Jie Zhao

Qihui Shi

Hongda Tian

Youzhi Li

Yan Liu

Zhen Xu

Anne Robert

Qiong Liu

Bernard Meunier

TDMQ20, a specific copper chelator, reduces memory impairments in ADmouse models

Beside targeting amyloid or tau metabolisms, regulation of redox metal ions is a recognized therapeutic target for Alzheimer's disease (AD). Based on the bioinorganic chemistry of copper, we designed specific chelators of copper(II) (TDMQs) in sight to regulate copper homeostasis in the brain, and to inhibit the deleterious oxidative stress catalyzed by copper-amyloid complexes.

An oral treatment by TDMQ20 was able to fully reverse the cognitive and behavioral impairment in three different murine models, two non-transgenic models mimicking the early stage of AD, and a transgenic representing a more advanced stage of AD. To our knowledge, such comparative study using the same molecule has never been performed. Regular C57 BL/6J mice received a single injection of human Cu-Aβ1-42 in the lateral ventricles (icv-CuAβ) or in the hippocampus (hippo-CuAβ). In both cases, mice developed a cognitive impairment similar to that of transgenic 5XFAD mice. Oral administration of TDMQ20 to icv-CuAβ or hippo-CuAβ mice within a 16-days period resulted in a significant improvement of the cognitive status. The 3-months treatment of transgenic 5XFAD mice with TDMQ20 also resulted in behavioral improvements. The consistent positive pharmacological results obtained using these different AD models correlate well with previously obtained physico-chemical data of TDMQ20.

The short-term novel object recognition (NOR) test was found particularly relevant to evaluate the rescue of declarative memory impairment. TDMQ20 was also able to reduce the oxidative stress in the mouse cortex. Due to its reliability and facile use, the hippo-CuAβ model can be considered as a robust non-transgenic model to evaluate the activity of potential drugs on the early stages of memory deficits.

INTRODUCTION

Alzheimer's disease (AD) affects millions of people worldwide, and the incidence of this agerelated disease is increasing due to the increase of life expectancy in many countries. [START_REF]Data on AD[END_REF][START_REF] Meunier | Age and Alzheimer's disease[END_REF] AD being mainly characterized by the formation of senile plaques of b-amyloid peptides and by neurofibrillary tangles due to the hyperphosphorylation of the tau protein, these phenomena have been considered as targets for the design of therapeutic agents.

Since memantine in 2003, no new drug has been approved, except sodium oligomannate (GV-971), recently approved by the National Medical Products Administration of China. [START_REF] Wang | Sodium oligomannate therapeutically remodels gut microbiota and suppress gut bacterial amino acids-shaped neuroinflammation to inhibit Alzheimer's disease progression[END_REF] Between 2003 and 2019, 100 % of drug-candidates failed in clinical trials. [START_REF] Cummings | Alzheimer's disease drug-development pipeline: few candidates, frequent failures[END_REF] The quasi-exclusive use of transgenic mice to select drug-candidates for a disease which has no genetic origin for sporadic cases, might be questioned. Predictive and easy to handle mouse AD models are needed. In an attempt of diversification, we developed a non-transgenic mice model for the evaluation of the chelator PA1637. [START_REF] Ceccom | Copper chelator induced efficient episodic memory recovery in a non-transgenic mouse model[END_REF] In this model, an episodic memory deficit was generated by a single intracerebroventricular (icv) injection in the brain of normal mice, of low molecular weight Ab1-42 oligomers without copper. Despite controversies on the role of soluble amyloids, [START_REF] Selkoe | The amyloid hypothesis of Alzheimer's disease at 25 years[END_REF] they are still considered as toxic agents contributing to synaptic alteration, abnormal tau phosphorylation, glial activation and neuronal loss in vivo. [START_REF] Tomiyama | A mouse model of amyloid β oligomers: Their contribution to synaptic alteration, abnormal tau phosphorylation, glial activation and neuronal loss in vivo[END_REF] More recently, the key role of Ab oligomers to trigger activation of the glycogen synthase kinase 3b (GSK3b), responsible for tau hyperphosphorylation, has been demonstrated. [START_REF] Zhang | bamyloid redirects norepinephrine signaling to activate the pathogenic GSK3b/tau cascade[END_REF] Despite the capacity of PA1637 to extract copper from copper-amyloid in vitro, and to inhibit the catalytic production of H2O2 by Cu(II)-amyloid [START_REF] Nguyen | Characterization of new specific copper chelators as potential drugs for the treatment of Alzheimer's disease[END_REF][START_REF] Nguyen | Transfer of copper from an amyloid to a natural copper-carrier peptide with a specific mediating ligand[END_REF] its development has been stopped due to its rather low bioavailability. Consequently, we designed a new series of specific chelators of Cu(II) named TDMQ, based on a 8-aminoquinoline skeleton with a chelating side-chain at C2 (Figure 1). Such tetradentate ligands have the required structure to discriminate between copper and zinc. [START_REF] Liu | Metals ions in Alzheimer's disease: a key role or not?[END_REF][START_REF] Zhang | Synthesis and characterization of copper specific tetradentate ligands as potential treatment for Alzheimer's disease[END_REF][START_REF] Zhang | Preparation of new tetradentate copper chelators as potential anti-Alzheimer agents[END_REF][START_REF] Huang | The TDMQ regulators of copper homeostasis do not disturb Cu,Zn-SOD and tyrosinase activity, nor the Co(III) cofactor vitamin B12[END_REF][START_REF] Li | Why is tetradentate coordination essential for potential copper homeostasis regulators in Alzheimer's disease?[END_REF] Two 8-hydroxyquinolines, clioquinol (CQ) and PBT2, failed in clinical trials to improve cognition and reduce amyloid plaques. [START_REF] Adlard | Metal and Alzheimer's disease: How far have we come in the clinic?[END_REF][START_REF]Prana Biotechnology announces preliminary results of Phase 2 IMAGINE trial of PBT2 in Alzheimer's disease[END_REF] This failure might be explained by their lack of specificity for copper chelation, [START_REF] Nguyen | The necessity of having a tetradentate ligand to extract copper(II) ions from amyloids[END_REF][START_REF] Nguyen | Structures of copper and zinc complexes of PBT2, a chelating agent evaluated as potential drug for neurodegenerative diseases[END_REF] and their inability to inhibit the production of reactive oxygen species (ROS) generated by the Cu-amyloids. [START_REF] Li | Why is tetradentate coordination essential for potential copper homeostasis regulators in Alzheimer's disease?[END_REF][START_REF] Zhang | N4-Tetradentate chelators efficiently regulate copper homeostasis and prevent ROS production induced by copperamyloid, even in the presence of an excess of zinc[END_REF] TDMQ ligands are able to extract copper(II) from Cu-amyloids, without disturbing the activities of Cu,Zn-SOD, tyrosinase or vitamin B12. 14 TDMQ20 has been selected as lead molecule for evaluation as anti-AD drugcandidate on three different mouse models, two non-transgenic-and a transgenic one. TDMQ22, a second chelator, differs from TDMQ20 by having a CF3 substituent at position 6 instead of Cl at C5 and C7. [START_REF] Zhang | Preparation of new tetradentate copper chelators as potential anti-Alzheimer agents[END_REF] Figure 1. Structure of the chelators TDMQ20, TDMQ22, clioquinol (CQ) and PBT2, and structure of the copper(II) complex of TDMQ20, for its X-ray structure data, see Reference 11. The deregulation of copper in AD brain and the associated memory deficits have been created by injection of the copper complex of amyloid Ab1-42 in the lateral ventricles (icv-CuAβ model) or hippocampus (hippo-CuAβ model) of regular mice. The third model was the transgenic mouse 5XFAD model. Data obtained with these three murine models indicated that the oral administration of TDMQ20 was able to inhibit memory deficits.

RESULTS AND DISCUSSION

Mouse AD models, and drug treatment.

In two non-transgenic mouse models, the cognitive deficit was created by injection of human Cu II -Ab1-42 complex either in the lateral ventricles (icv-CuAb model) or in the hippocampus (hippo-CuAb model) of regular young C57BL/6J mice (two months of age), at D0. The icv-CuAβ model of AD is similar to the icv injection of Aβ1-42 oligomers used to evaluate the tetradentate PA1637 chelator. [START_REF] Ceccom | Copper chelator induced efficient episodic memory recovery in a non-transgenic mouse model[END_REF] In the second non-transgenic model, hippo-CuAβ, the Cu-Aβ1-42 solution was injected in the hippocampus. This region of the brain is involved in declarative memory and learning as well as in spatial memory, and is particularly vulnerable in the early stages of AD. [START_REF] West | Regionally specific loss of neurons in aging human hippocampus[END_REF][START_REF] Mu | Adult hippocampal neurogenesis and its role in Alzheimer's disease[END_REF] Atrophy of hippocampus is strongly correlated with cognitive decline. [START_REF] Halliday | Pathology and hippocampal atrophy in Alzheimer's disease[END_REF] This non-transgenic hippo-CuAβ model can therefore contribute to mimic the early stages of AD.

