of the intervertebral disc[END_REF]. As soft tissues are structures with maximal energy gradients [START_REF] Violas | Quantification of intervertebral disc volume properties below spine fusion, using magnetic resonance imaging, in adolescent idiopathic scoliosis surgery[END_REF][START_REF] Noailly | How does the geometry affect the internal biomechanics of a lumbar spine bi-segment finite element model? Consequences on the validation process[END_REF][START_REF] Swider | Substructuring and poroelastic modelling of the intervertebral disc[END_REF], multiple studies focused more specifically on the IVD characterization. Information about effective mechanical properties of IVD [START_REF] Newell | Biomechanics of the human intervertebral disc: a review of testing techniques and results[END_REF]) and some components of segment effective stiffness are available [START_REF] Schultz | Mechanical properties of human lumbar spine motion segments[END_REF][START_REF] Stokes Ian | Measurement of a spinal motion segment stiffness matrix[END_REF][START_REF] Meng | Incorporating six degree-of-freedom intervertebral joint stiffness in a lumbar spine musculoskeletal model-method and performance in flexed postures[END_REF]. However, these approaches involved ex vivo experimental measurements and none of them concerned the adolescent. Another strategy could be to identify in vivo properties at tissue scale (Jebaseelan et al. 2012;Langlais et al. 2018;[START_REF] Grace | Human internal disc strains in axial compression measured noninvasively using magnetic resonance imaging[END_REF]) but pediatric functional imaging are rarely available.

To address the lack of physical parameters availability and the numerical limitations, we aimed at developing a new mechanical and numerical model in order to characterize the overall scoliotic spine deformation. The progression of scoliosis is targeted after diagnosis and the cause of scoliosis initiation is not explored in this study. A 3D wired model that overcomes the previous limitations based on original key points was constructed. To achieve this modeling, it was hypothesized that the quasi-static evolution of scoliosis followed an energetic minimization law, and that an effective stiffness tensor which models deformable structure interacting at a vertebral segment scale existed. The values of this tensor were reconstructed based on clinical data using reverse analysis.

Materials and methods

In this section, we develop the wireframe model and detail the theoretical formulation based on energy minimization and numerical methods. This methodology is evaluated on a test case and applied to a clinical application on a 2 years follow-up of a 13-year-old scoliotic patient.

Parameterization of the vertebral segment

Parameterization of the spine is described in Fig. 1a, b associated with EOS ® exams used in the clinical application of paragraph 2.4.2. Vertebral bodies follow rigid body motions and by contrast, disks, vertebral endplates, facet joints, ligaments and other soft tissues connected with the vertebral segments are considered as deformable tissues. Figure 1c shows a model of vertebral segment S i including the deformable structure between two rigid vertebral bodies, V i and V i+1 .

To establish the kinematic description of the wireframe model, a unique orthonormal basis B i was associated to each vertebra i as shown in Fig. 1d. The vector i joined the centers O i and O ′ i of vertebral endplates of the vertebra i. The second vector i was defined by the orientation of the spinous process. The vector i warranted the condition of a direct orthonormal reference frame. A similar method defined B i+1 attached to i + 1.

Considering now a vertebral segment, composed of two vertebrae (i and i + 1 ) and an IVD, the two reference frames of vertebra i were R i (O i , B i ) and R i � (O i � , B i ) , and the ones of the vertebra i + 1 were R i+1 (O i+1 , B i+1 ) and R i � +1 (O i � +1 , B i+1 ).

The displacement continuity at the boundary of a vertebra and an IVD ensured the uniqueness of the points O i , O ′ i and led to the following linking equations for the local reference frames:

where i was a rotation matrix, expressing the orientation of a base B i+1 in B i , and i , ′ i , i+1 were the vector formed with the points O i , O ′ i , O i+1 and the point O 0 . Vector i depended on the deformation of the IVD and i∕R i = (l vi , 0, 0) , l vi being the length of a vertebrae. We considered that the matrix i was the result of 3 successive rotations of 3 angles noted i , i , i (details in "Appendix 1").

The kinematics of the vertebral body i + 1 was described, relative to i, in the reference frame R i by the position vector i∕R i involving the 6 components introduced in i∕R i and i :

(1)

� i -i = i , i+1 -� i = i , and B i+1 = i B i , (2) i∕R i = (T xi , T y i , T zi , i , i , i ) .
The concatenation of vectors i∕R i , noted ∕R i allowed the kinematics of the whole spine to be obtained iteratively.

To ease further developments and, especially management of spine boundary conditions and gravity loading (Sect. 2.2), the kinematic fields were also written into an absolute reference frame R A (O A , B A ) . The unique ortho- normal basis B A defined the frontal plane of the trunk in anatomical position, with O A located at the top of the first thoracic vertebra (T1). The basis B i of each vertebra was related to the global basis B A by: In Eq. ( 3), i was a unique rotation matrix resulting from cumulative rotations of subjacent vertebral bodies k = 1 … i.

(3)

B i = i ∏ k=1 k B A = i B A , with i = i ∏ k=1 k .
Fig We had, in the global base B A , the expression of the vec- tor i :

We called i∕R A the vector characterizing the displacement of the IVD such that: However, the position of the points O i , O i ′ allowed us to only know the location of each vertebral body. To keep track of the information concerning the orientation of the vertebrae (torsion), we also introduced i∕R A :

For further use, we called ∕R A the concatenation of the vectors i∕R A and i∕R A .

