Cation Transporter Blocker

Alejandro Orrico

Laetitia Chausset-Boissarie

Rodolphe Alves De Sousa

Sara Rezai Amin

Vincent Vialou

Franck Louis

Assia Hessani

Patrick M Dansette

Teodoro Zornoza

Carole Gruszczynski

Bruno Giros

Bruno P Guiard

Francine Acher

Nicolas Pietrancosta
email: nicolas.pietrancosta@parisdescartes.fr

Sophie Gautron
email: sophie.gautron@upmc.fr

LONG-TERM ANTIDEPRESSANT EFFICACY OF A SELECTIVE ORGANIC

Keywords: organic cation transporter, prodrug, antidepressant, depression model, behavior

Current antidepressants act principally by blocking monoamine reuptake by high-affinity transporters in the brain. However, these antidepressants show important shortcomings such as slow action onset and limited efficacy in nearly a third of patients with major depression disorder. Here, we report the development of a prodrug targeting organic cation transporters (OCT), atypical monoamine transporters recently implicated in the regulation of mood. Using molecular modeling, we designed a selective OCT2 blocker, which was modified to increase brain penetration. In a rodent model of chronic depression induced by corticosterone exposure, daily administration of this compound, H2-cyanome, induced positive effects on several behaviors mimicking symptoms of depression, including anhedonia, anxiety, social withdrawal, and memory impairment. Importantly, in this validated model, H2-cyanome compared favorably with the classical antidepressant fluoxetine, with a faster action on anhedonia and better anxiolytic effects. Integrated Z-scoring across these depression-like variables revealed a lower depression score for mice treated with H2-cyanome than for mice treated with fluoxetine for 3 weeks. Repeated H2-cyanome administration increased VTA dopaminergic neuron firing, which may underlie its rapid action on anhedonia. H2-cyanome also modulated in a similar way than fluoxetine several intracellular signaling pathways previously involved in antidepressant response. Our findings provide proof-of-concept of long-term antidepressant efficacy of an OCT blocker, and a mechanistic framework for the development of new classes of antidepressants and therapeutic alternatives for resistant depression and other psychiatric disturbances such as anxiety.

Introduction

Depression is a common and disabling disorder worldwide, with considerable clinical, social, and economic impact [START_REF] Kessler | Lifetime prevalence and age-of-onset distributions of mental disorders in the World Health Organization's World Mental Health Survey Initiative[END_REF] . For over a half century, major depressive disorder has been managed primarily with medications modulating aminergic neurotransmission. This has provided the underlying rationale for the enduring monoamine hypothesis, which stipulates that depression results from an imbalance in serotonin (5-hydroxytryptamine, 5-HT), norepinephrine (NE), and/or dopamine (DA) signaling in the brain, which may be restored by long-term antidepressant treatment [START_REF] Wong | Research and treatment approaches to depression[END_REF] . Most standard antidepressants, such as selective serotonin reuptake inhibitors (SSRIs) and norepinephrine-serotonin reuptake inhibitors (NSRIs), target highaffinity transporters for 5-HT and NE located in the plasma membrane of nerve terminals.

However, these current treatments show important limitations, such as slow action onset and adverse side-effects. More importantly, they do not always lead to positive outcomes, and about a third of major depression patients do not respond satisfactorily to antidepressant therapy [START_REF] Rush | Acute and longer-term outcomes in depressed outpatients requiring one or several treatment steps: a STAR*D report[END_REF] . In this context, we speculated that targeting atypical monoamine transporters such as the organic cation transporters (OCT) could constitute a relevant strategy for the discovery and development of innovative antidepressant drugs.

OCT are facilitated diffusion transporters that participate in the absorption and clearance of various endogenous compounds and xenobiotics, by mediating their vectorial transport in renal, hepatic and placental cells [START_REF] Koepsell | Polyspecific organic cation transporters: structure, function, physiological roles, and biopharmaceutical implications[END_REF] . These transporters can transport with low affinity (in the micromolar range) the biogenic monoamines DA, 5-HT, NE and histamine [START_REF] Grundemann | Transport of monoamine transmitters by the organic cation transporter type 2, OCT2[END_REF][START_REF] Wu | Identity of the organic cation transporter OCT3 as the extraneuronal monoamine transporter (uptake2) and evidence for the expression of the transporter in the brain[END_REF][START_REF] Amphoux | Differential pharmacological in vitro properties of organic cation transporters and regional distribution in rat brain[END_REF] .

Two subtypes in particular, OCT2 and OCT3, are expressed in the central nervous system [START_REF] Wu | Identity of the organic cation transporter OCT3 as the extraneuronal monoamine transporter (uptake2) and evidence for the expression of the transporter in the brain[END_REF][START_REF] Amphoux | Differential pharmacological in vitro properties of organic cation transporters and regional distribution in rat brain[END_REF][START_REF] Busch | Human neurons express the polyspecific cation transporter hOCT2, which translocates monoamine neurotransmitters, amantadine, and memantine[END_REF][START_REF] Bacq | Organic cation transporter 2 controls brain norepinephrine and serotonin clearance and antidepressant response[END_REF] , where they contribute to modulate mood-related functions such as anxiety, response to stress and antidepressant efficacy [START_REF] Bacq | Organic cation transporter 2 controls brain norepinephrine and serotonin clearance and antidepressant response[END_REF][START_REF] Vialou | Altered aminergic neurotransmission in the brain of organic cation transporter 3-deficient mice[END_REF][START_REF] Wultsch | Decreased anxiety in mice lacking the organic cation transporter 3[END_REF][START_REF] Courousse | Brain organic cation transporter 2 controls response and vulnerability to stress and GSK3beta signaling[END_REF][START_REF] Courousse | Role of organic cation transporters (OCTs) in the brain[END_REF] . Brain OCT have been proposed to be an alternate monoamine clearance system, prevailing in areas lacking the high-affinity transporters, or when these transporters are saturated or inhibited, for instance after antidepressant administration [START_REF] Bacq | Organic cation transporter 2 controls brain norepinephrine and serotonin clearance and antidepressant response[END_REF][START_REF] Courousse | Role of organic cation transporters (OCTs) in the brain[END_REF][START_REF] Daws | Unfaithful neurotransmitter transporters: focus on serotonin uptake and implications for antidepressant efficacy[END_REF] .

2

In this study, we developed a novel prodrug targeting OCT, on the assumption that it could exert antidepressant effects by engaging mechanisms differing from high-affinity transporter blockade.

Known OCT inhibitors cover a wide range of compounds, of varied efficacy and often low selectivity [START_REF] Koepsell | Polyspecific organic cation transporters: structure, function, physiological roles, and biopharmaceutical implications[END_REF][START_REF] Courousse | Role of organic cation transporters (OCTs) in the brain[END_REF] . We selected disprocynium 24 (D24), an isocyanine derivative, as lead compound for pharmacomodulation [START_REF] Russ | Isocyanines and pseudoisocyanines as a novel class of potent noradrenaline transport inhibitors: synthesis, detection, and biological activity[END_REF] . D24 is a potent and relatively selective OCT inhibitor, interacting with a putative high-affinity binding site of these transporters [START_REF] Gorbunov | High-affinity cation binding to organic cation transporter 1 induces movement of helix 11 and blocks transport after mutations in a modeled interaction domain between two helices[END_REF] and affinity constants ranging from 15 to 280 nM for brain OCT [START_REF] Grundemann | Molecular identification of the corticosterone-sensitive extraneuronal catecholamine transporter[END_REF][START_REF] Amphoux | Inhibitory and facilitory actions of isocyanine derivatives at human and rat organic cation transporters 1, 2 and 3: a comparison to human alpha 1-and alpha 2adrenoceptor subtypes[END_REF] . Nevertheless, this derivative has limitations for preclinical studies or clinical use. Cyanine dyes are believed to diffuse poorly across the brain-blood barrier and have significant effects on peripheral organs [START_REF] Amphoux | Inhibitory and facilitory actions of isocyanine derivatives at human and rat organic cation transporters 1, 2 and 3: a comparison to human alpha 1-and alpha 2adrenoceptor subtypes[END_REF][START_REF] Russ | Cyanine-related compounds: a novel class of potent inhibitors of extraneuronal noradrenaline transport[END_REF][START_REF] Graefe | 1,1'-Diisopropyl-2,4'cyanine (disprocynium24), a potent uptake2 blocker, inhibits the renal excretion of catecholamines[END_REF][START_REF] Trendelenburg | The extraneuronal uptake and metabolism of catecholamines[END_REF] . Specifically, D24 inhibits potently α-adrenergic receptors [START_REF] Amphoux | Inhibitory and facilitory actions of isocyanine derivatives at human and rat organic cation transporters 1, 2 and 3: a comparison to human alpha 1-and alpha 2adrenoceptor subtypes[END_REF] , and affects OCT-mediated catecholamine clearance in sympathetically-innervated tissues [START_REF] Russ | Cyanine-related compounds: a novel class of potent inhibitors of extraneuronal noradrenaline transport[END_REF][START_REF] Trendelenburg | The extraneuronal uptake and metabolism of catecholamines[END_REF] . We addressed these issues by combining molecular modeling and a prodrug delivery approach, to generate a compound with increased selectivity for OCT2. This prodrug was evaluated for its long-term antidepressant efficacy in a validated model of chronic depression, in comparison with the classical antidepressant fluoxetine. The robust behavioral effects of the prodrug, and its action on ventral tegmental area (VTA) dopaminergic neuron firing and intracellular signaling pathways demonstrates a high therapeutic potential for treatment of depressive disorders.