In these non-transgenic AD mice, the drug treatment consisted of oral gavage with TDMQ20, TDMQ22 or CQ used as comparator, 8 times between D4 and D20 (each dose was 10 mg kg -1 ).

We also evaluated TDMQ20 on the transgenic 5XFAD transgenic mouse model (C57BL6 genetic background), that expressed human APP and PSEN1 transgenes with a total of five AD- linked mutations: the Swedish (K670N/M671L), Florida (I716V) and London (V717I) mutations in APP (the precursor of amyloids) and the two mutations in PSEN1 (M146L and L286V) (PSEN1 is the gene coding for presenilin-1, a constituent of g-secretase). This model recapitulates several AD-related aspects of the disease, [START_REF] Oakley | Intraneuronal b-amyloid aggregates, neurodegeneration, and neuron loss in transgenic mice with five familial Alzheimer's disease mutations: Potential factors in amyloid plaque formation[END_REF][START_REF] Jawhar | Motor deficits, neuron loss, and reduced anxiety coinciding with axonal degeneration and intraneuronal Aβ aggregation in the 5XFAD mouse model of Alzheimer's disease[END_REF] with early formation of amyloid plaques and gliosis at two months. Cognitive and motor deficits start at about 6 months, and develop with age in parallel with neuron degeneration in multiple brain regions. [START_REF] Schneider | Behavioral and EEC changes in male 5xFAD mice[END_REF][START_REF] Urano | Icariin improves memory impairment in Alzheimer's disease model mice (5xFAD) and attenuates amyloid b-induced neurite atrophy[END_REF] For 5XFAD mice, the drug treatment was carried out once every two days, from 2 to 5 months of age, and consisted of oral gavage with TDMQ20, or CQ used as comparator, (45 times, each drug dose: 10 mg kg -1 ). The mouse cognitive status was evaluated using the standardized behavioral tests: novel object recognition (NOR), elevated plus maze test (EPM), open field test (OFT), and Y-maze test (YMT), at the end of the treatment (between D22 and D26 for the icv-CuAb model and the hippo-CuAb model), and between 5.2 and 5.6 months of age for the 5XFAD mice.

TDMQ20 inhibits of the cognitive impairment of AD mice.

Novel object recognition (NOR)

The NOR test, based on the spontaneous propensity of rodents to explore new objects, allows the evaluation of short-term-or long-term memory, [START_REF] Morroni | Early effects of Aβ1-42 oligomers injection in mice: involvement of PI3K/Akt/GSK3 and MAPK/ERK1/2 pathways[END_REF] assessing cognitive deficits in a wide range of pathological conditions, in transgenic and non-transgenic murine models of AD. [START_REF] Grayson | Assessment of disease-related cognitive impairments using the novel object recognition (NOR) task in rodents[END_REF] This assay is particularly relevant to AD, and consequently for the evaluation of the efficacy of potential drugs since it is dependent on the integrity of the temporal regions affected in the early stages of AD.

The recognition index is calculated as the percentage of explorations of a novel object with respect to the total number of explorations for short-term memory or long-term memory {[(nY/nX+Y)´100] or [(nZ/nX+Z)´100], respectively, where nX, nY, nZ stand for the number of explorations of familiar object X, novel object Y in short-term memory evaluation, or novel object Z in long-term memory evaluation, respectively}. The recognition index is reported in Figure 2 for the icv-CuAβ (panel A), the hippo-CuAβ (panel B), and the 5XFAD (panel C) mouse models.

First of all, for short-term memory evaluation, the recognition index was significantly lower for the icv-CuAβ mice or hippo-CuAβ mice compared to the healthy controls (Figure 2, panel A or B, respectively), indicating that this test was relevant to discriminate between healthy and nontransgenic AD mice, a necessary condition to evaluate the activity of potential drugs on these models. Contrary, the difference of the recognition index was not statistically significant between 5XFAD mice and their wild type littermates (panel C), consistent with bibliographic data indicating that, in transgenic models of AD, the performance of mice in the NOR is age-dependent and deficits appear only after 6 months of age, [START_REF] Grayson | Assessment of disease-related cognitive impairments using the novel object recognition (NOR) task in rodents[END_REF] or even later. [START_REF] Ardestani | Modulation of neuroinflammation and pathology in the 5XFAD mouse model of Alzheimer's disease using a biased and selective beta-1 adrenergic receptor partial agonist[END_REF] This result suggests that icv-CuAβ-and hippo-CuAβ mice are mimicking early cognitive impairment of AD, whereas such damages are probably undetectable in young 5XFAD mice. non-transgenic mouse models, and in the transgenic 5XFAD (panel C) model. nX, nY, nZ stand for the number of explorations of familiar object X, novel object Y in short-term memory evaluation, or novel object Z in long-term memory evaluation, respectively. In each case, the recognition index of AD mice which received no drug-treatment (untreated group) was compared to that of healthy mice (control group), and to the recognition index of AD mice treated with TDMQ20 or clioquinol [8 × 10 mg kg -1 in 16 days (panels A and B), or 10 mg kg -1 , one every two days during 3 months (panel C)]. In panels A or B, the control/untreated groups were brain-injected with CuAb/PBS. In panel C, the untreated and control groups were the transgenic 5XFAD mice and their non-transgenic littermates (C57BL6), respectively. All control and untreated mice received oral gavage with aqueous NaCl 0.9 w/v instead of drug. For details, see SI. Each mark represents the result obtained with a single mouse; horizontal lines represent After a short oral treatment by TDMQ20 icv-CuAβ mice spent significantly more time exploring the novel object in the short-term memory test, compared to untreated AD mice. A similar trend was evidenced for hippo-CuAβ mice. In fact, the recognition index of treated mice was similar to the score of control mice. Then, TMDQ20 significantly reduced the cognitive injuries created by the intracerebral injection of the Cu-Aβ1-42 complex. By comparison, CQ did not significantly improved the short-term memory of icv-CuAβ mice and hippo-CuAβ mice.

Duplication of these experiments using icv-CuAβ-and hippo-CuAβ mice provided fully consistent results provided as Supporting Information (Figure S1). The ability of TDMQ22 to restore the short-term memory of icv-CuAβ mice was close to that of TDMQ20 (SI, Figure S1, panel A).

Contrary to the short-term memory, the long-term recognition index was not significantly impaired in icv-CuAβ-and hippo-CuAβ mice (panels A and B, right). This result suggests that these models efficiently mimic the early stage of AD, in which the short-term memory is the first to be impaired, while the long-term memory, including semantic and procedural memories are not yet significantly impacted. We should note that the development of efficient drugs requires the use of mouse models of the early stages of the disease, and not a model for the late stages, where cognitive and anatomic damages are irreversible.

Y-maze test

The Y-maze test is a measure of spatial working memory driven by the innate curiosity of rodents to explore previously unvisited areas. [START_REF] Kraeuter | The Y-maze for assessment of spatial working and reference memory in mice[END_REF] The spontaneous alternation of mice in the Y-maze has been validated as a hippocampal relevant spatial task. [START_REF] Conrad | Chronic stress impairs rat spatial memory on the Y maze, and this effect is blocked by tianeptine pretreatment[END_REF][START_REF] Ohno | BACE1 deficiency rescues memory deficits and cholinergic dysfunction in a mouse model of Alzheimer's disease[END_REF] The spontaneous alternation ratios of hippo-CuAβ-and 5XFAD mice in the Y-maze are reported in Figure 3, panels A and B, respectively. The 5XFAD mice exhibit a tendency to reduced spontaneous alternation ratio compared with wild-type mice (Figure 3), consistent with previous reports indicating that alternation ratio was impaired after the age of 6 months. [START_REF] Oakley | Intraneuronal b-amyloid aggregates, neurodegeneration, and neuron loss in transgenic mice with five familial Alzheimer's disease mutations: Potential factors in amyloid plaque formation[END_REF][START_REF] Jawhar | Motor deficits, neuron loss, and reduced anxiety coinciding with axonal degeneration and intraneuronal Aβ aggregation in the 5XFAD mouse model of Alzheimer's disease[END_REF] The oral treatment by TDMQ20 restored the alternation ratio of 5XFAD mice (panel B) up to the level of control mice, with nearly the same efficacy than CQ. A similar trend was obtained with hippo-CuAβ mice. Due to the result lack of statistical significance, we suggest that the spatial memory impairment is not yet established in these non-transgenic early AD mice. Therefore this test is poorly suitable to evaluate the cognitive impairment and the efficacy of drugs in 2-3 month old hippo-CuAβ mice.

Open field test

In the open-field assay, the unsupported rearings and travel distances of the hippo-CuAβ mice in the open-field were found significantly lower than that of age-matched controls, indicating that the locomotor deficit of hippo-CuAβ mice was well established (Figure 4A). Here also, the treatment by TDMQ20 for a short period of 16 days resulted in full rescue of the mouse abilities, with both rearings and travel distances similar to that of controls.