Governing equations of spine balance by minimization of total potential energy

The spine dynamics was studied in a time scale long enough to smooth daily variations and short enough to assume a closed and non dissipative system. Consequently, transient effects due to diffusion, convection, and structural damping could be averaged in the studied time window during which all forces could be considered conservative. The model achieves an extraction of mechanical parameters from clinical data at the specific time of the X-ray image capture. No growth model has been integrated. For each different clinical data, i.e., time point, a new numerization is performed from which we can deduce the length of the vertebrae and of the IVD, although the IVD growth is very limited for the patient studied as she is older than 12. The first hypothesis was that the spine mechanical behavior could be described by a succession of quasi-static states, at equilibrium in . The second hypothesis was that the nonlinear mechanical response eventually due to nonlinear material properties, large displacements or large strains, could be searched in the form of successive piecewise linear equilibria. This implied that associated forces derived from a local potential. The total potential energy V was the sum of internal strain energy of the isolated segment generated by the strain of the deformable structure between one vertebra relatively to the adjacent one and the work of external loading taking into account the distributed body weight (gravity) and the role of muscles and ligaments. Components of vector were nondimensionalized by the height of thoracic and lumbar spine of the patient.

The segment displacement field i was defined by the difference between the kinematics i and a reference energy

(4) i∕R A = l vi i ⋅ i . (5) i∕R A = i∕R A = i ⋅ i∕R i . (6) i∕R A = i ⋅ i state 0
i . The independent symmetric tensors i ( 6 × 6 ) and i ( 6 × 6 ) were, respectively, associated with local strain energy and potential energy of external mechanical forces. Therefore, the contribution of forces inside the vertebral segment and due to disk, muscles or any other tissue was included into the effective tensor . The contribution of forces outside the vertebral segment such as dorsal muscle, gravity, or other components such as braces, was included into the effective tensor . Vector i ( 1 × 6 ) represented the contribution of gravity forces. In the following, the variable will correspond to the components of the effective tensors ( and ) and to the reference state 0 . The dependence of the total potential energy V to and was defined as:

When considering energy from internal deformation of a vertebral segment, the displacement was expressed in local coordinates, with a relative unconstrained position of one vertebra to the adjacent one [Eq. ( 8)]. When considering the work of external forces, the description of the spine was developed in the absolute reference frame [Eq. ( 9)].

Using Eqs. ( 8) and (9), Eq. ( 7) became: Kinematics boundary conditions were imposed at the limits of the region of interest. Locations and orientations of extremal vertebral bodies were obtained from X-ray clinical exams and used as input into the absolute reference frame.

Numerical resolution

Resolution of the direct problem

Knowing a set of parameters , the admissible stationary kinematic field which minimized V was called eq ( ) and described the static equilibrium of the system. The stability conditions were described as:

(7) V( , ) = ∑ i i ⋅ i ⋅ i + ∑ i i ⋅ i ⋅ i - ∑ i i ⋅ i . (8) i∕R i = i∕R i -i 0 ∕R i , (9) i∕R A = i∕R A -i 0 ∕R A . (10) V( , ) = ∑ i i ∕R i ⋅ i ⋅ i∕R i + ∑ i i ∕R A ⋅ i ⋅ i∕R A - ∑ i i ⋅ i∕R A .
The problem of minimum search was strongly nonlinear, and the surface response of the function V(., ) ∶ u ↦ V( , ) was non convex. Solutions were found by implementing a trust-region algorithm and a Newton algorithm [Scipy library, Virtanen et al. (2019)] sequentially to accelerate convergence. Gradient ∇ V and hessian matrix ∇ ∇ V were also analytically calculated to minimize computation time.

Resolution of the inverse problem

For the clinical problem, we did not have access to mechanical tensors nor initial reference states. To identify the parameters and to test the robustness of our approach, we implemented an inverse problem methodology (Tarantola 2005). This was formulated by the set of Eqs. ( 12), ( 13) and ( 14) where eq ( ) was the kinematic equilibrium, c was the observed clinical equilibrium (EOS ® X-ray) and sol was the converged solution of mechanical parameters:

with Kk,k the mean value defined as:

Kk,k = 1 m ∑ i K k,k,i
and m the number of vertebral segment. The formulation was adapted to full effective tensors and . The problem size was reduced when clinical applications were considered and only diagonal terms of stiffness tensor were taken into account. Therefore, only those terms appeared in the penalty function c( ) described by Eq. ( 14).

The objective function f involved the distance function d and the penalty function c. The distance function weighted the norm of discrepancies between predicted equilibrium eq ( ) and clinical data c . The direct problem resolution provided eq ( ) at each incremental step. Matrix was used to attenuate the impact of values with smaller precision from the clinical data (rotation of vertebrae). Penalty function c( ) aimed at deforming the image surface of the objective function f in order to give more weight to the set of parameters minimizing the penalty function c. Mean values and (11) eq ( ) = argmin (V( , )).

(12) sol = argmin f ( eq ( ), ) with f ( eq ( ), ) = d( eq ( )) + c( ), (13) d( eq ( )) = ( eq ( ) -c ) ⋅ ⋅ ( eq ( ) -c ), (14) c( ) = 𝜖 1 6 � k=1 ( Kk,k -K prior k,k ) 2 + 𝜖 2 6 � k=1 m � i=1 (K k,k,i -Kk,k ) 2 + 𝜖 3 (‖ 0 -0 prior ‖ 2 ),
variances of tensors coefficients were, respectively, penalized in first term and second term of equation ( 14) using prior values as controls.

The inverse problem was solved using a Newton conjugate gradient algorithm (Scipy library) which required the first and second derivatives of the objective function f. The first derivation was obtained using the adjoint method detailed in "Appendix 2". The second derivation was evaluated numerically using finite differences, exploiting the multi-core potential of the computer to fill the hessian matrix ∇ ∇ f faster (linear acceleration).

The nondimensionalized difference a ec of eq and c expressed by Eq. ( 15) was an indicator of the methodology accuracy with a 0 value for a perfect fit.