Material and methods

Homology modeling and drug design

Three-dimensional (3D) OCT and adrenoceptor models were built from sequence alignments and crystallographic atomic coordinates of XylE (PDB code 4GBY) and a combination of β1 adrenoceptor (PDB codes 3ZPR and 3D4S) and 5-HT1B receptor (PDB code 4IAR) using MODELER 9.0 (Discovery Studio 2016, BIOVIA, Dassault Systèmes, Vélizy-Villacoublay, France). The models were minimized and docking calculations were performed using CDOCKER [START_REF] Wu | Detailed analysis of grid-based molecular docking: A case study of CDOCKER-A CHARMm-based MD docking algorithm[END_REF] . The poses showing the lowest energy were retained and clustered according to their binding mode. 3D snapshots of the protein-ligand complexes were generated using BIOVIA DS Visualizer. Synthesis protocols, compounds characterization and full modeling and drug design procedures are detailed in supplementary materials and methods.

In vivo detection of H2-cyanome

Brain or plasma samples were homogenized in 33% acetonitrile. Analyses were performed on a high-performance liquid chromatography-mass spectrometry (HPLC-MS/MS) system consisting of an AmaZon SL ion trap mass spectrometer (Bruker Daltonics GmbH, Bremen, Germany) equipped with an ESI interface and an Ultimate 3000 device (Dionex, Thermo Fisher Scientific, Sunnyvale, CA), a thermostated autosampler and column compartment and an UV detector. Data were acquired and processed using Hystar and the extension QuantAnalysis (version 3.2, Bruker Daltonics GmbH). HPLC separation was performed with an A/B gradient (A: 10 mM ammonium acetate, pH 4.6, and B: CH 3 CN/HCOOH [999:1, vol/vol]). The column was equilibrated with 20% B for 5 min, followed by a linear gradient to B over 10 min, and 4 min in B. The column was then equilibrated with a second linear gradient from 100 to 20% B over 1 min and an isocratic concentration of 20% B over 8 min.

The analysis was performed in the reversed phase mode (ACE Excel 2 C18-Amide, 2 µm, 2.1 ×150 mm, Ref. EXL-1012-1502U, AIT, Houilles, France), and detection was carried out in MS mode with UV wavelength scaled at 220 nm.

Behavioral studies

Animals

Male C57BL6/J mice were used for the experiments. Most behavioral studies were performed during the inactive phase (09:00-13:00) with age-matched (8-16 weeks) mice. Animal care and experiments were conducted in accordance with the European Communities Council Directive for the Care and the Use of Laboratory Animals (2010/63/UE) and approved by the French ethical committee (#5786-2016062207032685).

Model of chronic depression

To induce a chronic depression-like state, individually housed mice were administered corticosterone in drinking water (35 µg ml-1; Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) dissolved in 0.45% (wt/vol) hydroxypropyl-b-cyclodextrin (Sigma-Aldrich) as previously reported [START_REF] Bacq | Organic cation transporter 2 controls brain norepinephrine and serotonin clearance and antidepressant response[END_REF] . Fluoxetine (15 mg/kg/day; LKT laboratories, St Paul, MN, USA) or H2cyanome (0.1 mg/kg/day) was administered intraperitoneally (i.p.) daily during the last 3 weeks of the corticosterone regimen. Mice were tested for sucrose consumption, social interaction, open-field, elevated O-maze and object location test, before antidepressant treatment and after 10 days and 3 weeks of treatment. The coat state was assessed weekly as a measure of motivation toward self-care. It was evaluated as the sum of the score of different parts of the body, ranging between 0 for a well-groomed coat and 1 for an unkempt coat for head, neck, dorsal/ventral coat, tail, and forepaws/hindpaws. For the sucrose preference test, singled-house mice were first habituated for 48 h to drink water from two bottles. On the following 3 days, the mice could choose between a water bottle and a 1% (wt/vol) sucrose solution bottle, with the position switched daily. Sucrose solution intake for 24 h was measured during the last 2 days and expressed as a percentage of the total amount of liquid ingested. The elevated O-maze consisted of an annular runway positioned 40 cm above the floor and divided into two opposing 90º closed sectors and two 90º open sectors. Mice were individually placed in the closed sector and their behavior recorded over a 5-min period. The time spent in each sector and the number of sector entries (a sector entry was defined as all four paws being placed in a sector) were determined by video tracking (Viewpoint, Lyon, France). The social interaction test was performed in a white open-field (42 x 42 cm) containing an empty wire mesh cage (10 x 6.5 cm) located at an extremity of the field in a low luminosity environment (25 lux). Individual mice were allowed to explore the open-field for two consecutive sessions of 2.5 min. During the second session, an unfamiliar mouse was introduced into the wire mesh cage. Between the two sessions, the test mouse was placed back into its home cage for approximately one minute. The time spent by the test mouse in the interaction zone, defined as an 8-cm-wide region surrounding the mesh cage, was measured in both sessions by video tracking (Viewpoint). For the object location test, the mice were habituated during two successive days to an open-field containing an intra-field cue (one wall covered with black and white stripes). Each mouse was allowed to freely explore the open-field for a 30-min period on day 1 and for two 10-min sessions separated by 5 h on day 2. On the third day, the test mouse was allowed to explore for 5 min two identical objects (5 x 2.5 cm) positioned in two adjacent corners of the open-field (acquisition phase) then returned to its home cage for 1 h. For the sample phase trial, one of the two objects was of sucrose intake in the sucrose preference test, time of interaction in social interaction test, and coat state). Test z values were calculated by averaging individual Z-scores. Z-score methodology was used to combine results across the different tests and the data was normalized as previously described [START_REF] Guilloux | Integrated behavioral z-scoring increases the sensitivity and reliability of behavioral phenotyping in mice: relevance to emotionality and sex[END_REF] . Integrated z-scores were calculated after 3-week treatment with fluoxetine or H2-cyanome.

Behavioral evaluation of acute treatment with H2-cyanome

For the evaluation of its acute effects in the open-field, the elevated-O maze and forced-swim tests, various doses H2-cyanome were dissolved in saline (0.9% NaCl [wt/vol]) and administered intraperitoneally at a volume of 10 ml/kg 30 min before each test. For the forced-swim test, the mice were placed individually in a glass beaker filled with 25±1 °C water to a depth of 12 cm. The duration of immobility (time during which the mice make only minimal movements to stay afloat) was recorded during the last 4 min of the 6-min testing period, after 2 min of habituation.

In vivo electrophysiological recordings of ventral tegmental area (VTA) DA neurons

Mice were anesthetized with chloral hydrate (400 mg/kg i.p.) and placed into a stereotaxic frame. Extracellular recordings of DA neurons in the VTA were performed using singlebarreled glass micropipettes (Stoelting, Dublin, Ireland) preloaded with a 2 M NaCl solution to obtain an impedance ranging between 6-9 MΩ. Electrical signals were amplified by a high-impedance amplifier (Bak Electronics, Umatilla, OR, USA) and monitored visually with an oscilloscope (Tektronix, Beaverton, OR, USA) and audibly through an audio monitor (A.M. Systems Inc., Sequim, WA, USA). The signal was digitalized, sampled at 25 kHz and recorded on a computer using Spike2 software (Cambridge Electronic Design, Cambridge, UK). To record VTA DA neurons, the glass micropipette was positioned using the following coordinates (in mm from bregma): AP: 3.5 to 3.1 mm, L: ±0.5 to 0.7 mm, V: -3.5 to -4.5 mm. Spontaneously active dopaminergic neurons were identified on the basis of previously established electrophysiological in vivo criteria [START_REF] Grace | Intracellular and extracellular electrophysiology of nigral dopaminergic neurons--3. Evidence for electrotonic coupling[END_REF] . At least 2 minutes of spontaneous baseline electrophysiological activity were recorded before systemic injection of H2-cyanome alone or in combination with cocaine (30 mg/kg in a final volume of 100 µl). The firing rate of VTA DA neurons was then determined until neuronal activity stabilization, thereby allowing the determination of the percent of change of basal firing rate.

Western blots

Whole tissue extracts were prepared from bilateral punches (1-1.

Results

Development of a selective OCT prodrug

To develop a new potent and selective OCT ligand, 3D homology models of the human α2adrenergic receptor (Figure1b), an unwanted target, and of human OCT2 (Figure 1c) were generated from selected templates (Figure b-d). The OCT2 model generated was coherent with previous models [START_REF] Dakal | Structural modeling of human organic cation transporters[END_REF][START_REF] Pan | A new chemotype inhibitor for the human organic cation transporter 3 (hOCT3)[END_REF] , and interactions defined in functional studies [START_REF] Gorbunov | High-affinity cation binding to organic cation transporter 1 induces movement of helix 11 and blocks transport after mutations in a modeled interaction domain between two helices[END_REF][START_REF] Popp | Amino acids critical for substrate affinity of rat organic cation transporter 1 line the substrate binding region in a model derived from the tertiary structure of lactose permease[END_REF][START_REF] Keller | Rat Organic Cation Transporter 1 Contains Three Binding Sites for Substrate 1-Methyl-4phenylpyridinium per Monomer[END_REF] . By comparing positioning and interactions of D24 in both models, we modified this compound to introduce a bulky substituent hindering interaction with the α2C adrenoceptor, thereby generating the analog cyanome (Figure 1a). Cyanome showed a similar docking pattern of interaction to D24 with hOCT2 (Figure 1c). The relevance of the interactions predicted for cyanome was verified through binding and uptake experiments. Binding experiments showed that cyanome inhibited less potently the human α2C and the α2A adrenoceptors than D24 (Supplementary figure 1f).