Conversely, the locomotor activity of 5XFAD mice was found rather close to that of the corresponding healthy mice, with no significant difference in the rearings between WT and 5XFAD mice (Figure 4B). This is consistent with the fact that the exploratory behavior and locomotor activity of 5XFAD mice were reported as being normal at least until 6 months, [START_REF] Schneider | Behavioral and EEC changes in male 5xFAD mice[END_REF][START_REF] Urano | Icariin improves memory impairment in Alzheimer's disease model mice (5xFAD) and attenuates amyloid b-induced neurite atrophy[END_REF] or 9-12 months old. [START_REF] Jawhar | Motor deficits, neuron loss, and reduced anxiety coinciding with axonal degeneration and intraneuronal Aβ aggregation in the 5XFAD mouse model of Alzheimer's disease[END_REF][START_REF] Ardestani | Modulation of neuroinflammation and pathology in the 5XFAD mouse model of Alzheimer's disease using a biased and selective beta-1 adrenergic receptor partial agonist[END_REF] However, in our hands, the grid crossings by 5XFAD animals was significantly higher than that of their WT littermates (Figure 4B), suggesting that, at 5.4 months, brain damage were already installed. In these conditions, the treatment by TDMQ20 significantly normalized the grid crossing by transgenic AD mice.

Elevated plus maze

The elevated plus maze assay has been described for assessing anxiety response of rodents, and screening of drugs for treatment of anxiety-related disorders. [START_REF] File | Factors controlling measures of anxiety and responses to novelty in the mouse[END_REF][START_REF] Walf | The use of the elevated plus maze as an assay of anxietyrelated behavior in rodents[END_REF] The score of 5XFAD mice in the elevated plus maze is reported in mice (Figure 5), that may correspond to dis-inhibitory tendencies seen in AD patients. [START_REF] Jawhar | Motor deficits, neuron loss, and reduced anxiety coinciding with axonal degeneration and intraneuronal Aβ aggregation in the 5XFAD mouse model of Alzheimer's disease[END_REF][START_REF] Schneider | Behavioral and EEC changes in male 5xFAD mice[END_REF] Oral treatment of these mice with TDMQ20 fully reversed their behavior to to the score of WT, more efficiently than the CQ treatment did. As a summary of behavioral tests (Table 1), both 2.5-2.7 month hippo-CuAβ or icv-CuAβ mice exhibited significant deficits of short-term memory in the NOR assay. The score of the hippo-CuAβ mice was also impaired in the open-field assay, validating these non-transgenic models to evaluate the activity of potential drugs on the early stages of the memory deficits. The short oral treatment with TDMQ20 was efficient to fully restore the abilities of mice in these reference tests. The score of hippo-CuAβ mice in the Y-maze was less impaired than in transgenic 5XFAD mice, but nevertheless confirmed that TDMQ20 was able to reverse the ongoing impairment of the spatial memory on young mice affected by intracerebral injection of copperamyloids.

Conversely, the deficits of 5XFAD mice observed in the open field, Y-maze or elevated plus maze assays, are rather weak below 6 months of age. Cognitive deficits of these transgenic mice are reported to correlate well with an increasing burden of amyloid plaques in the cortex and hippocampus, axonal degeneration, spinal cord pathology and neuron loss in specific cortical layer, mimicking an advanced AD pathology. [START_REF] Jawhar | Motor deficits, neuron loss, and reduced anxiety coinciding with axonal degeneration and intraneuronal Aβ aggregation in the 5XFAD mouse model of Alzheimer's disease[END_REF] Similarly, in APP/PS-1 mouse model, the cognitive deficits in both recognition memory and spatial memory become apparent only at 11-24 months of age, when the disease damages are definitively installed. [START_REF] Webster | Comprehensive behavioral characterization of an APP/PS-1 double knock-in mouse model of Alzheimer's disease[END_REF] In these conditions, such transgenic models should be considered more relevant to describe advanced stages of AD, but are probably not really adapted to evaluate drug-candidates that should be efficient on the early stages of the disease. Table 1. Improvement of the scores of AD mice treated by TDMQ20, with respect to untreated AD controls.

TDMQ20 inhibits cortex oxidative injuries

The ability of TDMQ20 to rescue cognitive deficits is consistent with the capacity of this copper homeostasis regulator to reduce in vitro the formation of ROS. [START_REF] Zhang | Preparation of new tetradentate copper chelators as potential anti-Alzheimer agents[END_REF][START_REF] Zhang | N4-Tetradentate chelators efficiently regulate copper homeostasis and prevent ROS production induced by copperamyloid, even in the presence of an excess of zinc[END_REF] In fact, neuro-inflammation is considered a key factor of the loss of neurons and synapses in AD. [START_REF] Ardestani | Modulation of neuroinflammation and pathology in the 5XFAD mouse model of Alzheimer's disease using a biased and selective beta-1 adrenergic receptor partial agonist[END_REF] The molecular origin of brain inflammation might be linked to the oxidative stress initiated by the reductive activation of dioxygen catalyzed by copper-amyloids. It is therefore relevant to evaluate the oxidative status of the mouse brains. For this purpose, the malondialdehyde (MDA) level is significant of the potential occurrence of peroxide induced lipid injuries. [START_REF] Tsikas | Assessment of lipid peroxidation by measuring malondialdehyde (MDA) and relatives in biological samples: analytical and biological challenges[END_REF] The level of MDA, measured by the thiobarbituric reactive species (TBARS) assay, in the cortex of icv-CuAβ-and 5XFAD mice is reported in Figure 6A and 6B, respectively. The concentration of MDA in the cortex of AD mice, transgenic (5XFAD) or not (icv-CuAβ), was slightly higher than that of the controls (Figure 6), supporting the involvement of an oxidative stress in these mice. In both cases, the treatment with TMDQ20 reduced the MDA level in the cortex until the level of control mice, indicating a normalization of the oxidative status in the cortex. This encouraging data is consistent with the ability of TDMQ20 to inhibit in vitro the production of ROS induced by Cu-Aβ. [START_REF] Zhang | Preparation of new tetradentate copper chelators as potential anti-Alzheimer agents[END_REF][START_REF] Li | Why is tetradentate coordination essential for potential copper homeostasis regulators in Alzheimer's disease?[END_REF] 2.4. Preliminary safety and druggability studies of TDMQ20

Single dose

In an acute toxicity assay in WT C57BL/6J mice, TDMQ20 was found completely safe after administration of an oral dose 150 or 200 mg kg -1 , corresponding to 15-20 times the dose used to treat AD mice, with all mice surviving without any detectable deficit (Table 2). The drug exhibited some delayed toxicity by ip route at 150 mg kg -1 : two mice over 11 died at D3 and D15, respectively, indicating an LD50 value higher than 150 mg kg -1 . By comparison, clioquinol was found significantly more toxic, with LD50 values close to 200 mg kg -1 and 150 mg kg -1 , by oral and ip route, respectively (2/5 and 7/11 mice died on the day of drug administration).

Multiple doses

The toxicity of TDMQ20 was then evaluated after on oral administration of 100 mg kg -1 every day to WT C57BL/6J mice. Two mice died at D8 and D9 (survival 8/10), ie. after administration of 10-11 times the total drug dose used in the AD treatment (80 mg kg -1 in total). The assay was then discontinued at D9. Mice (n = 9) were then treated with TDMQ20 by oral route at 80 mg kg - 1 , 3 times a week during 2 weeks, corresponding to a total dose of 480 mg kg -1 . In these conditions, with a schedule similar to that used for the evaluation of the pharmacological activity of TDMQ20 (80 mg kg -1 in total), all mice survived without any detectable sign of toxicity during the treatment, suggesting a safety index ≥ 8. In addition, the treatment of 5XFAD mice clearly indicated that this drug-candidate was well tolerated upon long-term oral treatment (45 doses, 450 mg kg -1 in total), even in animals weakened by the disease.

The aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine and urea were measured in the serum of 5XFAD mice, treated or not by TDMQ20 (45 × 10 mg kg -1 in 3 months, total dose = 450 mg kg -1 ), at the end of the treatment period and compared to the levels found in the WT C57BL/6J controls. There was no significant difference between the levels obtained for the WT controls, the 5XFAD mice and the 5XFAD mice treated by TDMQ20, indicating the lack of any detectable damage on liver and kidneys upon long-term oral treatment with TDMQ20 in these conditions (45 doses, 450 mg kg -1 in total).

Druggability

In vitro preliminary metabolic studies indicate that the TDMQ20 significantly inhibits CYP 2D6, but not CYP 3A (< 20 %) and 2C9 (< 5 %) (SI). We have to remember that the drug prozac is also an inhibitor of CYP2D6. The measured pKa values indicate that, at physiological pH, TDMQ20 should exist as a mixture of species, due to mono-and diprotonation of the side chain, [START_REF] Li | Why is tetradentate coordination essential for potential copper homeostasis regulators in Alzheimer's disease?[END_REF] like many CNS drugs containing protonable nitrogen sites. [START_REF] Pajouhesh | Medicinal chemical properties of successful central nervous system drugs[END_REF] The calculated log P value for TDMQ20 is 2.5, 39 and topological polar surface area (tPSA) is 53 Å 2 , 39 in the range consistent for passive transport across the blood-brain barrier. [START_REF] Pajouhesh | Medicinal chemical properties of successful central nervous system drugs[END_REF] The membrane crossing capacity has been confirmed by the Caco-2 data. In standard conditions at 10-5 M, A-B permeability was 8.4 with a recovery of 35% and B-A permeability was 10.1 with a recovery of 74%. In addition, TDMQ20 was Ames negative on TA98, TA100, TA1535 and TA1537, even after microsomal activation (SI), indicating that this ligand was not mutagenic in bacteria.