Beyond the determination of the kinematic field, the methodology allowed us to determine the distribution patterns of energies using Eq. ( 7). The model proposed an energy method to circumvent the ignorance of forces, tissue properties, detailed anatomy and local boundary conditions, by assuming the existence of energy local potentials. As a result, distribution patterns of energies were determined using Eq. ( 10). In addition, forces considered as model output measures, could be calculated using Eq. ( 16) in post-processing thanks to equilibrium eq and effective parameters obtained from the inverse problem. However, we will not work with forces distribution in the spine in the following and only the energetic approach will be exploited as it includes more information, it circumvents the lack of loading knowledge at the boundary conditions and it is the starting point of our model.

Applications

Numerical test case of a 34 elements simplified spine

The vertical wireframe model was made of 34 elements corresponding to m = 17 vertebral bodies and deformable ele- ments as shown in Fig. 2a. Diagonal terms of were fixed to 1 and all other components were nil. The reference state was defined by i 0 ∕R i = (1, 0, 0, 0, 0, 0) , i = 1.17 in the local reference frames. This set of parameters was called ref in the following parts. Boundary conditions in the absolute reference frame were ∕R A (1) = (0, 0, 0, 0, 0, 0) and ∕R A (17) = (25, 0, 0, 0, 0, 0) . We solved the direct problem and verified its convergence. We ran it multiple times to obtain many possible ( 15)

a ec = ‖ eq -c ‖ ‖ c ‖ (16) i = i i∕R i + i ⋅ i∕R A + i equilibrium positions.
We then chose one of the equilibrium position, and used it as reference position c for the test on the inverse problem. We solved the inverse problem using this reference geometry and verified the convergence of the numerical solver first. Then we verified that the solution geometry eq and c were the same. Ultimately, we looked for a correspondence between the solution parameters and the reference ones.

Clinical application

Clinical data were extracted from a clinic radiological prospective monocentric study approved by the Institutional Review Board of the Ethic Committee (Toulouse University Hospital). Spine geometry in frontal and sagittal planes were obtained in vertical position by a low-dose X-ray EOS ® system. The rotation of vertebrae was calculated using the remoteness of the spinal process. The selected patient was A python ® custom-made script was implemented in ImageJ ® to build the wireframe description of the scoliotic spine from T1 to L5 and to build c for 34 elements. The boundary conditions were fixed in term of location and orientation in T1 and L5. The reference state was defined assuming an isotropic level dependent compression of deformable elements in standing position, i.e 0 = × c . This approximation of 0 was based on the mean size variation of healthy patient between morning and night [START_REF] Ludescher | T2-and diffusion-maps reveal diurnal changes of intervertebral disc composition: an in vivo MRI study at 1.5 Tesla[END_REF]Zhu et al. 2015).

The terms of effective mechanical tensors i and i were reduced to the diagonal terms and associated priors of Eq. ( 12) were adapted from the literature [START_REF] Meng | Incorporating six degree-of-freedom intervertebral joint stiffness in a lumbar spine musculoskeletal model-method and performance in flexed postures[END_REF]; [START_REF] Newell | Biomechanics of the human intervertebral disc: a review of testing techniques and results[END_REF]. The gravity loading Gi, followed the values introduced in [START_REF] Villemure | Biomechanical simulations of the spine deformation process in adolescent idiopathic scoliosis from different pathogenesis hypotheses[END_REF]. These choices still led to an inverse algorithm dealing with a size of 153 parameters. In this clinical application, the direct problem numerical resolution was starting close to u c contrary to the simplified spine model in Sect. 2.4.1 which explored the multiplicity of solutions. This methodology, in addition to asymmetric boundary conditions, lead to the uniqueness of equilibrium geometry Üe q • A sensitivity study bas been performed to evaluate the impact of errors on parameter calculations. Its main aspects are described in "Appendix 3".

Results

Numerical test case of a 34 elements simplified spine

The direct problem was geometrically under-constrained and several admissible equilibria for 11eq have been obtained from energy minimization. Four solutions or deformation modes, Üe q l• lle q2 , u eq 3 , lleq4, have been selected in Fig. 2b. The convergence criterion of energy balance minimization was lower than 10-12 • Calculations lasted between 0.2 s and 1 min CPU rime on a desk computer (Intel Xeon ® 3.6GHz).

The inverse problem algorithm was applied using u eq 1 as the targeted kinematic mode. It also played the role of uc previously introduced in paragraph 2.3.2. The initial vector of effective properties p, i.e., K i , B i and uf, was randomly generated with values between 0.5 X P re f and 1.5 X Pref• Without using the penalty fonction c(p), the converged solution Psol slightly differed from the target Pref as shown in Fig. 3. However, the converged solution geometry Üe q ! was perfect with a ec !::! O. When c(p) was used with priors corresponding with Pref • then both kinematic balance Üe q ! Fig. 3 Test case with 34 wire frame elements: comparison of a resolution of the inverse problem (IP) with and without penalty and effective properties Psol were predicted with a ec !::! 0 and the energy distribution fi tted the reference energy (Fig. 3). A good convergence was obtained, as shown in Fig. 2c with computation rimes lower than 1 h CPU time.

0 6'ô 1-< Q) "id 0 ....

Clinical application

For the clinical application, the fitting of spine balance Üe q on clinical data uc was excellent since a ec was lower than 3 x 10-3 (Fig. 4a-c). As shown in Fig. 4d the convergence of the inverse problem was obtained for calculations lasting between 2 and 8 h (CPU rime) with a convergence criterion lower than 10-8 • When the impact of distance fonction d and regularization fonction c (identical in 2014 and 2016) was exarnined, it was found that d(p 501 ) was always lower than one third of c(p 501 ). It showed that the penalty fonction was properly chosen and did not altered the main purpose of the algorithm to fit clinical and modeled geometries.