The observed inhibition constant (K i ) values were 185 ± 42 nM (mean ± s.e.m.) for the α2A adrenoceptor and 252 ± 32 nM for the α2C adrenoceptor (Supplementary figure 1f), compared to 28 nM and 66 nM for D24 for α2A and α2C adrenoceptors, respectively [START_REF] Amphoux | Inhibitory and facilitory actions of isocyanine derivatives at human and rat organic cation transporters 1, 2 and 3: a comparison to human alpha 1-and alpha 2adrenoceptor subtypes[END_REF] .

Cyanome also retained the ability to block the transport activity mediated by hOCT2 in transfected cells, with an IC 50 value of 123 ± 26 nM (Supplementary figure 1f), a value comparable to that of D24 [START_REF] Amphoux | Inhibitory and facilitory actions of isocyanine derivatives at human and rat organic cation transporters 1, 2 and 3: a comparison to human alpha 1-and alpha 2adrenoceptor subtypes[END_REF] . These results suggest that modification of D24 into cyanome markedly increased selectivity at hOCT2, as predicted by molecular modeling. Next, we speculated that charged cyanine analogs such as cyanome might have limited potential for use as antidepressants, due to low brain penetration. By exploiting a redox brain delivery system previously described [START_REF] Bodor | A strategy for delivering peptides into the central nervous system by sequential metabolism[END_REF] , cyanome was modified into a prodrug that could more readily diffuse into the brain parenchyma and be activated therein, H2-cyanome (Figure 1a). The fate of H2cyanome in vivo after systemic administration was determined by HPLC and mass spectrometry analysis of mice plasma and brain extracts, at different time points after a single i.v. injection. These analyses showed that after i.p. injection H2-cyanome diffuses into the brain and is rapidly cleared from the soluble fractions of both plasma and brain (Table 1).

Furthermore, to identify the potential metabolites of H2-cyanome in a physiological context, we investigated the nature of the derivatives generated after incubation with human liver microsomal preparations. Four main metabolites were identified within the first minutes of incubation, the most abundant metabolite of which was cyanome (Supplementary figure 2).

Antidepressant effects of H2-cyanome in a chronic depression model

H2-cyanome was evaluated for its behavioral effects in mice, in particular antidepressant efficacy. In a model of chronic depression induced by corticosterone administration [START_REF] Bacq | Organic cation transporter 2 controls brain norepinephrine and serotonin clearance and antidepressant response[END_REF][START_REF] David | Neurogenesis-dependent and -independent effects of fluoxetine in an animal model of anxiety/depression[END_REF] , daily administration of a low dose of H2-cyanome (0.1 mg/kg, i.p.) induced rapid positive effects on several behaviors mimicking symptoms of depression, such as anhedonia, anxiety, social withdrawal, and memory impairment (Figure 2 and supplementary figure 3). The action of this compound on these parameters was as robust as those of the classical antidepressant fluoxetine (15 mg/kg). Specifically, H2-cyanome efficiently reversed the action of corticosterone on anhedonia, evaluated by sucrose preference (Figure 2b), on social interaction (Figure 2c), on anxiety level, evaluated by time spent in the anxiogenic zone of the elevated O-maze (Figure 2d), by latency to enter this zone (Figure 2e) and by time and activity in the center of the open-field (Supplementary figure 3b), and on short-term memory, evaluated by performance in the object location test (Figure 2f). This last effect, similar to that exerted by fluoxetine, was confirmed in the novel object test (Supplementary figure 3c).

Importantly, H2-cyanome showed an accelerated onset of action on anhedonia compared to fluoxetine at 11 days (Figure 2b) and better effects on anxiety level at 21 days (Figure 2d-e and supplementary figure 3b). Finally, H2-cyanome also improved partially coat state (Figure 2g) and induced no weight loss over time (Supplementary figure 3d), as fluoxetine. To evaluate more finely the effects of H2-cyanome, we applied z-normalization across measures of these depression-related variables, i.e., anxiety and anhedonia-like behaviors and coat state. Interestingly, individual z-scores of mice treated with H2-cyanome were significantly lower than those of mice treated with fluoxetine (Figure 2h), suggestive of a better antidepressant effect of H2-cyanome.

Contrasting with the effects of repeated administration, acute injection of various doses of H2-cyanome had no effect on locomotor activity or anxiety level in the open-field (Figure 3a) and elevated O-maze (Figure 3b). However, acute injection elicited a moderate but reproducible antidepressant-like effect on immobility in the forced-swim test, which appeared to reach a maximum at the dose of 0.1 mg/kg (Figure 3c).

Effects of H2-cyanome systemic administration on the firing of VTA neurons

H2-cyanome was initially designed to influence monoamine neurotransmission in the brain through blockade of OCT. Based on its potent action on anhedonia in the sucrose preference test, we speculated that H2-cyanome might have an impact on dopaminergic signaling. Dopaminergic neurons of the VTA encode reward, motivation and learning [START_REF] Morales | Ventral tegmental area: cellular heterogeneity, connectivity and behaviour[END_REF] , functions profoundly impaired in depressed patients. We thus investigated the effects of both acute and repeated H2-cyanome administration on the firing of this neuronal population. The acute injection of H2-cyanome alone did not alter VTA dopaminergic neuron firing, but potentiated the inhibitory effect of cocaine, a DA transport blocker (Figure 4a). Since VTA neuron firing is negatively controlled by local extracellular DA, this observation reflects an inhibitory effect of H2-cyanome on DA clearance. Remarkably, a 10-day repeated administration of H2cyanome alone exerted an opposite action, i.e., a significant increase in the firing of VTA neurons (Figure 4b). This increased activity may underlie the antidepressant efficacy of H2cyanome in our chronic model of depression.

Effects of H2-cyanome on intracellular signaling pathways in the brain

ERK1/2, GSK3β and mammalian target of rapamycin (mTOR) signaling have been implicated in antidepressant response in depressed patients and in animal models [START_REF] Duman | A role for MAP kinase signaling in behavioral models of depression and antidepressant treatment[END_REF][START_REF] Beaulieu | Akt/GSK3 signaling in the action of psychotropic drugs[END_REF][START_REF] Li | mTOR-dependent synapse formation underlies the rapid antidepressant effects of NMDA antagonists[END_REF] . To gain further insight into the mechanisms of action of H2-cyanome, we investigated its influence on these signaling pathways in the brain, by Western blot analysis in the corticosterone depression model. We found that all three pathways (ERK1/2, GSK3β and mTOR) were strongly modulated by chronic corticosterone treatment in mouse prefrontal cortex and hippocampus. The phosphorylation levels of ERK1/2 (Figure 5a), Akt at Ser-473 (Figure 5c) and p70S6 (Figure 5d) were significantly decreased in both brain areas after corticosterone, reflecting inhibition of ERK1/2, TORC2 and TORC1 pathways, respectively.

Phosphorylation of GSK3β at Ser-9, a site controlling negatively its activity (Figure 5b), was also decreased, reflecting activation of this pathway. All these alterations in activity induced by corticosterone were reversed in both brain areas after a 21-day treatment with either fluoxetine or H2-cyanome (Figure 5a-d). Thus, H2-cyanome restored basal state ERK1/2, GSK3β and mTOR signaling in prefrontal cortex and hippocampus as efficiently as fluoxetine in this depression model.

Discussion

There is at present a pressing need for novel well-tolerated antidepressants combining better efficacy and quicker action onset. In spite of interesting leads [START_REF] Harmer | How do antidepressants work? New perspectives for refining future treatment approaches[END_REF] , there has been no significant paradigm shift over the last years in the pharmacological management of major depression disorder in current practice. Important shortcomings in standard treatments include poor efficacy, slow action onset, adverse side effects and unpredictable resistance, with one-third of patients not responding adequately to two or more successive trials of treatment. We demonstrate here proof-of-concept that targeting the atypical monoamine transporters OCT is a promising strategy for the development of novel antidepressant drugs.

OCT have been drawing increasing attention within the last decade, as evidence accumulated that they subserve major central functions, controlling anxiety, stress response and antidepressant action [START_REF] Bacq | Organic cation transporter 2 controls brain norepinephrine and serotonin clearance and antidepressant response[END_REF][START_REF] Vialou | Altered aminergic neurotransmission in the brain of organic cation transporter 3-deficient mice[END_REF][START_REF] Wultsch | Decreased anxiety in mice lacking the organic cation transporter 3[END_REF][START_REF] Courousse | Brain organic cation transporter 2 controls response and vulnerability to stress and GSK3beta signaling[END_REF][START_REF] Courousse | Role of organic cation transporters (OCTs) in the brain[END_REF] . OCT2 and OCT3 specifically have been proposed to act as a low-affinity monoamine clearance system, regulating aminergic tonus and complementing the better-known high-affinity transporters in the brain [START_REF] Courousse | Role of organic cation transporters (OCTs) in the brain[END_REF][START_REF] Baganz | Organic cation transporter 3: Keeping the brake on extracellular serotonin in serotonintransporter-deficient mice[END_REF] . However, until now, these potential therapeutic targets have been seldom exploited for drug development [START_REF] Pan | A new chemotype inhibitor for the human organic cation transporter 3 (hOCT3)[END_REF][START_REF] Krause-Heuer | Evaluation of the antidepressant therapeutic potential of isocyanine and pseudoisocyanine analogues of the organic cation decynium-22[END_REF] . The present study describes a novel OCT ligand with a high therapeutic potential, developed by combining molecular modeling with a prodrug brain delivery strategy.