In conclusion, TDMQ20 efficiently reversed the cognitive or behavioral impairment using two non-transgenic mouse models of the early stage of AD and in a transgenic model. With the icv-CuAβ and hippo-CuAβ models, the short-term NOR test is specially robust to evaluate the impairment of the declarative memory in mice, making such models easy to select molecules that should be active on the early stages of AD.

The efficacy of TDMQ20 by oral administration of low doses on three different mouse AD models is an encouragement for further pharmacological evaluations of this specific copper chelator.

METHODS

Materials.

Human Aβ1-42 (CF3COO -as counter-ions) was obtained from Aladdin, China. According to a protocol adapted from Reference 40, Aβ1-42 (1 mg) was dissolved in 1,1,1,3,3,3hexafluoroisopropanol (HFIP, 1 mL) and sonicated for 15 minutes. The solution was centrifugated at 12,000 rpm for 15 min. The concentration of the Aβ1-42 solution was determined using Micro BCA Protein Assay kit from ThermoFisher Scientific™ (code 23235), working with BSA (bovine serum albumin) as a calibration protein. HFIP was then removed by lyophilization, and the solid Aβ1-42 was then stored at -20 °C until used. The chelators TDMQ20 and TDMQ22 were synthesized as previously reported. [START_REF] Zhang | Synthesis and characterization of copper specific tetradentate ligands as potential treatment for Alzheimer's disease[END_REF][START_REF] Zhang | Preparation of new tetradentate copper chelators as potential anti-Alzheimer agents[END_REF] Clioquinol (5-chloro-7-iodo-8-hydroxyquinoline, CQ), CuCl2, DMSO and usual chemicals were purchased from Sigma. Phosphate buffered saline 10X (PBS 10X) was prepared as follows: NaCl 137 mmol, KCl 2.7 mmol, Na2HPO4 10.0 mmol, KH2PO4 1.76 mmol, sterile water qs. 100 mL. PBS 1X was prepared by dilution of PBS 10X in sterile water. Isoflurane was supplied by Shenzhen RWD life Science Co.

Animals.

Specific pathogen free (SPF) C57 BL/6J mice, weighting at 20-25 g (8 weeks) were purchased from the Guangdong Medical Experimental Animal Center. 5XFAD mice (strain name: B6.Cg-Tg(APPSwFlLon,PSEN1*M146L*L286V)6799Vas/Mmjax), with genetic background C57BL/6

x SJL, were purchased from the Jackson Laboratory (Stock# 34848-JAX|5XFAD, Bar Harbor, ME, USA) and propagated in the College of Life Sciences and Oceanography in Shenzhen University. Their offsprings were assessed by PCR detection (Mei5 Biotechnology, China) of the AD pathogenic genes. Their non-transgenic littermates were screened out as healthy controls.

Animals were separated in groups containing males and females in a 50/50 ratio. They were housed in standard laboratory conditions: air-conditioned room, temperature 20-25ºC, 12 h light/dark illumination cycle, free access to food and water. Drinking water was distilled water obtained from a Millipore Direct-Q 5 UV water purification system (Millipore SAS, France) and food was purchased from the Guangdong Medical Experimental Animal Center. Before icv and intra-hippocampal injections of Cu-Aβ, mice were anesthetized with isoflurane, using a R500 Series Compact Small Animal Anesthesia Machine (Shenzhen RWD Life Science Co.) according to the supplier instructions. Mice were killed by cervical dislocation after anesthesia with isoflurane. All animal experiments were conducted in accordance with ethical animal research standards of the Chinese Council on Animal Care, and approved by the Animal Ethical and Welfare Committee of Shenzhen University (permit number: AEWC-20140615-002). All efforts were made to minimize animal suffering.

Experimental methods for the icv-CuAβ and hippo-CuAβ models.

Preparation of the Cu-Ab1-42 solution

Preparation of the Ab1-42 solution. Human Aβ1-42 monomer powder obtained after HFIP treatment (0.1 µmole measured by BCA titration) was dissolved in DMSO (50 µL). This mother solution was then diluted by addition of 200 µL of deionized water. The final solution (400 µM measured by BCA assay, H2O/DMSO = 80/20, v/v) was stirred using a vortex (1800 rpm, 1 minute) and sonicated for 1 minute. This limpid solution was aliquoted in seven parts of 30 µL each, stored at -20 ºC away from light, covered with an aluminum foil (Solution A).

Preparation of the CuCl2 solution.

A 40 mM stock solution of copper(II) chloride was prepared by dissolution of CuCl2 (2.70 mg) in DMSO (500 µL). This stock solution was diluted 20 times in DMSO, to provide a 2 mM solution (Solution B).

Preparation of the Cu-Ab1-42 solution. The copper-Aβ1-42 complex was prepared in a 1/1 metal/peptide ratio, as follows. An aliquot of the human Aβ1-42 (above Solution A, 30 µL) was kept at room temperature for 30 min. One mole equivalent of CuCl2 in DMSO (Solution B, 6 µL), was then added in the flask containing the solution A, the mixture was vortexed for 30 sec, and then allowed to stand at 37 °C for 15 min. Then, H2O (18 µL) was added and vortexed, followed by addition of PBS 10X (6 µL), then vortexed for 30 sec. The solution was kept at 37 °C for 15 min. It was again vortexed for 30 sec before each injection in the mouse brains. The absence of precipitate was carefully checked before each injection. The final ratio H2O/DMSO was 80/20, v/v. This solution (Solution C) was freshly prepared each day, before the intracerebroventricular (icv-CuAβ model) or intrahippocampal (hippo-CuAβ model) injections.

Injection of Cu-Aβ1-42 in mouse brain ventricle. (icv-CuAβ model).

Injections were performed using the protocol adapted from Reference 5. The 35 mice were randomly assigned to one of the five following groups (7 mice in each group): control mice, mice which received a single bilateral intracerebroventricular injection of Cu-Aβ1-42 complex (icv-CuAβ group), icv-CuAβ mice treated with TDMQ20 (TDMQ20 group), icv-CuAβ mice treated with TDMQ22 (TDMQ22 group), and icv-CuAβ mice treated with clioquinol (CQ group). The intracerebroventricular injections were made at 2 months (mo) of age, with PBS 1X containing 20 vol% of DMSO for the control group, or Cu-Aβ1-42 Solution C, the four other groups. The coordinates of the injection sites in each lateral ventricle were: (AP) = -0.5 mm from bregma;

(L) = +/-1 mm from bregma; and (D) = -2.5 mm from skull. The skull was drilled, and an injector (27 G, 35 mm) was guided successively into the two lateral ventricles, using a mouse brain localization injector (RWD, Shenzhen, China). Then, 2.5 µL of solution C or PBS 1X containing 20 % DMSO was injected directly into each of the two lateral ventricles (total volume of injection = 5 µL per mouse), using a 5 µL syringe mounted on an automated pump. In each ventricle, the solution was delivered at a rate of 0.5 µL/min. After each injection, the injector was let in place for 5 min to avoid flow back of the solution.

Injection of Cu-Aβ1-42 in mouse hippocampus (hippo-CuAβ model).

The mice were randomly assigned to one of the four following groups (7

Preparation of drug solutions, drug dose, and treatment.

Mother solutions of TDMQ20, TDMQ22 were prepared by dissolving 25 mg of each drug in 10 mL of NaCl 0.9 % (w/v) solution. Clioquinol (25 mg) was dissolved in 10 mL of DMSO. 100 µL of such drug solutions contained 0.25 mg of drug. While the injections of Cu-Ab1-42 into ventricles or hippocampus were carried out on D0, the mice were treated intragastrically 8 times (at D4, D6, D9, D11, D13, D16, D18, and D20) as follows: NaCl 0.9% (w/v) in distilled water (100 µL) for the control groups, the icv-CuAβ group, and the hippo-CuAβ group, TDMQ20 in NaCl 0.9 % (100 µL) for the TDMQ20 groups, TDMQ22 in NaCl 0.9% (100 µL) for the TDMQ22 groups, and CQ in DMSO (100 µL) for the CQ group. Each drug dose was therefore 10 mg kg -1 for TDMQ20, TDMQ22, and CQ. Mice were individually weighted on the day of the intracerebral injection of Cu-Ab (2 months of age) for the icv-CuAb-and hippo-CuAb models, and the exact volume of drug solution for each mouse was calculated accordingly. For 5XFAD mice, animals were weighted at the age of two months (beginning of treatment) and the volumes of drug solutions were calculated accordingly.

Experimental methods for the 5XFAD transgenic model.