Table 1 summarizes the contribution of energy compo nents, i.e., compression, lateral bending, sagittal bending and torsion, to the total energy of the spine. Components were calculated into the segment local reference frame. In 2014, bending energy was predominant and that of lateral bending in the frontal plane greater than that in the sagit tal plane. The compression energy and torsion energy were found with a smaller percentage. The evolution in 2016 did not modify the prominence of bending but compressive energy decreased to the benefit of lateral and torsion energy.

The distribution patterns of energies along the spine are plotted in Fig. 5. Figure 5a shows an oscillation of the lateral bending, especially marked in the apical zone (vertebral segment S8, S9, S10, corresponding to vertebrae T8-Tl l) in 2014. For the evolving scoliosis in 2016, the energy peak strongly emerged and a noticeable increase was predicted in the lower junctional zone (S 14, cor responding to vertebrae L2-L3). The sagittal energy in Fig. 5b showed an almost steady distribution up to the junctional zone (S13, corresponding to Ll-L2) followed by a monotonie increase reaching its maximum value at the sacro-illiac junction (S 17, corresponding to L5-Sacrum). Scoliotic deformation exacerbated energy concentration in 2016. Concerning torsion energy Fig. 5c, the distribu tion was disrupted by a sudden increase before the apex (S5-S6, corresponding to T5-T7) and particularly marked in 2016. Maximum values were reached in the upper junc tional zone (S6, corresponding to T6-T7). It was also observed that the lower junctional zone (S 12, S 13, cor responding to Tl2-L2) showed a noticeable increase of torsion energy in 2016.

We initially hypothesized that a wireframe model based upon mechanical energy minimization might depict the spine balance in scoliosis time course. The governing equations and the associated numerical direct and inverse methods were first evaluated with a test case mimicking a simplified spine architecture, and second with a 2 years evolving scoliosis. The nonlinear response was searched in the form of successive piece-wise linear equilibria and in the vicinity of the equilibrium all forces derived from a local potential. Progressive results supported the proposed methodology and confirmed our central hypothesis.

We proposed the concept of mechanical effective tensor to condense, in a reduced number of scalars, the complex behavior of spine segments involving bones and soft tissues. Additionally, patient medical images constrained the inverse algorithm to check kinematic and mechanical balances to warrant the clinical relevance of the methodology. The penalization process guided the inverse problem resolution among multiple numerical admissible solutions.

Despite the concept of effective tensors, the amount of unknowns was still diminished to diagonal components because they played major roles. While accelerating numerical resolution, this choice provided convincing results compared to clinical diagnosis. However, the set of equations can predict the influence of coupled mechanical loading (i.e bending, torsion, compression) with enhanced clinical relevance by exploring the impact of extra diagonal terms potentially asymmetric. Especially, this could improve predicting the structure nonlinear response due to loading history.

The time scale of the clinical follow-up, allowed us to assume that the AIS evolution could be considered as being a steady-state mechanical problem at each time observation. Two snapshots at 2 year intervals were selected to establish the clinical relevance of our proposed methodology. The domain was restrained to thoracic and lumbar segments. Indeed, cervical spine balance is rarely deformed by AIS and it is mainly determined by the dynamical maintain of horizontal sight.

AIS deformation was interpreted in terms of mechanical energies and this objective quantification allowed completing the empirical clinical approach. As observed clinically, we found that the apical zone was playing a significant role in energy concentration, especially for lateral bending and torsion. Beyond that, the prominence of sagittal bending found in sacro-illiac junction was also corroborated by previous clinical studies [START_REF] Brink | Anterior-posterior length discrepancy of the spinal column in adolescent idiopathic scoliosis-a 3d CT study[END_REF]. The junction zones located on both sides of the apex observed in clinics by curve inversion showed intrinsic variations of energies and notably for torsion. Indeed, these zones are examined to establish clinical diagnosis, AIS classification and surgical planning.

In this article, the energy distribution obtained from available clinical data was calculated. This can be considered as the basis of further clinical studies on multicentric cohorts involving main types of AIS in Lenke classification and the concept of stable or unstable scoliosis. The implementation of specific algorithms will allow a prediction of scoliotic curve progression by the analysis of segments effective properties. In that case, the clinically enriched models will be considered as being predictive models.

  a 13-year-old girl diagnosed with a 1A scoliosis according to Lenke classification (Lenke et al. 2001) and a 40 • Cobb angle in 2014. The deformation progressed to 65 • and ended up to a T2-L2 surgical spinal fusion in 2016 (Fig. 1b for an illustration of Cobb angle).

Fig. 2

 2 Fig. 2 Numerical test case with 34 wireframe elements mimicking a simplified adolescent spine: a wireframe schematic view, b four solutions eq of the direct problem, c convergence of the inverse problem in term of objective function and residual

Fig. 4

 4 Fig. 4 Clinical application: 2 years follow-up of a 13-yearold girl (IA Lenke type, 40 ° Cobb angle in 2014, 65 ° Cobb angle in 2016). Spine balance obtained from clinical data and inverse problem resolution: a sagittal plane, b frontal plane, c transverse plane. Convergence of the inverse problem in d with residual on the Jeft and objective function/ values on the right

Table 1

 1 Clinical application: normalized percentage of total energy components in the wireframe mode) of the spine in 2014 and 2016 (13-year-old female patient, lA Lenke type)

	EOS ® exam date	Compressive Flexion	Laierai	Torsion
			extension bencling
	2014	18	52	19	10
	2016	10	54	25	12

Acknowledgements

The French Minister of Education and Research and The Children Hospital of Toulouse (France) are acknowledged for their assistance.

Compliance with ethical standards

Conflict of interest The authors declare that they have no conflict of interest.

Appendix 1: Rotation matrix

The derivative operator ∇ used on any function h( eq ( ), ) is defined as: Therefore, Calling ( , ) = (∇ V)( , ) , the definition of eq for every gives:

(20) ( eq ( ), ) = 0 therefore ∇ ( ( eq ( ), )) = 0, leading to the following equation: Using this result in Eq. ( 19) gives:

The vector -f eq eq -1

, often called , is the adjoint vector. It is the solution of a linear system, faster to solve than an explicit finite difference calculation to access the gradient ∇ f .