In a two-step process, we generated starting from the OCT inhibitor D24 a ligand with increased selectivity for OCT2, which we further modified into an analog that could efficiently penetrate the brain, H2-cyanome [START_REF] Bodor | A strategy for delivering peptides into the central nervous system by sequential metabolism[END_REF] . To evaluate its antidepressant potential, we took advantage of a rodent model of chronic depression with strong construct, face and predictive validity [START_REF] Bacq | Organic cation transporter 2 controls brain norepinephrine and serotonin clearance and antidepressant response[END_REF][START_REF] David | Neurogenesis-dependent and -independent effects of fluoxetine in an animal model of anxiety/depression[END_REF][START_REF] Surget | Drugdependent requirement of hippocampal neurogenesis in a model of depression and of antidepressant reversal[END_REF] , which surpasses acute behavioral despair tests commonly used for antidepressant screening [START_REF] Bacq | Organic cation transporter 2 controls brain norepinephrine and serotonin clearance and antidepressant response[END_REF][START_REF] Krause-Heuer | Evaluation of the antidepressant therapeutic potential of isocyanine and pseudoisocyanine analogues of the organic cation decynium-22[END_REF][START_REF] Horton | Decynium-22 enhances SSRI-induced antidepressant-like effects in mice: uncovering novel targets to treat depression[END_REF] . In this model, prolonged corticosterone exposure induces a panel of behavioral modifications that mimic various symptoms of depression. As with human depression, these persistent depression-like anomalies can be improved by long-term, but not acute, antidepressant treatment, triggering on a longer time-scale specific mechanisms [START_REF] Levinstein | Mechanisms underlying the antidepressant response and treatment resistance[END_REF][START_REF] Krishnan | The molecular neurobiology of depression[END_REF] . In this depression model, H2-cyanome compared positively to the classical antidepressant fluoxetine, with a faster action on anhedonia, evaluated by sucrose preference, and better anxiolytic effects at a low dose (0.1mg/kg; Figure 2). Integrated Z-scores across depression-like variables revealed a significant difference of H2-cyanome compared to fluoxetine after 3-week treatments, supporting a stronger antidepressant effect. The parameters evaluating potential toxicity were encouraging (Supplementary figures 3 and4, and supplementary table 1). Daily H2-cyanome at 0.1 mg/kg was as well tolerated as fluoxetine, with no weight loss after prolonged administration (Supplementary figure 3d).

Blood parameters after 3 weeks of treatment were comparable to those after fluoxetine, with more favorable effects on hepatic enzymes (Supplementary table 1). In the brain, no evident neurotoxicity was detected in various areas including dopaminergic pathways (Supplementary figure 4).

The precise mechanisms of action of H2-cyanome in vivo remain to be clarified. A main issue is the nature of the metabolites of H2-cyanome present in the brain and their full impact on central processes, in particular after prolonged exposure. OCT blockers may be suspected of acting differently than current antidepressants like SSRI and NSRI. As opposed to the high-affinity transporters gathered in aminergic terminals, OCT are highly expressed in the cell bodies and processes (neurons and occasionally astrocytes) throughout the brain, in the principal regions receiving aminergic projections. This broad distribution and the high capacity properties of OCT, which compensate for their low-affinity, may explain their importance in the removal of extracellular monoamines [START_REF] Bacq | Organic cation transporter 2 controls brain norepinephrine and serotonin clearance and antidepressant response[END_REF][START_REF] Courousse | Role of organic cation transporters (OCTs) in the brain[END_REF][START_REF] Daws | Unfaithful neurotransmitter transporters: focus on serotonin uptake and implications for antidepressant efficacy[END_REF] . Thus, in contrast with SSRI and NSRI, molecules targeting OCT could regulate NE, 5-HT and DA tone, in a widespread manner throughout the brain [START_REF] Courousse | Role of organic cation transporters (OCTs) in the brain[END_REF] . In addition, as the lead compound D24 shows a modest affinity for the SERT [START_REF] Amphoux | Inhibitory and facilitory actions of isocyanine derivatives at human and rat organic cation transporters 1, 2 and 3: a comparison to human alpha 1-and alpha 2adrenoceptor subtypes[END_REF] , potential modulation of this transporter must also be considered.

Supporting an unconventional mode of action, H2-cyanome induced few behavioral modifications when administered acutely. In particular, this compound showed only moderate antidepressant-like effects in the forced-swim test, contrarily to SSRIs and NSRIs which exert robust anti-immobility effects in behavioral despair paradigms [START_REF] Cryan | The tail suspension test as a model for assessing antidepressant activity: review of pharmacological and genetic studies in mice[END_REF] .

In vivo electrophysiological recordings revealed a complex action of H2-cyanome on DA signaling, on two distinct time scales. Our experiments showed that acute H2-cyanome could potentiate the inhibitory effects of cocaine, a dopamine transporter inhibitor, on the firing of VTA neurons. Extracellular DA levels in the VTA have been shown to negatively modulate VTA DA neuron firing [START_REF] Einhorn | Electrophysiological effects of cocaine in the mesoaccumbens dopamine system: studies in the ventral tegmental area[END_REF] , via inhibitory somatodendritic D2 receptors [START_REF] Guiard | Characterization of the electrophysiological properties of triple reuptake inhibitors on monoaminergic neurons[END_REF] . Our results suggest therefore that acute H2-cyanome could dampen DA neuron activity synergistically with cocaine by increasing local DA concentrations, through the inhibition of OCT-mediated DA clearance in this brain region. To gain a full understanding of the action of the prodrug, additional experiments in vivo and ex vivo are required to determine how H2-cyanome affects NE and 5-HT as well as DA clearance throughout the brain.

Electrophysiological recording of DA neurons of the VTA also revealed significant effects of prolonged H2-cyanome administration. Contrarily to acute injection, a 10-day H2cyanome treatment was associated with an increased firing frequency of this neuron population, a process that may underlie its rapid action on anhedonia. VTA dopaminereleasing neurons display remarkable functional heterogeneity, with distinct subpopulations controlling motivated behaviors in a complex manner [START_REF] Morales | Ventral tegmental area: cellular heterogeneity, connectivity and behaviour[END_REF] . Notwithstanding, their activation has been consistently associated with reward and motivation [START_REF] Morales | Ventral tegmental area: cellular heterogeneity, connectivity and behaviour[END_REF] , functions often altered in MDD patients. The phasic activity of VTA DA neurons was also shown previously to be inhibited by chronic stress, a classical depression paradigm, while their optogenetic activation could reverse the depression-like behaviors induced in this paradigm [START_REF] Tye | Dopamine neurons modulate neural encoding and expression of depression-related behaviour[END_REF] . Thus, activation of these DA neurons could play a key role in the efficacy of H2-cyanome on behaviors reflecting anhedonia, in particular its early effects on sucrose preference at 11 days of treatment, when fluoxetine is still inactive (Figure 2).

At another level, our study discloses main effects of H2-cyanome on intracellular signaling pathways involved in the regulation of mood and in antidepressant efficacy. The MAP kinase ERK1/2 was previously shown to be inhibited in prefrontal cortex and hippocampus of depressed suicide victims [START_REF] Dwivedi | ERK MAP kinase signaling in postmortem brain of suicide subjects: differential regulation of upstream Raf kinases Raf-1 and B-Raf[END_REF] and activated by long-term antidepressant treatment in animal models of depression [START_REF] Gourley | Regionally specific regulation of ERK MAP kinase in a model of antidepressant-sensitive chronic depression[END_REF] . Abnormal GSK3β activity has been found in prefrontal cortex of individuals with major depressive disorder [START_REF] Karege | Protein levels of beta-catenin and activation state of glycogen synthase kinase-3beta in major depression. A study with postmortem prefrontal cortex[END_REF][START_REF] Karege | Alteration in kinase activity but not in protein levels of protein kinase B and glycogen synthase kinase-3beta in ventral prefrontal cortex of depressed suicide victims[END_REF] , and inhibition of GSK3β shown to play a role in antidepressant action [START_REF] Beaulieu | Akt/GSK3 signaling in the action of psychotropic drugs[END_REF] . The mTOR intracellular pathway has been proposed to underlie the fast-acting antidepressant effects of ketamine in humans [START_REF] Li | mTOR-dependent synapse formation underlies the rapid antidepressant effects of NMDA antagonists[END_REF] . In our model, all three pathways responded to corticosterone, in parallel with the emergence of depression-related behavioral anomalies. Remarkably, the beneficial effects on these behaviors of a 3-week treatment with either fluoxetine or H2-cyanome were associated with a return to basal activation states, similar to those before corticosterone treatment. Altogether, these experiments indicate that H2-cyanome modulates these intracellular regulatory pathways in a similar way than fluoxetine, suggesting at least in part common regulatory mechanisms within the brain, downstream of aminergic signaling.