At the age of 2 months, mice were weighted and randomly assigned to one of the following groups: untreated 5XFAD mice, 5XFAD mice treated with TDMQ20, 5XFAD mice treated with CQ, compared to wild type C57BL/6 x SJL mice. The drugs were dissolved in 0.9% NaCl (TDMQ20) or DMSO (CQ), and were delivered to the stomach of mice once every two days during the three following months (45 treatments, total drug dose: 450 mg kg -1 ). Each drug dose consisted of 10 mg kg -1 of mouse weight contained in 100 µL of solvent. All control and untreated mice received oral gavage with aqueous NaCl 0.9% instead of drug. The behavioral tests were performed at the end of the treatment, between 5.0 and 5.5 months of age.

Behavioral tests.

Novel object recognition test (NOR).

The test was carried out as previously reported. [START_REF] Morroni | Early effects of Aβ1-42 oligomers injection in mice: involvement of PI3K/Akt/GSK3 and MAPK/ERK1/2 pathways[END_REF] Mice were tested in an uncovered square box (40 cm ´ 40 cm ´ 40 cm). This open arena is divided into 25 equal squares. At D22 after the injection of Cu-Ab in the ventricles or hippocampus (two days after the last administration of drug), each mouse was placed for 5 min in the empty arena of the box placed in the test room equipped with daylight lamp, in order to habituate it to the environment and to the apparatus (habituation phase). At D23, the home cages were left for acclimation in the test room for one hour prior to the beginning of the test session (acclimation phase). Each mouse was then placed for 5 min in the arena containing two identical objects (X and X') (acquisition phase). After a 90 min retention interval in its home cage, the mouse was placed back into the arena and exposed to one of the familiar objects (X) and to a novel object (Y), for a short-term (90 min) recognition memory test. The mouse was initially placed in the apparatus facing the wall and allowed to explore the objects for 5 min. When the mouse's nose pointed or touched an object within 1 cm, it was recorded as an exploration behavior. The number of times the mouse explored one of the two objects was recorded. At D24, each mouse was similarly placed into the arena for 5 min, in the presence of a different pair of dissimilar objects (a familiar and a novel one; X and Z, respectively), for a long-term (1 day) recognition memory test. The recognition index, defined as the ratio of the number of times the novel object was explored to the number of times both objects were explored, was used to measure the memory function. So, the recognition indexes were calculated as nY/n(X+Y) and nZ/n(X+Z) for the short-term memory and long-term memory, respectively, where nX, nY, nZ stand for the number of explorations of objects X, Y or Z, respectively. Between trials, the arena was cleaned with 75% ethanol to eliminate animal clues, and then with water. For the 5XFAD mice, the schedule of the test was the same as described above, and the test begun at 5.3 ± 0.1 months of age.

Elevated plus maze test (EPM).

The elevated plus maze test was carried out using the protocol adapted from References 41 and Between trials, the arena was cleaned with 75% ethanol to eliminate animal clues, and then with water.

Open field test (OFT).

The test was carried out at D26 after intracerebral injection, using the protocol adapted from References 43 and 44. The home cages were left for acclimation in the test room equipped with daylight lamp, for one hour prior to the beginning of the test session. The test field consisted of an empty square box of 100 ´ 100 ´ 30 cm, with 25 square grids on the floor. Each mouse was initially put at the center of the field, and allowed to explore it for 5 min. A digital camera was placed 2 m above and its field of view covered the entire experimental field. The number of rearings, crossings of the floor grid, and walking distances of mice were recorded. For 5XFAD mice, the test was carried out at 5.4 ± 0.1 months of age.

Y-maze test (YMT).

The Y-maze test was carried out using the protocol adapted from Reference 45. The home cages were left for acclimation in the test room equipped with daylight lamp, for one hour prior to the beginning of the test session. Spontaneous alternation behavior test has been performed in a symmetrical black plexiglas Y-maze with three arms (30 cm long × 10 cm wide × 30 cm high) at 120° angles. For 5XFAD mice, the test was carried out at 5.5 ± 0.1 months of age. The mice were initially placed in the center of the maze and allowed to explore the maze for 5 min. A successful alternation was defined as any three consecutive choices of three different arms without reexploration of a previously visited arm. Alternation rate (%) was calculated as follows: number of triads containing entries into all three arms/maximum possible alternations (the total number of arms entered -2)´100.

Biochemical analyses.

Titration of malondialdehyde (MDA).

After all behavioral experiments have been performed, all the mice had a rest for one day and then were sacrificed. The concentration of malondialdehyde (MDA) was determined in cortex of C57 BL/6J control mice, and icv-CuAb mice ± TDMQ20 or CQ, using the thiobarbituric assay according to the manufacturer's instructions (Nanjing Jiancheng Bioengineering Institute, China).

The cortex (ca. 100 mg was homogenized in 1 mL of phosphate buffer (pH 7.4, 10 mM). After centrifugation for 10 min (3,000 g), the MDA content in the supernatant was measured by UVvisible spectrometry at 532 nm. The levels of MDA are indicated as nmol/mg protein.

Titration of AST, ALT, creatinine and urea.

After all behavioral experiments have been performed, all 5XFAD mice had a rest for one day and then were sacrificed (5.6 months of age). The concentration of AST, ALT, creatinine and urea in the serum were measured using the automatic biochemical analyzer iMagic-M7, the assay kits and the software of the iCubio company (Shenzhen, China).

Preliminary toxicologic experiments

Scheme for Table of Contents only

Figure 2 .

 2 Figure 2. Declarative memory evaluated by the recognition index [(nY/nX+Y)´100] for short term memory or [(nZ/nX+Z)´100] for long-term memory in the NOR task in the icv-CuAβ (panel A) and the hippo-CuAβ (panel B)

  SEM [panels A and B: n = 7 (controls) or 6-7 (untreated-or treated AD mice); panel C: n = 9 (controls) or 7-8 (untreated-or treated 5XFAD mice)]. **p < 0.01, *p < 0.05 vs. the untreated AD group (ANOVA).

Figure 3 .

 3 Figure 3. Spatial memory evaluated by the spontaneous alternation ratio in the in the Y-maze task for the hippo-CuAβ (panel A) non-transgenic mouse model, and the transgenic 5XFAD (panel B) model. In each case, the spontaneous alternation rate of AD mice which received no drug-treatment (untreated group) was compared to that of healthy mice (control group), and to the alternation ratio of AD mice which received TDMQ20 or clioquinol (8 × 10 mg kg -1 in 16 days, panel A, or 10 mg kg -1 , one every two days during 3 months, panel B). In panel A, the control/untreated groups were brain-injected with CuAb in PBS. In panel B, the untreated and control groups were the transgenic 5XFAD mice and their non-transgenic littermates, respectively. All control and untreated mice received oral gavage with aqueous NaCl 0.9 w/v instead of drug. For details, see SI. Each mark represents the result obtained with a single mouse; horizontal lines represent mean values ± SEM [(A): n = 5; (B): n = 9 (controls) or 7-8 (untreated-or treated 5XFAD mice]. p values in the ANOVA are indicated.

Figure 4 .

 4 Figure 4. Locomotion ability evaluated by the crossings, travel distances, and rearings, in the open field test for the hippo-CuAβ (panel A) non-transgenic mouse model, and the transgenic 5XFAD (panel B) model. In each case, the scores of AD mice which received no drug-treatment (untreated group) was compared to that of healthy mice (control group), and to the scores of AD mice which received TDMQ20 or clioquinol (8 × 10 mg kg -1 in 16 days, panel A, or 10 mg kg -1 , one every two days during 3 months, panel B). In panel A, the control/untreated groups were braininjected with CuAb in PBS. In panel B, the untreated and control groups were the transgenic 5XFAD mice and their non-transgenic littermates, respectively. All control and untreated mice received oral gavage with aqueous NaCl 0.9 w/v instead of drug. For details, see SI. Each mark represents the result obtained with a single mouse; horizontal lines represent mean values ± SEM. [(A): n = 9 (control), or 6-7 (untreated or treated hippo-CuAβ mice; (B): n = 10 (control), or 7-9 (untreated or treated 5XFAD mice]. ***p < 0.001, **p < 0.01, *p < 0.05 vs. untreated AD mice (ANOVA).

Figure 5 .

 5 5XFAD mice exhibited an increased number of closed arm entries, and a decreased proportion of time spent in closed arms, compared to their WT littermates. Normally, mice tend to avoid the open arms, seeking protection in the closed arms. A decreased proportion of time spent in closed arms indicate a decreased anxiety of 5XFAD

Figure 5 .

 5 Figure 5. Anxiety evaluated by the closed arm entries and time spent in closed arms, in the elevated plus maze test for the transgenic 5XFAD model. The scores of AD mice which received no drug-treatment (untreated group) was compared to that of wild-type mice (control group), and to that of AD mice which received TDMQ20 or clioquinol (10 mg kg -1 , one dose every two days during 3 months). The untreated and control groups were the transgenic 5XFAD mice and their non-transgenic littermates, respectively. All control and untreated mice received oral gavage with aqueous NaCl 0.9 w/v instead of drug. For details, see SI. Each mark represents the result obtained with a single mouse; horizontal lines represent mean values ± SEM. (controls: n = 9; untrated-or treated 5XFAD mice: n = 7-8). ***p < 0.001, **p < 0.01, *p < 0.05 vs. the untreated 5XFAD mice (ANOVA).