Appendix 3: Sensitivity study

In the proposed methodology, the spine balance was found by minimizing the total mechanical energy using EOS ® medical images from patient follow-up as input data. We assessed the impact of uncertainties of clinical data numerization on numerical prediction. The measurements errors were evaluated using ten numerizations of a single clinical image. The locations and orientations of seventeen vertebral bodies have been computed for each numerization. Inspired by Bayesian methodology, the geometry space was described with a probability law chosen to be normal, i.e., defined by mean value and standard deviation. The first and last decile of the distribution provided the envelops of the clinical geometry. The maximum distance between the envelops was evaluated at 5% of the maximum displacement from vertical spine in both frontal and sagittal direction.

The computation cost for the uncertainties propagation through the inverse problem was prohibitive. Therefore, we investigated the propagation of hypothetical uncertainties on the parameters, through the direct problem. The parameters uncertainties were chosen to be independent and following a normal distribution with a fixed mean and an arbitrary initial standard deviation (few percents of the mean). The deciles distribution of the computed equilibrium geometry eq was then compared with the deciles of clinical data from image numerization. The parameters standard deviation was iteratively updated to obtain a good match between clinical measurements envelops and equilibrium geometry envelops. After computation, the discrepancies between clinical envelops and equilibrium envelops was lower than 6% and was obtained with uncertainties on parameters characterized by standard deviation of 5% of the mean values. .

(22) ∇ f = -f eq eq -1

+ f

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Vincent Le Moigne
email: vincent.le-moigne@uvsq.fr

Clément Raynaud

Flavie Moreau

Christian Dupont

Jérôme Nigou

Olivier Neyrolles

Laurent Kremer

Jean-Louis Herrmann

Efficacy of bedaquiline, alone or in combination with imipenem, against Mycobacterium abscessus in C3HeB/FeJ mice

Mycobacterium abscessus lung infections remain difficult to treat. Recent studies have recognized the power of new combinations of antibiotics such as bedaquiline and imipenem although in vitro data have questioned this combination. We report that the efficacy of the bedaquiline plus imipenem treatment relies essentially on the activity of bedaquiline in a C3HeB/FeJ mice model of infection with a rough variant of M. abscessus. The addition of imipenem contributed at clearing the infection in the spleen.

Note (1074 words)

Mycobacterium abscessus is a rapidly growing mycobacterial species, whose infections remain very difficult to treat, due to the limited panel of available antibiotics (1). Among them, the -lactams, imipenem (IPM) and cefoxitin (FOX), are part of the M. abscessus multidrug therapy along with amikacin (AMK) and clarithromycin (CLR) [START_REF] Griffith | An official ATS/IDSA statement: diagnosis, treatment, and prevention of nontuberculous mycobacterial diseases[END_REF][START_REF] Haworth | British Thoracic Society guidelines for the management of non-tuberculous mycobacterial pulmonary disease (NTM-PD)[END_REF][START_REF] Floto | US Cystic Fibrosis Foundation and European Cystic Fibrosis Society consensus recommendations for the management of non-tuberculous mycobacteria in individuals with cystic fibrosis: executive summary[END_REF][START_REF] Lefebvre | Inhibition of the β-Lactamase Bla Mab by Avibactam Improves the In Vitro and In Vivo Efficacy of Imipenem against Mycobacterium abscessus[END_REF]. In addition, the development of specific -lactamase inhibitors, enhancing the efficacy of IPM in vitro and in vivo, broadens the use of IPM in M. abscessus drug therapy [START_REF] Kaushik | In Vitro Activity of the New β-Lactamase Inhibitors Relebactam and Vaborbactam in Combination with β-Lactams against Mycobacterium abscessus Complex Clinical Isolates[END_REF][START_REF] Dubée | β-Lactamase inhibition by avibactam in Mycobacterium abscessus[END_REF][START_REF] Dubée | Impact of β-lactamase inhibition on the activity of ceftaroline against Mycobacterium tuberculosis and Mycobacterium abscessus[END_REF]. Other studies highlighted the potential of testing new drug combinations that include IPM and are associated with increased efficacy against M. abscessus (6, 9, 10), yet questioning the relevance of the bedaquiline (BDQ) plus IPM combination [START_REF] Lindman | Bedaquiline eliminates bactericidal activity of β-lactams against Mycobacterium abscessus[END_REF]. BDQ targets the ATP synthase and exhibits activity against a wide panel of M. abscessus clinical isolates in vitro and in infected zebrafish, although its effect is bacteriostatic only [START_REF] Dupont | Bedaquiline inhibits the ATP synthase in Mycobacterium abscessus and is effective in infected zebrafish[END_REF]. A recent study suggested that, by reducing the intracellular pool of ATP in M. abscessus, BDQ suppresses the effect of IPM and FOX, although the effect of the BDQ plus IPM combination was considered additive [START_REF] Lindman | Bedaquiline eliminates bactericidal activity of β-lactams against Mycobacterium abscessus[END_REF]. This led the investigators to conclude that the addition of BDQ to a -lactam-containing regimen may negatively affect the treatment outcome [START_REF] Lindman | Bedaquiline eliminates bactericidal activity of β-lactams against Mycobacterium abscessus[END_REF]. In comparison, data from the hollow fiber model highlight that -lactam is the most active and important part of the M. abscessus regimen [START_REF] Ferro | Failure of the Amikacin, Cefoxitin, and Clarithromycin Combination Regimen for Treating Pulmonary Mycobacterium abscessus Infection[END_REF]. That these studies focused exclusively on the interaction of β-lactams and BDQ in vitro, confirmatory results in a pre-clinical animal model are warranted.