We speculate that both limited interactions with α adrenoceptors and efficient brain penetration contribute to the robust antidepressant effects of H2-cyanome. α adrenoceptors play important roles in controlling blood pressure, heart rate and contractility [START_REF] Docherty | Subtypes of functional alpha1-adrenoceptor[END_REF][START_REF] Hein | Two functionally distinct alpha2-adrenergic receptors regulate sympathetic neurotransmission[END_REF][START_REF] Brede | alpha2-adrenergic receptor subtypesnovel functions uncovered in gene-targeted mouse models[END_REF] . Based on the metabolic profile of H2-cyanome in microsomal fractions, two charged metabolites of H2cyanome, cyanome and its demethylated derivative, are plausible candidates for mediating the antidepressant effects of the prodrug. Our experiments suggest that these two compounds should interact less potently than D24 [START_REF] Amphoux | Inhibitory and facilitory actions of isocyanine derivatives at human and rat organic cation transporters 1, 2 and 3: a comparison to human alpha 1-and alpha 2adrenoceptor subtypes[END_REF] with α2A and 2C adrenoceptors in vivo, while remaining capable of blocking OCT. Central α2A adrenoceptors and α2C adrenoceptors in the adrenal medulla both exert a negative control over blood pressure and heart rate, by mediating respectively sympathoinhibition [START_REF] Makaritsis | Sympathoinhibitory function of the alpha(2A)-adrenergic receptor subtype[END_REF] and feedback control of epinephrine release [START_REF] Brede | alpha2-adrenergic receptor subtypesnovel functions uncovered in gene-targeted mouse models[END_REF] . Thus, restricting the antagonism at α2 adrenoceptors may be beneficial to avoid unwanted cardiovascular effects. Importantly, interactions with central α2 adrenoceptors could also play a part in the antidepressant efficacy of H2-cyanome, but it is at this stage difficult to predict to what extent α2 blockade is a disadvantage. Some previous studies suggest that restricting α2A antagonism might improve antidepressant action and anxiety [START_REF] Schramm | The alpha(2a)-adrenergic receptor plays a protective role in mouse behavioral models of depression and anxiety[END_REF][START_REF] Wang | Effects of alpha2A Adrenoceptors on Norepinephrine Secretion from the Locus Coeruleus during Chronic Stress-Induced Depression[END_REF] , while others suggest on the contrary that α2 adrenoceptor blockade could contribute to the pharmacological activity of antidepressants [START_REF] Fernandez-Pastor | Chronic citalopram administration desensitizes prefrontal cortex but not somatodendritic alpha2adrenoceptors in rat brain[END_REF] , or potentiate their action [START_REF] Yanpallewar | Alpha2-adrenoceptor blockade accelerates the neurogenic, neurotrophic, and behavioral effects of chronic antidepressant treatment[END_REF] . Finally, we also believe that choice of D24 as lead scaffold was critical for favoring interactions with central OCT and limiting potential adverse side-effects. Other cyanine dyes such as decynium 22 (D22) can also inhibit OCT, with overall less affinity than D24 [START_REF] Koepsell | Polyspecific organic cation transporters: structure, function, physiological roles, and biopharmaceutical implications[END_REF][START_REF] Amphoux | Inhibitory and facilitory actions of isocyanine derivatives at human and rat organic cation transporters 1, 2 and 3: a comparison to human alpha 1-and alpha 2adrenoceptor subtypes[END_REF][START_REF] Fraser-Spears | Comparative analysis of novel decynium-22 analogs to inhibit transport by the low-affinity, high-capacity monoamine transporters, organic cation transporters 2 and 3, and plasma membrane monoamine transporter[END_REF] . However, while both D24 and D22 inhibit peripheral α1 adrenoceptors [START_REF] Russ | Pharmacokinetic and alpha 1-adrenoceptor antagonistic properties of two cyanine-type inhibitors of extraneuronal monoamine transport[END_REF] , D22 has a 50-fold higher affinity for these receptors than D24 [START_REF] Amphoux | Inhibitory and facilitory actions of isocyanine derivatives at human and rat organic cation transporters 1, 2 and 3: a comparison to human alpha 1-and alpha 2adrenoceptor subtypes[END_REF] or cyanome (data not shown), restricting its use in vivo due to its pronounced hypotensive action [START_REF] Russ | Pharmacokinetic and alpha 1-adrenoceptor antagonistic properties of two cyanine-type inhibitors of extraneuronal monoamine transport[END_REF] . These mechanisms may also have a bearing on the adverse effects of D22 on locomotor activity [START_REF] Krause-Heuer | Evaluation of the antidepressant therapeutic potential of isocyanine and pseudoisocyanine analogues of the organic cation decynium-22[END_REF] . Considering the diversity of functions attributed to α adrenoceptors both at periphery and in the brain, in-depth studies with compounds with refined selectivity are required to identify the combinations of binding properties at OCT and α adrenoceptor subtypes optimal for clinical use.

In conclusion, we have devised through molecular modeling and pharmacomodulation a novel OCT-targeted drug with improved selectivity, which can penetrate the brain. Its longterm antidepressant efficacy in a validated depression model demonstrates a high potential for therapeutic use, compared to the conventional antidepressant fluoxetine. Our findings provide a conceptual and mechanistic framework for the design of selective OCT ligands, which may open a field of discovery for new classes of antidepressants with improved properties. with hOCT2, with two additional interactions, between its aromatic ring and W218, and between its 6-methoxy substituent and N157. Position 6 of D24 was substituted in cyanome with a bulky substituent hindering interaction with the fourth helix of the α2C adrenoceptor and cyanome was reduced in H2-cyanome to facilitate brain penetration. Unpaired 2-tailed Student's t-test (n=4-7) reveals a significant effect of corticosterone treatment (*P < 0.05, **P < 0.01, ***P < 0.001), and significant increases after fluoxetine or H2-cyanome treatments compared to corticosterone-treated groups (#P < 0.05, ##P < 0.01, ###P < 0.001). Results are given as mean of phosphorylated over non-phosphorylated protein ratio ± s.e.m, or for p70S6 as mean of phosphorylated protein over β-actin ratio ± s.e.m.

  displaced to the opposite corner of the open-field. The time spent exploring both objects was recorded over a 5-min session by video tracking. The open field consisted of a white Plexiglas field (42x42 cm) with the center brightly illuminated (100 lux). General locomotor activity in the center and periphery of the open field were scored for 9 min. The time and number of entries in the center zone (20x20 cm) were evaluated as an index of anxiety-related response. Integrated z-scores of depression-related variables were calculated to compare the effects of H2-cyanome with those of fluoxetine in the corticosterone depression model. Individual depression-related z-scores were generated with data from five tests (time and activity in the center of open-field, time and latency to enter the open zone of elevated O maze, percentage

  5 mm diameter; Miltex, York, PA, USA) of brain regions from adult mice at basal state, treated with corticosterone or with corticosterone plus H2-cyanome or fluoxetine. Samples were homogenized by sonication in 2 vol of ice-cold phosphate-buffered saline containing 1% Triton X-100, protease inhibitors (Complete Protease Inhibitor Cocktail, Roche Diagnostics, Meylan, France) and phosphatase inhibitors (Phosphatase Inhibitor Cocktail 3; Sigma-Aldrich). Protein concentrations were determined by Bradford's method. Protein samples (15 µg) suspended in NuPage LDS sample buffer (Invitrogen, Carlsbad, CA, USA) were separated by Bis-Tris sodium dodecyl sulfate polyacrylamide gel electrophoresis (10% gels) and transferred onto nitrocellulose membranes (Invitrogen). Transfer efficacy was controlled by Ponceau S staining. Unspecific binding sites were blocked with Tris-buffered saline containing 0.1% Tween-20 and 5% nonfat milk and membranes were immunoprobed with antibodies against glycogen synthase kinase-3β (GSK3β, 1/2000) from Millipore (Billerica, MA, USA), phosphorylated GSK3β (p GSK3β, 1/1000), Akt (1/2000), pThr308 or Ser473 Akt (1/200), phosphorylated extracellular-signal regulated kinase1/2 (pErk1/2, 1/1000) and p-P70S6 (1/500) from Cell Signaling (Danvers, MA, USA), Erk1/2 (1/1500) from Santa Cruz, or β-actin (1/2500) from Sigma-Aldrich. Membranes were incubated with infrared-labeled secondary antibodies (IRDye 700DX and IRDye 800CW; 1/5000; Rockland, Gilbertsvillle, PA, USA). Immunoblotting was quantified with an Odyssey Infrared Imaging System and Application Software version 3.0 (LI-COR Biosciences, Lincoln, NE, USA). Statistics PRISM (GraphPad Software, San Diego, CA, USA) was used for statistical calculations. For sucrose preference, social interaction, open-field, O-maze, object location test, coat state and forced swim-test data were analyzed using one-way or two-way analysis of variance (ANOVA), followed by Fisher's LSD test. Student's t-test was used for analysis of z-scores after H2-cyanome and fluoxetine treatment, firing of VTA neurons, and Western blot experiments. Statistical significance was set at P < 0.05.

Figure 1

 1 Figure 1 Strategy for prodrug development. Design of a disprocynium (D24) analog with

Figure 2

 2 Figure 2 Antidepressant efficacy of H2-cyanome in a model of chronic depression. (a-h)

Figure 3 .

 3 Figure 3. Acute behavioral effects of H2-cyanome. One-way analysis of variance

Figure 4

 4 Figure 4 Action of H2-cyanome on dopaminergic neuron firing in the ventral tegmental

Figure 5 .

 5 Figure 5. Action of H2-cyanome and fluoxetine on brain intracellular signaling

Table 1 . Detection of H2-cyanome in plasma and brain

 1

	Time after Injection (i.v.)	5 min	15 min	2 h	24 h
	Plasma (ng/ml) 3.65 ± 1.69	2.15 ± 0.31	< 0.5	< 0.5
	Brain (ng/g)	3.97 ± 2.3	0.56 ± 0.56	< 0.5	ND

Data are expressed as means ± s.e.m. (n=6). ND: not detectable

Acknowledgements

We thank J-P Guilloux (UMRS 1178) for help with z-scoring, F. Machulka for expert assistance with animal care, and C. Betancur for critical reading of the manuscript. Financial support was provided by the Agence Nationale de la Recherche (ANR-13-SAMENTA-0003-01) and SATT Lutech. S.R. was a recipient of a fellowship from the French Ministry for Research.