Figure 6 .

 6 Figure 6. Effect of TMDQ20 on the level of malondialdehyde in the cortex of icv-CuAβ AD mice (A) and 5XFAD mice (B). (A): The control mice were brain-injected with PBS 1X/DMSO, 80/20, v/v; untreated mice were brain injected with CuAb in 1X/DMSO, 80/20, v/v. (B): The controls were WT C57BL/6J mice. All control and untreated

  : Statistically significant with p-value ≤ 0.05 (ANOVA); b + Means: tendency, but with p-value > 0.05; c Not determined; d Not relevant (no significant difference between WT controls and 5-month old 5XFAD mice; e Only for grid crossing. Behavioral test mice received oral gavage with aqueous NaCl 0.9 w/v instead of drug. Each mark represents the result obtained with a single mouse; horizontal lines represent mean values ± SEM of n = 5 experiments. Relevant p values are indicated (ANOVA).

  mice in each group): control mice, mice which received a single bilateral intrahippocampal injection of Cu-Aβ1-42 complex (hippo-CuAβ), hippo-CuAβ mice treated with TDMQ20, hippo-CuAβ mice treated with clioquinol (CQ group). The injections in the two CA3 hippocampus regions were made at 2 months of age, with PBS 1X containing 20 vol% of DMSO for the control group, or Cu-Aβ1-42 Solution C, for the three other groups. The coordinates of the injection sites were: (AP) = -2.18 mm from bregma; (L) = +/-2.3 mm from bregma; and (D) = -2.15 mm from skull. The skull was drilled, and an injector (27 G, 35 mm) was guided successively into the bilateral CA3 hippocampal regions. The delivery of the Cu-Aβ1-42 (solution C, 2 x 2.5 µL of the Cu-Aβ1-42 solution C, or the PBS 1X/DMSO, 80/20, v/v into the bilateral hippocampal CA3 region was carried out in the same way than icv injections, except that the flow rate was 0.25 µL/min (compared to 0.5 µL/min for cerebroventricular space, in order to avoid damage to the smaller hippocampus).

42 .

 42 The experiment was started at D26 from intracerebral injection. The home cages were left for acclimation in the test room equipped with daylight lamp, for one hour prior to the beginning of the test session. The elevated plus maze apparatus consisted of four arms of 30 cm length ´ 5 cm width, two of them with 30 cm high walls, elevated at 60 cm above the floor. The mouse was initially placed in the center of the maze, facing an open arm and allowed to explore the maze for 5 min. The number of times the mouse entered the open and closed arms was recorded, as well as the time spent in the open and closed arms. If the mouse fell off an open arm, the trial was stopped and excluded from the analysis. For 5XFAD mice, the test was carried out at 5.4 ± 0.1 mo. of age.

Table 2 .

 2 Preliminary

toxicological evaluation of TMDQ20 compared to CQ on C57BL/6J mice, after a single oral or intraperitoneal (ip) dose.

Funding Sources

This work was financially supported by the Guangdong Provincial Key S&T Program (2018B030336001), NSFC (grants 21977019 and 21877081), the Guangdong Province (Program for Innovative Research Teams and Leading Talents Introduction, grant 2050205), the Open Project of Guangdong Provincial Key Laboratory of New Drug Screening (No.GDKLNDS-2018OF004), and the University of Technology of the Guangdong (GDUT) (grant 220418037) of the P. R. China, and by the CNRS, France.

Single dose (acute toxicity).

TDMQ20 and CQ were administered by gavage or by intraperitoneal injection, at 150 or 200 mg kg -1 , to C57 BL/6J mice (groups of 5-11 mice each), using 25 mg mL -1 solutions in 0.9% NaCl (for TDMQ20) or in DMSO (for CQ). Mice were observed for 16 days after the administration.

Multiple doses.

C57 BL/6J mice received TDMQ20 (25 mg mL -1 solution in 0.9% NaCl) by intragastric administration at 100 mg kg -1 per day during 9 days (10 mice), or at 60 mg kg -1 per day, three times per week during two weeks (12 mice). Mice were weighted each day.

Statistical analysis.

Figures 23456and Figure S1 are presented as univariate scatterplots of all the individual data points obtained, with mean and SEM superimposed. Data with multiple independent experiments were analyzed by one-way ANOVA, followed by multiple comparisons LSD test when the ANOVA was significant (p < 0.05). p-levels < 0.05, 0.01 and < 0.001 were noted *, ** and ***, respectively. For each test, each mice was evaluated only once (no repeated measures). The factor of the treatment was entered as a within-subjects (independent) factor. The F-statistic, degrees of freedom, p-value and the magnitude of the difference were calculated. All statistical calculations are provided as Supporting Information (SI).

ASSOCIATED CONTENT

The Supporting Information is available free of charge at https://pubs.acs.org/doi/...

The Supporting Information contain:

-Figure S1 (duplicate of Figure 2), -Raw data and statistical analysis of results reported in Figures 23456, -ADME-toxicological data of TDMQ20.

AUTHOR INFORMATION

Corresponding Authors

Anne Robert -Laboratoire de Chimie de Coordination du CNRS (LCC-CNRS), 205 route de Narbonne, 31077 Toulouse, cedex 4, France; orcid.org/0000-0002-9092-6776; anne.robert@lcc-


Caroline Jobin

Pascal Le Masson

Sophie Hooge

What does proof-of

come

Introduction

As pointed out by the Richelieu's Committee, a network of French innovative companies, for which "proof of concept has been the subject of special attention for several years", there has been an increasing use of this term in practice over the last years (Comité Richelieu and Médiateur des entreprises, 2019). From start-ups and large groups, to research centres, through public institutions, all these typologies of organisation have embraced this terminology inherited from the Technology Readiness Levels developed at NASA in 1970-1980s [START_REF] Mankins | Technology Readiness Levels: A White Paper, Advanced Concepts Office[END_REF][START_REF] Sadin | The NASA technology push towards future space mission systems[END_REF]. Such an enthusiasm has to be considered in the context of contemporary innovation challenges that suppose the ability to explore unknowns ever more complex and vaster [START_REF] Le Masson | Strategic management of innovation and design[END_REF]. Establishing and sustaining original and specific exploratory communities, including actors with heterogeneous expertise who belong to various institutions and often without previous collaboration experience, is an increasingly studied way to face these challenges [START_REF] Chesbrough | Open business models: How to thrive in the new innovation landscape[END_REF][START_REF] Star | Institutional ecology, translations' and boundary objects: Amateurs and professionals in Berkeley's Museum of Vertebrate Zoology, 1907-39[END_REF]. Although practitioners recognise in proof of concept (PoC) some virtues such as being a "flexible object in its relations between economic actors" and "a step towards innovation and value creation, a learning step that is often decisive", they also regret that "insufficiently or poorly apprehended, the POC can result in disappointments for both [parts]" (Comité Richelieu and Médiateur des entreprises, 2019). If we pay attention to what the scientific literature tells us about PoC, we realise that the latter is very poor and quite entirely related to the Technology Readiness Levels [START_REF] Héder | From NASA to EU: The evolution of the TRL scale in Public Sector Innovation[END_REF][START_REF] Mankins | Technology readiness assessments: A retrospective[END_REF] which today only represents a small facet of its practical uses. Furthermore, this notion anecdotally appears or is even absent in handbooks where we would expect to find it, for instance, in research on new product development (Golder and Mitra, 2018) or on design thinking (Plattner et al., 2018). Therefore, in view of the practical issues highlighted and the sharp existing gap between literature and practice of PoC, we would like to investigate in this paper a basic question that is: what does proof-of-concept really prove or do? The study aims at clarifying the polysemous practices around PoC and helping stakeholders to know why and how PoC is a design tool to establish and sustain collaborative exploratory communities through PoC. To achieve these contributions, we will proceed in two steps: first, an historical investigation of the different branches that constitute the genealogical tree of PoCs, then a case study of a rich practice of PoC in a design agency called 'the Sismo'. We will compare PoC practices in the light of an analytical framework based on two performance dimensions that are generativity and robustness [START_REF] Hatchuel | A systematic approach of design theories using generativeness and robustness[END_REF]. In other words, we will investigate (1) what kind of effects are produced and ( 2) to what extent are these effects of quality? In particular, we will use the scientific method and more precisely the statistical method that follows a codified and widespread learning model [START_REF] Hacking | Representing and intervening: Introductory topics in the philosophy of natural science[END_REF][START_REF] Radder | The philosophy of scientific experimentation: a review[END_REF] to more precisely define this analytical framework and describe how the Sismo's PoCs diverge from it, follow it or do a little both. The plan of this paper is as follows. First, we will conduct a quick literature review to position the research and present the analytical framework before introducing our methodology and developing the analytical framework. Then, we will conduct the systematic comparison of PoC practices through two steps: an historical study and a case study of the Sismo's PoCs. Finally, we will enlighten the main findings and return to the research question to discuss the contributions.