Herein, we explored the therapeutic efficacy of BDQ or IPM, alone or in combination, using the immunocompetent C3HeB/FeJ mouse model of M. abscessus infection. C3HeB/FeJ mice are highly susceptible to mycobacterial infections, particularly to Mycobacterium tuberculosis due to a deletion on the Ipr1 (Intracellular pathogen resistance 1) gene located in the locus called sst1 [START_REF] Kramnik | Genetic control of resistance to experimental infection with virulent Mycobacterium tuberculosis[END_REF][START_REF] Pan | Ipr1 gene mediates innate immunity to tuberculosis[END_REF]. All animal experiments were performed according to ethical guidelines and with ethical committee (N°047 with agreement A783223) agreement APAFIS#11465.

First, we evaluated, the in vitro interaction between BDQ and several β-lactams or CLR against M. abscessus CIP104536 strain in cation-adjusted Mueller-Hinton broth (CaMHB) (Becton-Dickinson, Le Pont-de-Claix, France) using a 2-dimensional microdilution checkerboard method, as previously described [START_REF] Ruth | A bedaquiline/clofazimine combination regimen might add activity to the treatment of clinically relevant non-tuberculous mycobacteria[END_REF][START_REF] Odds | Synergy, antagonism, and what the chequerboard puts between them[END_REF][START_REF] Li | Synergistic Interactions of MmpL3 Inhibitors with Antitubercular Compounds In Vitro[END_REF][START_REF]Susceptibility Testing of Mycobacteria, Nocardiae spp., and Other Aerobic Actinomycetes[END_REF]. Our results confirm that the -lactam plus BDQ combinations are indifferent, as it is the case with the CLR plus BDQ combination (Table 1).

Next, the performances of pulmonary and intravenous (IV) infection routes were compared in C3HeB/FeJ mice. Mice were infected intratracheally using agar bead-embedded bacteria to maintain a persistent infection, as reported previously for Pseudomonas aeruginosa [START_REF] Cigana | Tracking the immunopathological response to Pseudomonas aeruginosa during respiratory infections[END_REF]. A significant increase in mortality was noticed when mice were infected intratracheally with a solution of agar beads containing 2.10 5 CFUs/mouse in 50 µl, leading only to 40% of mouse survival at 14 days post-infection (dpi) (see Fig. S1A in the supplemental material) correlated with an important increase in the CFU at 14 dpi suggesting accelerated bacterial growth in the lungs (Fig. S1B). In contrast, persistence occurs for up to 25 days after IV infection with 10 6 CFU/mouse as evidenced by CFU counting after plating of the organ homogenates (Fig. 1 and Fig. S2A andS2B) although as soon as the injected dose is less than 10 6 CFU, persistence in the organs is reduced (Fig. S2B). This represents an important asset over previously described murine models, characterized by a more rapid bacterial clearance [START_REF] Bernut | In vivo assessment of drug efficacy against Mycobacterium abscessus using the embryonic zebrafish test system[END_REF][START_REF] Moigne | Bacterial phospholipases C as vaccine candidate antigens against cystic fibrosis respiratory pathogens: the Mycobacterium abscessus model[END_REF][START_REF] Ordway | Animal model of Mycobacterium abscessus lung infection[END_REF].

The IV route of infection was subsequently used to evaluate and compare the activity of BDQ and AMK. Because AMK is bactericidal against M. abscessus while BDQ is bacteriostatic in vitro, we wondered whether BDQ would be more effective than AMK in an in vivo infection model. CFU were significantly reduced in mice receiving 30 mg/kg BDQ (oral administration) as compared to mice treated with 150 mg/kg AMK (subcutaneous administration) in the lungs and the spleen at 12 and 25 dpi (Fig. 2A and2B). No significant differences were observed between the BDQ-or AMK-treated animals in the spleen at 12 dpi, but bacterial loads in these two groups were significantly lower compared to the control group (oral administration of DMSO) (Fig. 2C).

The efficacy of BDQ in this infection model prompted us to compare it with IPM (subcutaneous administration) either alone or as a companion drug, for 15 days of treatment. No significant differences were noticed between the animals treated with BDQ alone and the animals treated with BDQ plus IPM at 12 and 20 dpi, with the exception of the liver at 12 dpi (Fig. 3A to 3C), indicating that the overall activity of the BDQ plus IPM combination was mainly due to the intrinsic activity of BDQ. In general, BDQ alone or in combination with IPM exhibited an increased activity as compared to IPM in the liver and spleen but not in the lungs (Fig. 3). The spleens of treated and untreated mice were weighed as an additional marker of the effectiveness of the various treatments. These measures indicated that only treatments with BDQ plus IPM or IPM alone were associated with lower spleen weights, as compared to those of the untreated or BDQ-treated mice (Fig. 3D). Collectively, the reduced bacterial burden, together with the lower spleen weights represent a marker for improved outcome of the infection.