Author contributions

N.P., F.A., and S.G. conceived the study. N.P. conceived and performed molecular modeling and designed the compounds. N.P., A.H., and P.D performed microsomal fraction analysis.

Conflict of interest

The authors declare no conflict of interest.

Supplementary information is available at MP's website


Matthieu Peurois
email: matthieu.peurois@univ-angers.fr

Aline Ramond-Roquin

Céline Bouton

Cyril Bègue

Natacha Fouquet

Najia Adjeroud

Cécile Raber

Eline Bouton

Cyril B Egue

Ecile Raber

Influence of low back pain characteristics on the healthcare procedures prescribed by general practitioners for adult patients: ancillary analysis of the French ECOGEN study

Keywords:

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

Original article

Influence of low back pain characteristics on the healthcare procedures prescribed by general practitioners for adult patients: ancillary analysis of the French ECOGEN study -Les douleurs lombaires non sp ecifiques sont un motif fr equent de consultation, mais on en sait peu sur la mani ere dont les m edecins g en eralistes g erent ces plaintes.

Objectif. -D ecrire les soins prodigu es ou prescrits par les m edecins g en eralistes en France pour les douleurs lombaires.

Mat eriel et m ethodes. -Il s'agit d'une analyse secondaire de l' etude ECOGEN, une etude observationnelle et transversale men ee entre novembre 2011 et avril 2012 aupr es de 128 m edecins g en eralistes. Étaient inclus les adultes de moins de 65 ans qui consultaient pour des douleurs lombaires. Les caract eristiques des patients et des praticiens, le r esultat de la consultation (diagnostics) et les soins prodigu es ou prescrits etaient collect es et cod es selon la classification internationale en soins primaires. Les analyses ont distingu e les consultations initiales ou de suivi, en ajustant pour l'âge, le sexe et la cat egorie socio-professionnelle. R esultats et discussion. -Sur 11510 consultations, 845 (7,3 %) etaient motiv ees par des douleurs lombaires. Parmi celles-ci, 776 (79,5 %) ont conduit a un examen clinique, 634 (73,4 %) a une ordonnance de m edicament et 203 (

Introduction

Non-specific low back pain (LBP) is a musculoskeletal condition of multifactorial origin. It is defined as pain between the ribcage and the lower inter-gluteal fold, which can be associated with radiating pain, particularly of crural or sciatic nerve origin, without signs of trauma, inflammation, infection, neoplasia, metabolic or congenital disease [START_REF] Maher | Non-specific low back pain[END_REF]. LBP has the sixth highest global burden and causes more disability worldwide than any other condition [START_REF] Hoy | The global burden of low back pain: estimates from the Global Burden of Disease 2010 study[END_REF].

Although non-specific LBP lasting less than four weeks has a good prognosis, the probability of a favourable outcome decreases markedly after this time lapse [START_REF] Costa | The prognosis of acute and persistent low-back pain: a meta-analysis[END_REF]. After 12 weeks, subacute LBP becomes chronic and is commonly associated with physical deconditioning syndrome, which must be managed with physical exercise [START_REF] Maher | Non-specific low back pain[END_REF]. It is also associated with psychosocial difficulties, prolonged absenteeism [START_REF] Bouton | Prise en charge de la lombalgie commune et int erêt de l'ost eopathie: analyse de la pratique des m edecins g en eralistes en[END_REF][START_REF] Hartvigsen | What low back pain is and why we need to pay attention[END_REF] reduced quality of life and major costs for society [START_REF] Tymecka-Woszczerowicz | Indirect costs of back pain -Review[END_REF]. In recent decades, a biopsychosocial model of LBP has emerged, highlighting the importance of psychosocial (psychological, psychiatric, occupational, social. . .) risk factors in the transition to chronicity [START_REF] Truchon | Determinants of chronic disability related to low back pain: towards an integrative biopsychosocial model[END_REF].

Up until recently, patient pathway guidelines were unclear, and meaning referrals to specialists were often late or non-existent. Late referral to occupational health services is commonly a barrier to job retention, which is a major problem insofar as LBP has become the leading cause of absenteeism before the age of 45 in France [START_REF] Maher | Non-specific low back pain[END_REF]. That is one reason improved coordination between clinical services and occupational health professionals is called for [START_REF] Michel | Return to work after rehabilitation in chronic low back pain workers. Does the interprofessional collaboration work?[END_REF][START_REF] Buchbinder | Low back pain: a call for action[END_REF].

Published in the early 2000s, previous guidelines recommend multidimensional management: prescribing pain relief (both pharmacological and non-pharmacological), promoting physical exercise, identifying psychosocial factors and preventing occupational disability [START_REF] Airaksinen | Chapter 4. European guidelines for the management of chronic nonspecific low back pain[END_REF][START_REF] Top | Guideline for the Evidence-Informed Primary Care Management of Low Back Pain[END_REF][START_REF] Pillastrini | An updated overview of clinical guidelines for chronic low back pain management in primary care[END_REF]. However, a lack of clear, practical recommendations resulted in heterogeneous LBP management and treatment strategies, with General Practitioners (GPs) often adhering to traditional schemas based on biomechanical analysis, imaging, non-pharmacological therapies and pharmacological analgesia, even though medications are often being ineffective or even harmful [START_REF] Maher | Non-specific low back pain[END_REF][START_REF] Zheng | Stagnant Physical Therapy Referral Rates Alongside Rising Opioid Prescription Rates in Patients With Low Back Pain in the United States 1997-2010[END_REF][START_REF] Imagama | Impact of background factors on outcomes of pharmacological therapy for chronic low back pain: A nationwide multicenter prospective study[END_REF]. Heterogeneous and poorly evidence-based medical practices result in GPs feeling frustrated and experiencing difficulty caring for patients with LBP, especially those with subacute and chronic LBP [START_REF] Gremeaux | Do Teaching General Practitioners' Fear-Avoidance Beliefs Influence Their Management of Patients with Low Back Pain?[END_REF].

GPs are well-placed to influence patient prognosis, adherence, and outcome. In France, 77 % of adults aged 30 to 69 with LBP consult a GP compared to just 9 % consulting a rheumatologist and 3 % a surgeon [START_REF] Plenet | Seeking care for lower back pain in the French population aged from 30 to 69: the results of the 2002-2003 Decennale Sante survey[END_REF]. Moreover, healthcare reimbursement data research suggest that LBP management among GPs in France is suboptimal [START_REF] Lafuma | Management and cost of care for low back pain in primary care settings in France[END_REF].

To improve recommendations for LBP treatment and patient care pathways, this study aimed to describe the healthcare procedures French GPs prescribe for patients aged 18 to 65 years consulting for LBP, with or without radiating pain.

Methods

Study design

This study is an ancillary study to ECOGEN (Elements of Consultation in General practice), which is a French observational, cross-sectional, multicentre, national study conducted by the French National College of General practitioner Teachers (Coll ege National des G en eralistes Enseignants) between November 2011 and April 2012. The ECOGEN study design has previously been described [START_REF] Letrilliart | ECOGEN: etude des Él ements de la COnsultation en m edecine GEN erale[END_REF]. One hundred and twenty-eight GPs (internship supervisors) affiliated with 27 French medical schools participated and 54 trainee GPs collected the data. The trainees had undergone a 2-day data collection training course.

Inclusion criteria

Adult patients younger than 65 years were included in this ancillary study if a health problem managed during the consultation (hereafter called a consultation result) was coded L03 (low back symptom), L84 (low back syndrome without radiating pain) or L86 (low back syndrome with radiating pain), either as a single consultation result or as one of several.

ECOGEN Data collection

During the ECOGEN study, trainee GPs collected demographic and health data from each patient who consulted the GP one day a week. Age, gender, socio-professional category, workers' compensation and other benefits, initial or follow-up consultation, reason(s) for consultation, consultation result(s) and healthcare procedures were specified. A healthcare procedure was defined as a clinical examination, imaging or laboratory investigation, medication prescription, referral to other physicians or allied health professionals, certificate provision, sick leave prescription, advice, or recommendations.

Healthcare procedures were coded using ICPC-2 codes (International Classification in Primary Care, 2 nd edition) [START_REF]WONCA Classification Committee. International Classification of Primary Care[END_REF]. This classification was initially proposed by the WONCA (World Organization of Family Doctors) and developed by leading primary care experts worldwide. In 1987, the WHO recognized the ICPC as the reference classification for classification of patient data and clinical activity in primary care. It bears mentioning that reasons for consultation, consultation results, and healthcare procedures can be classified by body system according to a hierarchical structure. The ICPC-2 data codes were supplemented with verbatims from patient records. The data were then anonymized and filed in a centralized online database. Double data collection was performed in one day to evaluate reproducibility and the error rate for each investigator.

Analyses

The characteristics of patients consulting for LBP were initially compared with those consulting for other reasons. As regards patients who consulted for LBP, patient and GP characteristics and healthcare procedures were subsequently described and compared according to gender, age, presence of radiating pain, LBP persistence (initial or follow-up consultation), practice location and type. A multivariate marginal model was drawn up, adjusted on age, gender, socio-occupational category, and variables associated with physiotherapy prescription in univariate analyses.

Statistics

Quantitative variables were described by means and standard deviation (or medians and quartiles according to distribution) and compared using Student's t-test, or a non-parametric Mann Whitney Wilcoxon test if needed. Qualitative variables were described by percentages and compared using the Chi-square test (or a Fisher test if n<5). When relevant, a Bonferroni correction for multiple statistical comparisons was applied.