Literature review

'Proof-of-concept' genesis

As far as we have been able to trace back, the roots of 'proof-of-concept' came from the Technology Readiness Level (TRL) scale originally conceived at National Aeronautics and Space Administration (NASA) in 1970-80s [START_REF] Mankins | Technology readiness assessments: A retrospective[END_REF]. Following President Reagan's encouragement for more cooperation between NASA, and civil and commercial as well as international partners [START_REF] Reagan | Presidential Directive on National Space Policy 11[END_REF], Sadin and colleagues (1989) proposed a new technology development strategy. They introduced a paradigm shift in strategy (from pull to push strategy); instead of entirely driving technology development in relation to the needs of large space programs such as Apollo, the development of some technologies should be permitted regardless space program. Indeed, a framework was needed to assess "how proven a certain technology was" in a context where "they cannot necessarily prove their feasibility in an actual space mission at the time of development" [START_REF] Héder | From NASA to EU: The evolution of the TRL scale in Public Sector Innovation[END_REF]. "Proof-of-Concept demonstrated analytically and/or experimentally" described the third level of one of the first established scale [START_REF] Sadin | The NASA technology push towards future space mission systems[END_REF].

PoC as an experimental method

Whatever its context and field of application, PoCs in contemporary practice share a common property: its experimental dimension to provide evidence in order to support an idea. Bacon and Galileo are strong advocates of experimental method such as [START_REF] Bernard | Introduction à l'étude de la médecine expérimentale[END_REF] in medicine. Such a practice is today often associated to the scientific method [START_REF] Hacking | Representing and intervening: Introductory topics in the philosophy of natural science[END_REF] which experienced growth since the mid-1970s and is related to a "strong reliance in statistical methods" [START_REF] Radder | The philosophy of scientific experimentation: a review[END_REF].

Generativity and robustness of PoC

Generativeness (or generativity) and robustness are two performance dimensions that Hatchuel and colleagues (2011) used to compare a sampled of design theories. According to them, both notions "generalize two well known series of design criteria that are traditionally discussed in the literature" (Hatchuel et al., 2017). The word 'generativity' comes from the Latin generativus, which means "that causes, that creates, that produces" whereas the world 'robustness' comes from the Latin robustus, which means "solid, hard, strong, resistant".

Given the current diversity of uses of PoC, we would like to investigate in this paper the following research question: what does proof-of-concept really do?

Methodology

Our research approach consisted of two steps: (a) an historical investigation of the different branches that constitute the genealogical tree of PoCs (Technology Readiness Levels, medicine, software security, start-up and design) and (b) a case study of a particular rich practice of PoC in a design agency named 'the Sismo'. For the first step, data collection was ensured in scientific literature and grey literature and enriched by meetings with practitioners conducted in the context of the intervention research [START_REF] Hatchuel | Collaboration for management research[END_REF]Radaelli et al., 2012) that the main author carried out at the Sismo since October, 2018 as part of her PhD. For the second step, data was acquired through two complementary channels. On the one hand, an 1h30-interview was conducted with the Sismo's employee who had the most experience in the agency. She was also the project manager for two projects (A. and B.) and became the referent in the agency regarding PoC. Project A. aimed at designing a new governmental employment agency model to foster social dialogue and accelerate the return to work and Project B. aimed at, in client stores, rethinking the discovery of product and service lines and developing new services to their customers. During the interview, she was asked questions related to generativity and robustness of the Sismo's PoCs. On the other hand, this interview was mediated and enriched through the intervention research conducted at the Sismo. In both steps, a systematic comparison will be performed between PoC practices in the light of an analytical framework based on two performance dimensions that are generativity and robustness. (1) We define generativity as the ability to produce effects that go beyond the initial knowledge of the designer community. (2) We define robustness as the ability to produce "expected performances despite being subjected to uncertainties or hard-to-control disturbances" [START_REF] Hatchuel | A systematic approach of design theories using generativeness and robustness[END_REF]. In particular, we will use the scientific method and more precisely the statistical method that follows a codified and widespread learning model [START_REF] Hacking | Representing and intervening: Introductory topics in the philosophy of natural science[END_REF][START_REF] Radder | The philosophy of scientific experimentation: a review[END_REF]. Such a model will allow us to more precisely define this analytical framework that will be used to describe how the Sismo's PoCs diverge from it, follow it or do a little both. (1) In terms of generativity, statistical method allows to reject (or not reject) a null hypothesis if the observed significance level (pvalue) is less than or equal (more than) to the chosen value of alpha (type I error) [START_REF] Karlsson | Research methods for operations management[END_REF]. ( 2) In terms of robustness, we choose to use the main requirements of scientific method outlined by [START_REF] Sekaran | Research methods for business[END_REF]: (1) Purposiveness: The researcher/practitioner has been guided by and communicates a purpose for the research, (2) Rigour: A strong theoretical base and a sound methodology are needed to collect the appropriate information and to interpret it adequately; that is, to do research in a trusted manner, (3) Testability: The researcher/practitioner car reliably infer whether or not the data support conjectures and he or she controlled for what can influence the results, (4) Replicability: It is possible to repeat the study exactly. If controlled repetition of replicable studies is performed, conjectures will be neither supported (or discarded) merely by chance, nor due to differences in method, (5) Precision and confidence: These refer on the one hand to how close the findings are to 'reality' and, on the other, to the probability that estimations are correct, (6) Objectivity: The conclusions are based on all the relevant facts and are not influenced by the researcher/practitioner's subjective values, (7) Generalizability: Refers to the applicability of the research findings to settings different from the studied one, and ( 8) Parsimony: The use of small number of variables and relationships among variables to describe and explain a phenomenon generally makes research frameworks more manageable and useful. This framework was presented to the Sismo's project manager and was used to drive the interview.

Historical study of PoC

PoC in Technology Readiness Levels (TRLs)

As far as we have been able to trace back, the roots of 'proof-of-concept' came from the Technology Readiness Levels (TRLs) originally conceived at NASA [START_REF] Mankins | Technology readiness assessments: A retrospective[END_REF]. NASA was established in 1958 as an independent agency of the United States Federal Government responsible for the civilian space program to catch up with the advance taken by the Soviet Union. Following the observation that "new system developments suffer from cost overruns, schedule delays and the steady erosion of initial performance objectives", the idea of a 'technology readiness review' emerged [START_REF] Mankins | Technology readiness assessments: A retrospective[END_REF]. In the 1970s, the NASA's Office of Aeronautics and Space Technology department introduced the concept of 'technology readiness levels' (TRLs) "as a systematic tool that enables assessments of the maturity of a particular technology and the consistent comparison of maturity between different types of technology" [START_REF] Mankins | Technology readiness assessments: A retrospective[END_REF]. According to the inventors of the first TRL scale, S. Sadin and colleagues, such an instrument that we qualified as a 'boundary object' [START_REF] Star | Institutional ecology, translations' and boundary objects: Amateurs and professionals in Berkeley's Museum of Vertebrate Zoology, 1907-39[END_REF] was needed because "the differing perceptions of the researchers and the mission planners between the intended and actual proof of readiness was often the cause of an aborted handoff, or technology transfer, of ART [Advanced Supporting Research Technology] to the SRT [Supporting Research Technology] users" [START_REF] Sadin | The NASA technology push towards future space mission systems[END_REF]. Due to several events (terrible accident of Challenger Space Shuttle, Space Exploration Initiative, ...) that NASA encountered in the mid-to-later 1980s, the use of the TRL scale became more widely accepted and used [START_REF] Mankins | Technology readiness assessments: A retrospective[END_REF]. When the TRL scale spread in the NASA ecosystem, several versions coexisted that was detrimental to its use. In this context, John C. [START_REF] Mankins | Technology Readiness Levels: A White Paper, Advanced Concepts Office[END_REF] then wrote a paper in which he extended the scale to nine stages and proposed expanded descriptions along with examples for each TRL that is now the standard scale [START_REF] Mankins | Technology readiness assessments: A retrospective[END_REF]. Indeed, this scale has been progressively adopted by other U.S. agencies (Department of Defense, Department of Energy, …). In this 9-level TRL scale in which low levels are associated with low technology maturity and vice versa, 'proof-of-concept' occurred within TRL 3 characterisation ("Analytical and experimental critical function and/or characteristic proof-of-concept") and description [START_REF] Mankins | Technology Readiness Levels: A White Paper, Advanced Concepts Office[END_REF]. Even though 'proofof-concept' is related to early maturity and thus early development process phases, the claimed goal is the validation of analytical predictions about applications/concepts formulated at TRL 2 trough studies and experiments. This scale also inspired international space agencies and their contractors such as the European Space Agency (ESA) that adopted the TRL scale in the mid-2000s. ESA closely followed the NASA definition of TRLs [START_REF] Mankins | Technology Readiness Levels: A White Paper, Advanced Concepts Office[END_REF] canonised through the ISO standard 16290 Space systems established in October, 2014. This scale has globally gained popularity in numerous organisations [START_REF] Mankins | Technology readiness assessments: A retrospective[END_REF] in high-tech industry (aeronautical, defence, automobile, …) and become an innovation policy tool in international institutions such as the European Commission (Horizon H2020 program).