BDQ is a diarylquinoline approved by the Food and Drug Administration and the European Medicines Agency for the treatment of multidrug-resistant tuberculosis. It is bacteriostatic against M. abscessus in vitro, displaying MIC 50 of 0.125 µg/ml and a MIC 90 > 16 µg/ml, and ECOFF values demonstrates that BDQ exhibits moderate activity [START_REF] Ruth | A bedaquiline/clofazimine combination regimen might add activity to the treatment of clinically relevant non-tuberculous mycobacteria[END_REF][START_REF] Pang | In Vitro Activity of Bedaquiline against Nontuberculous Mycobacteria in China[END_REF]. Discordant results regarding the efficacy of BDQ were generated in various immunocompromised mouse models, raising the question of the influence of immunosuppression on antibiotic efficacy [START_REF] Lerat | In vivo evaluation of antibiotic activity against Mycobacterium abscessus[END_REF][START_REF] Obregón-Henao | Susceptibility of Mycobacterium abscessus to antimycobacterial drugs in preclinical models[END_REF]. However, efficient responses to BDQ were observed in other animal models, such as zebrafish [START_REF] Dupont | Bedaquiline inhibits the ATP synthase in Mycobacterium abscessus and is effective in infected zebrafish[END_REF]. Two studies reported poor or negative results for BDQ administration against NTM infected patients [START_REF] Philley | Preliminary Results of Bedaquiline as Salvage Therapy for Patients With Nontuberculous Mycobacterial Lung Disease[END_REF][START_REF] Zweijpfenning | Failure with acquired resistance of an optimised bedaquiline-based treatment regimen for pulmonary Mycobacterium avium complex disease[END_REF]. However, recent studies showed that the activity of BDQ can be potentiated with adjunctive therapy, by so improving BDQ-based treatments [START_REF] Ruth | A bedaquiline/clofazimine combination regimen might add activity to the treatment of clinically relevant non-tuberculous mycobacteria[END_REF][START_REF] Viljoen | Verapamil Improves the Activity of Bedaquiline against Mycobacterium abscessus In Vitro and in Macrophages[END_REF]. This study provides evidence that the BDQ plus IPM combination remains superior to IPM alone and equivalent to BDQ alone as judged by the comparable bacterial clearance in the spleens of the mice treated with BDQ plus IPM as compared to BDQ alone.

In summary, the IPM plus BDQ combination enhances the clearance of the infection. This supports also the importance of evaluating antibiotic activity in combination rather than separately against this highly drug-resistant mycobacterium. (£) Interaction between the two compounds was defined as synergistic when FICI value was ≤0.5, indifferent when 0.5< FICI ≤4, and antagonistic when FICI was >4.

(&) Values showed in the table are the mean of four independent experiments ±SD.

Legend to figures

Figure 1. Bacterial persistence of M. abscessus CIP 104536 T (rough variant) in the lungs, spleen and liver of C3HeB/FeJ mice after infection in the tail vein with 10 6 CFU/mouse in a total volume of 200 µl of water containing 0.9% sodium chloride. The following day, three mice were euthanized and whole organs were harvested to determine baseline bacterial burden. Mouse lungs, spleens and livers were homogenized, serially diluted and plated onto VCAT (Vancomycin, Colistin sulfate, Amphotericin B, and Trimethoprim) chocolate agar plates (BioMérieux, France) and incubated for 5-6 days at 37°C prior to CFU count. Results are expressed as the log 10 units of CFU at 1, 12 and 25 dpi.

(

  *) MIC were evaluated by REMA checkerboard assay in cation-adjusted Mueller-Hinton broth (CaMHB) (Becton-Dickinson, Le Pont-de-Claix, France). 10 5 bacteria were diluted in Mueller-Hinton media (Sigma-Aldrich). Plates were incubated for 3 days at 30°C then 20 µL (10% v/f) of Resazurin 0.025% were added to the wells and plates were incubated overnight at 30°C. ($) The fractional inhibitory concentration index (FICI) was calculated as follows: FICI = (MIC drug A in combination/MIC drug A alone) + (MIC drug B in combination/MIC drug B alone), where drug A was bedaquiline (BDQ) and drug B was clarithromycin (CLR, Sigma-Aldrich, France), imipenem (IPM, Mylan S.A.S, France), cefoxitin (FOX, Panpharma, France) or ampicillin (AMP, Euromedex, France).

Figure 2 .

 2 Figure2. M. abscessus R-infected C3HeB/FeJ mice (9.2×10 5 CFU/mouse) treated with Bedaquiline (BDQ) or Amikacin (AMK). Bacterial counts in the lungs (A), liver (B) and spleen (C) of C3HeB/FeJ mice infected IV, as described in Fig.1. Antibiotic treatment began at 2 dpi. Mice were treated starting on day 2 for 7 days (D12) or 17 days (D25) by daily subcutaneous injections of 150 mg/kg AMK (Mylan laboratories) in saline solution or daily oral gavage of 30 mg/kg BDQ at in a total volume of 200 µl (BDQ solution in DMSO was diluted in 20 % 2-hydroxypropyl--cyclodextrin). A control group received a daily subcutaneous injection of saline and oral gavage of DMSO containing 20 % 2-hydroxypropyl--cyclodextrin. All solutions were administered five times weekly for latter time point. Mice were euthanized 3 days after antibiotic cessation to allow antibiotic clearance. Furthermore, given the long half-life and high protein binding capacity of BDQ, spleens, livers and lungs from drug-treated and control mice were homogenized in water supplemented with 10% bovine serum albumin (30) before dilution. Experimental groups of mice were evaluated for bacterial burden on day 1 (before treatment started), 12 and 25 as described in Fig.1. n = 5 mice were used per experiment and bacterial load in each group are expressed as log 10 units of CFU (± SD) cells. Differences between means were analyzed by two-way ANOVA and the Tukey post-test, allowing multiple comparisons. n.s. = non-significant, * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001. Experiment was realized once. Figure3. M. abscessus R-infected C3HeB/FeJ mice treated (2.7×10 5 CFU/mouse) by Bedaquiline (BDQ), Imipenem (IPM) or BDQ plus IPM. Bacterial loads in the lungs (A), liver (B) and spleen (C) were determined as reported in Fig.1. Relative weight of spleen to mouse weight are shown in (D). Antibiotic treatment began 2 days after infection. Mice were treated starting on day 2 for 7 days (D12) or 13 days (D20) with twice daily (i.e. every 12 h) subcutaneous injection of IPM (MSD laboratories, France) in saline solution at 100 mg/kg or daily oral gavage of BDQ as described in Fig.2or both IPM plus BDQ. Experimental groups of mice were evaluated for bacterial burden on day 1 (before treatment started), 12 and 20 as described in Fig.1. (D) Mouse spleens were weighed at 1, 12 and 20 dpi. The value represents the relative weight of each spleen relative to the weight of the mouse from which they were collected. n = 5 mice were used per experiment and bacterial load in each group are expressed as log 10 units of CFU (± SD) cells. Differences between means were analyzed by two-way ANOVA and the Tukey post-test, allowing multiple comparisons. n.s. = non-

Figure 3 .