Probability of each healthcare procedure was modelled according to LBP characteristics (radiating pain and persistence) using a logistic marginal adjusted model based on Generalised Estimating Equations (GEE) with an exchangeable variance-covariance matrix, given the hierarchical data structure and the populationaverage approach. The marginal univariate analyses were first performed for each healthcare procedure, and only those associated with a statistical p-value < 0.20 were retained. A multilevel analysis adjusted on age and gender was then performed with the variables retained, and a statistical a threshold of 0.05 was used for the final model. Statistical analyses were performed on R software, version 1.1.463.

Ethical considerations

An ethics committee approved the ECOGEN study (CPP Sud-Est IV, registration number: L11-149, approval date: 10/11/2011) and included consent for ancillary studies on the ECOGEN database. A poster in the waiting room informed patients about the study and the GP collected verbal consent at the beginning of the consultation.

Results

Study population

Participating GPs were 53 years old on average and one third were women (33.6 %). More than half were in group practice (61.7 %) within an urban area (51.6 %). The median number of consultations per day was 20 and the median number of consultations annually was 5000 [Q1=3991; Q3=6125].

The number of ECOGEN consultations performed for patients aged 18 to 65 years was 11510 (Figure 1). While 60 % of the patients were women, their mean age was 44.1 (SD=13.26) and 93.78 % were previously known to the GP. On average, each consultation generated 2.1 consultation results and 4.6 associated healthcare procedures, with median consultation duration of 15 minutes [Q1=10; Q3=20] and a mean number of 2.21 procedures (SD=1.12) per consultation result.

LBP was a consultation result for 845 (7.3 %) patients. Compared with consultations for other health problems, patients consulting for LBP were less frequently female (55.6 % vs 60.5 %, p=0.006), aged 45 to 54 (26.1 % vs 21.4 %, p<0.001), manual workers (10.5 % vs 6.6 %, p<0.001) and receivers of benefits (9.6 % vs 4.1 %, p<0.001). The mean number of healthcare procedures was higher in consultations for LBP than in those for any other reason: 2.92 vs 2.19, p<0.001. (Table 1)

Among the 845 LBP consultations, 324 (38 %) patients presented radiating pain. There was no statistical difference between the presence or absence of radiating pain, in terms of age, gender, profession, workers' compensation and other benefits, practice location or consultation duration (Table 2). The mean number of healthcare procedures was slightly higher in consultations with radiating pain than in those without (3.06 vs 2.84; p=0.02).

Healthcare procedures for LBP according to patient characteristics

Among the patients who presented for LBP, 776 (79.5 %) underwent a clinical examination and 634 (73.4 %) were prescribed medication. Sick leave was prescribed for 203 (23.9 %), 220 (22.8 %) received advice and 190 (22.1 %) were referred to physiotherapists, while only 113 (12.9 %) were referred for imaging and 45 (5.2 %) to medical specialists (Table 3). Radiography was more frequent with 69 (8.1 %) patients being x-rayed compared with only 23 (2.7 %) having a CT and 18 (2.1 %) having an MRI scan (p<0.001). No significant difference for healthcare procedures was found according to patient age or gender.

Healthcare procedure according to physician characteristics

No significant association was found between healthcare procedures and GP age, gender, or practice type. There were 3.02 healthcare procedures prescribed per consultation result in rural and semirural areas and 2.84 in urban areas (p=0.048). Physiotherapy prescription was considerably more frequent in rural (26.9 %) and urban areas (24.7 %) than in semi-rural areas (13.1 %) (p<0.001) and referral to complementary therapies was decidedly more common in rural areas (12.6 %) compared with urban (2.8 %) and semi-rural areas (6.8 %) (p<0.001).

Discussion

Main findings

Our study found that on average, GPs delivered more healthcare procedures during LBP consultations than during consultations for other health problems. Around three quarters of LBP patients underwent clinical examinations and were prescribed medication, around one quarter were prescribed sick leave, but imaging and specialist referrals were rare. LBP clinical characteristics, including radiating pain or persistence, influenced the number and types of healthcare procedures more than patient or GP sociodemographic characteristics.

Results in relation to the existing literature

Patient and LBP characteristics

In this study, LBP accounted for 7.3 % of consultations for adults aged between 18 and 65. These results concur with the Observatory of General Medicine results, where LBP was the eighth most common reason for consultation in 2009 and accounted for 6.88 % of consultations [START_REF]Lombalgie commune en M edecine G en erale[END_REF]. More specifically, LBP consultations more frequently involved patients aged 45 to 54, women, manual workers, and salaried employees. These results are similar to ours and those reported in an epidemiological LBP study which found that LBP prevalence was 5.14 %, slightly higher among women than men (5.3 % vs 4.99 %) and more frequent in the 50 to 54 age group using the ICPC-2-classification (codes L03, L84 and L86) [START_REF] Bartholomeeusen | Higher incidence of common diagnoses in patients with low back pain in primary care[END_REF]. Furthermore, the French CON-STANCE study identified workers and employees in certain industries as being more susceptible to developing LBP symptoms during their professional lives [START_REF] Ngabirano | Comparison Between a Job-Exposure Matrix (JEM) Score and Self-Reported Exposures for Carrying Heavy Loads Over the Working Lifetime in the CONSTANCES Cohort[END_REF].

Healthcare procedures

We observed that more healthcare procedures were prescribed in LBP consultations than in other consultations. This is consistent with the biopsychosocial LBP model and the multidimensional therapeutic approach. GPs proposed a variety of healthcare procedures depending on the clinical situation. We found that more patients underwent a clinical examination during the initial consultation (n=363, 94.5 %) than in the follow-up consultations (n=309, 67 %) and were prescribed more medications (n=324, 84.4 % vs n=294, 64.2 %) (aOR=2.34; 95 % CI [1.65, 3.30]). These results are similar to those from a Dutch study, in which 85 % of patients underwent a clinical examination during the initial consultation compared with 65 % in follow-up consultations and were prescribed more medication in the initial consultation than in the follow-up consultations (53 % vs 41 %) [START_REF] Van Tulder | Chronic low back pain in primary care: a prospective study on the management and course[END_REF].

In agreement with our study, E. Volin found no link between LBP management and patient gender, but urban GPs prescribed less medication than rural GPs [START_REF] Volinn | The epidemiology of low back pain in the rest of the world. A review of surveys in low-and middle-income countries[END_REF]. Other studies in Spain, Sweden and the UK reported similar results for clinical examination, medication, referral to a specialist or physiotherapist and sick leave prescription [START_REF] Kovacs | Non-specific low back pain in primary care in the Spanish National Health Service: a prospective study on clinical outcomes and determinants of management[END_REF][START_REF] Werner | Primary care doctors' management of low back pain patients-ten years after[END_REF]. Sick leave prescription rates vary according to LBP diagnosis and persistence. In a Swedish cohort of 20251 patients, the sick leave prescription rate was higher (around 31 %) for persistent LBP [START_REF] Hubertsson | Sick leave patterns in common musculoskeletal disorders-a study of doctor prescribed sick leave[END_REF]. It has been suggested that regular medication use could be a prognostic factor associated with long-term sick leave [START_REF] Sundstrup | Regular use of medication for musculoskeletal pain and risk of long-term sickness absence: A prospective cohort study among the general working population[END_REF]. We found that two thirds of patients with persistent LBP received medication, suggesting a possible marker, and indicating that specific intervention such as active physiotherapy is called for.

European guidelines recommend imaging only for specific LBP cases with clinical red flags or persistent pain. In our study, radiography prescriptions are high compared to these recommendations. However, CT and MRI scans and referrals to specialists were more New guidelines on LBP management have recently been published, providing clear patient pathways and management recommendations [START_REF] Autorit E De Sant E | Prise en charge du patient pr esentant une lombalgie commune[END_REF]. Our study data are likely to be useful in discussion of changes in French GP clinical practice in light of these new guidelines. While GPs are well-placed to provide early LBP diagnosis and management, they need time and experience to integrate recommendations in their practice.

Strengths and limitations

To our knowledge, the 2011-2012 ECOGEN study is the first and only large-scale French study providing a detailed description of general practice consultations and exploring healthcare procedures according to clinical and sociodemographic patient characteristics. Similarly, as there are relatively few data available and since many are dated, it was difficult to find relevant comparative studies from other countries.

The ICPC-2 classification and observational design added value compared with previous studies using questionnaires, which can entail memory and declaration biases, or with studies using medico-socio-economic data restricted to reimbursed procedures [START_REF] Lafuma | Management and cost of care for low back pain in primary care settings in France[END_REF][START_REF] Hanney | The Influence of Physical Therapy Guideline Adherence on Healthcare Utilization and Costs among Patients with Low Back Pain: A Systematic Review of the Literature[END_REF].

The ECOGEN study describes reasons for consultation and healthcare procedures associated with the main health problems confronted in French general practice, thereby creating a degree of observer bias. A Hawthorne effect is also possible, either from the GP or the patient. However, French GP internship supervisors are used to being observed by a student in their practice, which should limit this effect. Another limitation is that LBP episode duration was not recorded (consultations could only be classified as initial or follow-up), and the ICPC-2 classification did not enable pain assessment.

As the ECOGEN study was conducted at a consultation level, it is possible that a given patient consulted several times during the study period. This risk is limited and would have had little impact on the study results. The double data collection assessing reproducibility covered around 5 % of all consultations and found a difference of 3.5 %, which was considered satisfactory. The low refusal rate (1 %) implies a low level of non-participation bias. Physician representativeness in the ECOGEN study was assessed according to sociodemographic variables (age, gender, practice location). The ECOGEN GP characteristics were not statistically different from those of French GPs generally [START_REF] Letrilliart | ECOGEN: etude des Él ements de la COnsultation en m edecine GEN erale[END_REF][START_REF] Bouton | Repr esentativit e des m edecins g en eralistes maîtres de stage universitaires[END_REF] meaning that the results can be generalised to GPs throughout France.

Conclusion

The healthcare procedures prescribed for LBP were not always in line with existing French guidelines. LBP characteristics, including radiating or persistent pain, influenced healthcare procedures more markedly than patient or GP characteristics.

Disclosure statement

The authors declare that they have no competing interest.

L

  'impact des caract eristiques des douleurs lombaires sur les soins prodigu es ou prescrits aux patients adultes par les m edecins

  23,9 %) a un arrêt de travail. La prescription d'imagerie et le renvoi vers un sp ecialiste etaient peu fr equents. La prescription d'imagerie etait plus fr equente en cas de douleurs irradiantes (Odds ratio (OR) ajust e = 1,61 ; IC95 % [1,07-2,42]), de même que l'adressage a un sp ecialiste (OR ajust e = 2,92 ; IC95 % [1,40-6,09]) et l'arrêt de travail (OR ajust e = 1,52 ; IC95 % [1,10-2,09]). En revanche, la prescription de kin esith erapie etait moins fr equente (OR ajust e = 0,55 ; IC95 % [0,38-0,82]). L'examen clinique (OR ajust e = 2,75 ; IC95 % [1,98-3,80]), l'imagerie (OR ajust e = 1,61 ; IC95 % [1,02-2,31]) et la prescription de m edicaments (OR ajust e = 2,34 ; IC95 % [1,65-3,30]) etaient plus fr equents en consultation initiale, mais l'adressage chez un sp ecialiste (OR ajust e = 0,16 ; IC95 % [0,05-0,47]) et l'arrêt de travail (OR ajust e = 0,68 ; IC95 % [0,48-0,97]) etaient moins fr equents. Conclusion. -Les caract eristiques des douleurs lombaires pourraient influencer les soins prodigu es ou prescrits plus fortement que les caract eristiques des patients ou celles des praticiens. © 2022 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Fig. 1 .

 1 Fig. 1. Flow diagram of patient inclusion process

Table 1

 1 Patient characteristics in consultations with or without LBP (N=11510 consultations)

		Consultation for low back pain* N=845	Consultations for any other reason N=10665	p-value
	Female gender, n (%)	470 (55.6)		6453 (60.5)	0.006
	Age, n (%)				< 0.001 »
	18-34 years old	168 (19.9)		3022 (28.4)
	35-44 years old	197 (23.3)		2167 (20.4)
	45-54 years old	221 (26.1)		2286 (21.4)
	55-65 years old	259 (30.7)		3190 (29.9)
	Socio-occupational category, n (%)				<0.001 »
	Farmer, self-employed, retail trades, business person	43 (5.1)		578 (5.4)
	Managerial and Intermediate professions	125 (14.8)		2096 (19.7)
	Salaried employee	362 (42.8)		3554 (33.3)
	Manual worker	89 (10.5)		703 (6.6)
	Retired	99 (11.8)		1401 (13.1)
	Unemployed	127 (15)		1333 (21.9)
	Workers' compensation and other benefits, n (%)	81 (9.6)		432 (4.1)	<0.001
	Consultation duration in minutes: mean (SD)	18.11 (15.20)		17.15 (16.35)	0.08
	Practice location, n (%)				0.11 »
	Rural	167 (19.8)		1876 (17.6)
	Semi-rural	221 (26.2)		2638 (24.7)
	Urban	457 (54.1)		6151 (57.7)
	Number of procedures by consultation result: mean (SD)	2.92 (1.39)		2.19 (1.28)	<0.001
	Table 2
	Patient characteristics in LBP consultations with or without radiating pain (N=845)
			Low back pain without radiating	Low back pain with radiating
			pain N=521		pain N=324	p-value
	Female gender, n (%)		293 (56.2)		177 (54.6)	0.7
	Age, n (%)				0.22 »
	18-34 years old		110 (21.1)		58 (17.9)
	35-44 years old		128 (24.6)		69 (21.3)
	45-54 years old		125 (24)		96 (29.6)
	55-65 years old		158 (30.3)		101 (31.2)
	Socio-occupational category, n (%)				0.42 »
	Farmer, self-employed, retail trades, business person	26 (5.0)		17 (5.2)
	Managerial and intermediate professions		71 (13.6)		54 (16.7)
	Salaried employee		220 (42.2)		142 (43.8)
	Manual worker		50 (9.6)		39 (12)
	Retired		68 (13.1)		31 (9.6)
	Unemployed		86 (6.5)		41 (12.7)
	Workers' compensation and other benefits, n (%)		45 (8.6)		36 (14.2)	0.53
	Consultation duration in minutes: mean (SD)		17.41 (8.5)		19.5 (21.9)	0.08
	Practice location, n (%)				0.33 »
	Rural		111 (21.3)		56 (17.3)
	Semi-rural		136 (26.1)		85 (26.2)
	Urban		274 (52.6)		183 (56.5)
	Number of healthcare procedures per consultation result: mean (SD) 2.84 (1.36)		3.06 (1.44)	0.02

LBP: low back pain, * either as single consultation result or as one of multiple consultation results, » global p-value (Fisher's Exact Test) LBP: low back pain, » global p-value (Fisher's Exact Test)

Table 3

 3 Healthcare procedures associated with LBP consultations, overall, and according to radiating pain status When LBP was associated with radiating pain, imaging investigations were more frequently prescribed (Adjusted OR (aOR) =1.61; 95 % CI [1.07-2.42]), especially CT and MRI scans, as were specialist referrals (aOR=2.92; 95 % CI [1.40-6.09]) and sick leave (aOR=1.52; 95 % CI [1.10-2.09]), while physiotherapist referral was less frequent (aOR=0.55; 95 % CI [0.38-0.82]).Patients were prescribed an average of 3.57 healthcare procedures in an initial LBP consultation and 2.38 procedures in follow-up consultations, with mean consultation durations of 17.

						Multivariate model*
					Univariate model (with /	(with / without radiating
		LBP consultations	LBP without radiating	LBP with radiating	without radiating pain)	pain) Adjusted OR (CI
		overall N=845	pain N=521	pain N=324	OR (CI 95 %)	95 %)
	Clinical examination, n (%)	776 (79.5)	410 (78.7)	262 (80.9)	0.85 (0.64-1.14)
	Imaging, n (%)	113 (12.9)	55 (10.6)	54 (16.7)	1.61 (1.08-2.39)	1.61 (1.07-2.42)
	Radiography	69 (8.1)	45 (8.6)	23 (7.1)
	CT scan	23 (2.7)	6 (1.2)	17 (5.2)
	MRI scan	18 (2.1)	4(0.8)	14 (4.3)
	Referral to a specialist, n (%)	45 (5.2)	16 (3.1)	28 (8.6)	3.51 (1.68-7.3)	2.92 (1.40-6.09)
	Medication, n (%)	634 (73.4)	371 (71.2)	249 (76.9)	1.25 (0.92-1.7)
	Equipment and devices, n (%)	30 (3.6)	19 (3.6)	11 (3.4)	1.46 (0.62-3.47)
	Physiotherapy, n (%)	190 (22.1)	133 (25.5)	54 (16.7)	0.49 (0.33-0.72)	0.55 (0.38-0.82)
	Recommendations, education, advice,	220 (22.8)	118 (22.6)	75 (23.1)	1.11 (0.79-1.56)
	n (%)
	Sick leave, n (%)	203 (23.9)	106 (20.3)	96 (29.6)	1.57 (1.14-2.15)	1.52 (1.10-2.09)

LBP: low back pain, * marginal logistical analysis adjusted on gender and age M. Peurois, C. Bouton, C. B egue et al.

Table 4

 4 Healthcare procedures associated with LBP consultations according to follow-up status

					Multivariate model* (first/
		Initial LBP	Follow-up LBP	Univariate model (first/follow-	follow-up LBP consultation) Adj
		consultation N=384	consultation N=461	up LBP consultation) OR (CI 95 %)	OR (CI 95 %)
	Procedures	N=1372	N=1099
	Clinical examination, n (%)	363 (94.5)	309 (67.0)	3.22 (2.36-4.38)	2.75 (1.98-3.80)
	Imaging, n (%)	60 (15.6)	49 (10.6)	1.54 (1.02-2.31)	0.16 (0.05-0.47)
	Radiography	43 (11.2)	26 (5.4)	0.15 (0.05-0.43)
	CT scan	13 (3.4)	10 (2.2)
	MRI scan	4 (1.0)	14 (3.0)
	Referral to a specialist, n (%)	7 (1.8)	37 (8.0)
	Medication, n (%)	324 (84.4)	296 (64.2)	2.69 (1.94-3.71)	2.34 (1.65-3.30)
	Equipment and devices, n (%)	9 (2.3)	21 (4.6)	0.89 (0.37-2.12)
	Physiotherapy, n (%)	80 (20.8)	107 (23.2)
	Recommendations, education, advice, n (%) 93 (24.2)	100 (21.7)	0.93 (0.67-1.35)
	Sick leave, n (%)	94 (24.5)	108 (23.4)	0.74 (0.54-1.01)	0.68 (0.48-0.97)

LBP: Low back pain. * marginal logistical analysis adjusted on gender and age frequent with radiating pain, which is consistent with recommendations.

Acknowledgments

The authors would like to thank all the patients, interns, GP tutors and ECOGEN researchers who contributed to this study.

Editorial assistance in the preparation of this article was provided by Speak the Speech Consulting.

Funding

The authors received no financial support for the research, authorship, and publication of this article.