 3 Figure2. M. abscessus R-infected C3HeB/FeJ mice (9.2×10 5 CFU/mouse) treated with Bedaquiline (BDQ) or Amikacin (AMK). Bacterial counts in the lungs (A), liver (B) and spleen (C) of C3HeB/FeJ mice infected IV, as described in Fig.1. Antibiotic treatment began at 2 dpi. Mice were treated starting on day 2 for 7 days (D12) or 17 days (D25) by daily subcutaneous injections of 150 mg/kg AMK (Mylan laboratories) in saline solution or daily oral gavage of 30 mg/kg BDQ at in a total volume of 200 µl (BDQ solution in DMSO was diluted in 20 % 2-hydroxypropyl--cyclodextrin). A control group received a daily subcutaneous injection of saline and oral gavage of DMSO containing 20 % 2-hydroxypropyl--cyclodextrin. All solutions were administered five times weekly for latter time point. Mice were euthanized 3 days after antibiotic cessation to allow antibiotic clearance. Furthermore, given the long half-life and high protein binding capacity of BDQ, spleens, livers and lungs from drug-treated and control mice were homogenized in water supplemented with 10% bovine serum albumin (30) before dilution. Experimental groups of mice were evaluated for bacterial burden on day 1 (before treatment started), 12 and 25 as described in Fig.1. n = 5 mice were used per experiment and bacterial load in each group are expressed as log 10 units of CFU (± SD) cells. Differences between means were analyzed by two-way ANOVA and the Tukey post-test, allowing multiple comparisons. n.s. = non-significant, * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001. Experiment was realized once. Figure3. M. abscessus R-infected C3HeB/FeJ mice treated (2.7×10 5 CFU/mouse) by Bedaquiline (BDQ), Imipenem (IPM) or BDQ plus IPM. Bacterial loads in the lungs (A), liver (B) and spleen (C) were determined as reported in Fig.1. Relative weight of spleen to mouse weight are shown in (D). Antibiotic treatment began 2 days after infection. Mice were treated starting on day 2 for 7 days (D12) or 13 days (D20) with twice daily (i.e. every 12 h) subcutaneous injection of IPM (MSD laboratories, France) in saline solution at 100 mg/kg or daily oral gavage of BDQ as described in Fig.2or both IPM plus BDQ. Experimental groups of mice were evaluated for bacterial burden on day 1 (before treatment started), 12 and 20 as described in Fig.1. (D) Mouse spleens were weighed at 1, 12 and 20 dpi. The value represents the relative weight of each spleen relative to the weight of the mouse from which they were collected. n = 5 mice were used per experiment and bacterial load in each group are expressed as log 10 units of CFU (± SD) cells. Differences between means were analyzed by two-way ANOVA and the Tukey post-test, allowing multiple comparisons. n.s. = non-

Figure 1 .

 1 Figure 1

Figure 2 .

 2 Figure 2

Figure 3 .

 3 Figure 3. M. abscessus R-infected C3HeB/FeJ mice (2.7×10 5 CFU/mouse) treated by Bedaquiline (BDQ), Imipenem (IMP) or BDQ plus IPM. Bacterial loads in the lungs (A), liver (B) and spleen (C) were determined as reported in Fig. 1. Relative weight of spleen to mouse weight are shown in (D). Antibiotic treatment began 2 days after infection. Mice were treated starting on day 2 for 7 days (D12) or 13 days (D20) with twice daily (i.e. every 12 h) subcutaneous injection of IPM (MSD laboratories, France) in saline solution at 100 mg/kg or daily oral gavage of BDQ as described in Fig. 2 or both IMP+BDQ. Experimental groups of mice were evaluated for bacterial burden on day 1 (before treatment started), 12 and 20 as described in Fig. 1. (D) Mouse spleens were weighed at 1, 12 and 20 dpi. The value represents the relative weight of each spleen relative to the weight of the mouse from which they were collected. n = 5 mice were used per experiment and bacterial load in each group are expressed as log 10 units of CFU (± SD) cells. Differences between means were analyzed by two-way ANOVA and the Tukey post-test, allowing multiple comparisons. n.s. = non-significant, * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001. Experiment was realized once.

Table 1 .

 1 Interaction between bedaquiline and other drugs against M. abscessus CIP104536 T

	Interaction with BDQ

ACKNOWLEDGMENTS

Bedaquiline was a kind gift from Dr C. Happel (NIH AIDS Reagent Program, USA) and from Dr N. Lounis (from Janssen Pharmaceuticals, Beerse, Belgium). We acknowledged Pr N.

Véziris and Pr J. van Ingen for helpful comments and critical reading of the manuscript. We thank the members of the Genotoul core facility ANEXPLO (IPBS, Toulouse) for animal experiments. This study was supported by INSERM, University of Versailles Saint Quentin en Yvelines, by the Association Gregory Lemarchal and Vaincre la Mucoviscidose (RIF20180502320) to LK and JLH, the Agence Nationale de la Recherche (MyCat ANR-15-CE18-0007-02) to LK, by CNRS, University of Toulouse, Agence Nationale de la Recherche /Programme d'Investissements d'Avenir (ANR-11-EQUIPEX-0003), and the Bettencourt Schueller Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

AUTHOR CONTRIBUTIONS STATEMENT

JLH, JN and ON designed the project and experiments; VLM, CR, FM and CD performed the experiments; VLM, CR, JN, ON, LK and JLH wrote and corrected the manuscript.

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