Generativity and robustness:

The creation of the TRLs and its associated scale echo what [START_REF] Star | Institutional ecology, translations' and boundary objects: Amateurs and professionals in Berkeley's Museum of Vertebrate Zoology, 1907-39[END_REF] call "generating a series of boundary objects which would maximize the autonomy and communication between worlds". The authors support their statement by sharing that "the creation and management of boundary objects is a key process in developing and maintaining coherence across intersecting social worlds". Indeed, presenting the TRL scale as a tool that fits "both for management it's own instrument technology programs, and also for communicating more effectively with technology researchers and organizations inside and outside" [START_REF] Mankins | Technology readiness assessments: A retrospective[END_REF] can, in a big part, explains the enthusiasm generated around it and its massive adoption. If at the beginning, TRLs did meet the definition of boundary objects defined by [START_REF] Star | Institutional ecology, translations' and boundary objects: Amateurs and professionals in Berkeley's Museum of Vertebrate Zoology, 1907-39[END_REF], namely "objects [that may be abstract or concrete] which are plastic enough to adapt to local needs and the constraints of the several parties employing them, yet robust enough to maintain a common identity across sites", its massive adoption by different institutions has inevitably reduced its robustness. Indeed, in order to ensure its suitability, its adoption was accompanied by an adaptation of the language to better fit organisation or technologies, production patterns, or management structures [START_REF] Marvel | Technology readiness levels for randomized bin picking[END_REF]. Moreover, as Héder's paper (2017) pointed out, "the concreteness and sophistication of the TRL scale gradually diminished as its usage spread outside its original context (space programs)", in particular because it does not take into account the existence of implicit related assumptions such as the fact that "the Technology Readiness is an analogue to Flight Readiness". Hence, the interpretation of technology maturity levels has been multifaceted and raised a non-alignment issue that was particularly studied in the technology transfer between universities and industry (McAdam et al., 2009;[START_REF] Jensen | Proofs and prototypes for sale: The licensing of university inventions[END_REF]. Finally, more than differences in appropriation of scale, different uses of this tool could be described. One example is the Government Acounting Office (GAO) case study reporting the first TRL experiments at the Department of Defense. The report showed that projects that had used immature technologies inherited various difficulties, conversely, programs that integrated TRL6-technologies no longer experienced any additional cost. The main message of this report was therefore that by formalising the maturity of technologies on the TRL scale and by refusing to support programs technologies that are too immature (below level 7 in any circumstances, with the preferred levels 8 and 9), DoD will improve the performance of these acquisition programs (Government Acounting Office, 1999; Jean, 2018).

PoC in medicine

Following the tradition of medicine as an evidence-based practice and responding to the excesses that have marked our history, the pharmaceutical and biomedical industries are today characterised by one of the most regulated environments related to safety and ethics. In that context, PoC became an 'obligatory passage point' [START_REF] Callon | Some elements of a sociology of translation: domestication of the scallops and the fishermen of St Brieuc Bay[END_REF] in drug and medical device development process following the philosophy of Cooper's stage-gate (1994). Indeed, PoC is a widely used term to describe the Phase IIa of clinical trials and even described "perhaps the key deliverable of exploratory drug development" [START_REF] Cartwright | Proof of concept: a PhRMA position paper with recommendations for best practice[END_REF]. During this stage, "the study drug is tested for the first time for its efficacy in patients with the disease or the condition targeted by the medication. These studies may have up to several hundred patients and may last from several months to a few years" [START_REF] Corr | The pathway from idea to regulatory approval: examples for drug development[END_REF]. [START_REF] Cartwright | Proof of concept: a PhRMA position paper with recommendations for best practice[END_REF] suggested an interesting alternative definition of PoC borrowing an expression from the code of federal regulations: "POC is the earliest point in the drug development process at which the weight of evidence suggests that it is 'reasonably likely' that the key attributes for success are present and the key causes of failure are absent". In addition of being a milestone in an internal development process to "allow drug developers to make 'Go/No Go' decisions about proceeding with larger, more expensive studies" [START_REF] Preskorn | The role of proof of concept (POC) studies in drug development using the EVP-6124 POC study as an example[END_REF], such studies are a "regulatory step" that must sufficiently convince the competent authorities to allow the release of their agreement to pursue the development process". Generativity and robustness: The goal of the PoC is not only to "demonstrates that the drug did what it was intended to do, that is, interacted correctly with its molecular target and, in turn, altered the disease", but also "help[s] determine the correct dosage, common shortterm side effects and the best regimen to be used in larger clinical trials" [START_REF] Corr | The pathway from idea to regulatory approval: examples for drug development[END_REF]. Contrary to later stages of drug development, PoC "typically involv[es] a small number of subjects and more latitude in statistical requirements" [START_REF] Preskorn | The role of proof of concept (POC) studies in drug development using the EVP-6124 POC study as an example[END_REF]. If we look at the scientific publications in this field and the structures which incubate biomedical technological innovations, we realise that PoC can play a completely different role than the one described above and overlap with the genealogy of the TRL. Thus, most scientific publications using the term PoC, across all disciplines, come from life science and healthcare researchers working in laboratories whose main objective seems to be the prospect of technology transfer. Moreover, for researcher, stating that he or she has done his or her PoC or achieved TRL3 allows him or her to get access to acceleration programs regarding support and/or budget. This dual system of PoC in this field is problematic and has been reported to us by a Technology Transfer Project Manager at SATT Lutech, a French structure that accompanies innovative projects from academic laboratories to the market. He told us that there were often misunderstandings between researchers and large groups looking to buy back licences. Thus, the researcher may consider having established his PoC by validating the mechanism of action on a few human cells while the industrialist thought that the drug had already been tested on several dozen sick patients.

PoC in (software) security

A genealogy of PoC also comes from the hacker movement, especially in the world of software. The purpose of PoC is to show that there is a security breach that allows a potential intrusion. This person can be benevolent and reports such defect to its manufacturer or even proposes corrective solutions in order to prevent malicious person to enter the same security breach. Such security breach can constitute an entry point for a virus capable of altering the DNA of the software or I.T. system and thus reprogramming it to divert its initial use or crashing the system. Indeed, due to our increasing reliance on I.T. systems and the new black gold that data represents, such attacks or at least attempts may unfortunately be more and more casual. Generativity and robustness: As clearly express in English Wikipedia regarding PoC in security, the PoC is a demonstration that "a system may be protected or compromised, without the necessity of building a complete working vehicle for that purpose". One can take the example of Winzapper "which possessed the bare minimum of capabilities needed to selectively remove an item from the Windows Security Log, but it was not optimized in any way".

PoC in start-up

One of the main reasons why start-ups perform PoC or declare having done is for its strong power of attraction. With such label, entrepreneurs hope to attract and convince stakeholders that the concept is worth investing in. Stakeholders or PoC targets may be potential internal or external partners, potential clients, investors, buyers, etc. Another reason for B2B and B2BC start-ups that explain why they carry out PoC is related to the widespread practice of open innovation in large groups (Chesbrough, 2003) that are increasingly looking at start-ups. As Bpifrance, a French public investment bank that supports French companies by offering financing solutions to support their projects and development points out, many start-ups and large groups enter into collaboration through PoC. According to Bpifrance, on the side of large groups, PoC "allows to assess the capacity of the solution to meet the expectations of the large group and to see whether collaboration between the two stakeholders can be considered on a larger scale". On the side of start-ups, PoC is an opportunity to "generate revenue by acquiring customers [and] to engage quickly with large groups, with few technical constraints, and demonstrate the value of a startup's services". In addition, PoC is a term that is increasingly found in support structures such as incubators and accelerators that may or may not be corporate, either as an exit or an entry point. Finally, PoC is no longer restricted to start-ups but is open to the notion of innovative company which could be all kind of organisation. Generativity and robustness: On the one hand, as we stress above, PoC has a strong power of attraction, and that is the reason why support structures such as incubators help entrepreneurs to achieve it. Experiment approach as promoted in Lean Startup [START_REF] Ries | The lean startup: How today's entrepreneurs use continuous innovation to create radically successful businesses[END_REF] then often serves as a reference. On the other hand, PoC in its collaborative shape, tends to generate "disappointments for both the person who delivers it and the person who commands it" (Comité Richelieu and Médiateur des entreprises, 2019) and thus does not lay the foundation for a sustainable relationship beyond PoC. According to Bpifrance, some companies are "promoting a reputation of 'serial Pocer'". We can interpret this trend as a (new) purpose of such collaboration that is not intended to provide validation prior deployment (as the start-up expects) but a new exploration strategy.

PoC in design

PoC in the design world is often associated with the prototype and prototyping stage. Indeed, it is not uncommon to see the term PoC in the form of 'proof-of-concept prototype' [START_REF] Yu | Prototyping in theory and in practice: A study of the similarities and differences between engineers and designers[END_REF]. It can be explained by the fact that the model, the prototype acts as a proof of feasibility through the notion of materiality (Blomkvist, 2014;Kirsh, 2010). In the same vein, Gay and Szostak ( 2016) declare: "one of the strengths of design thinking lies, in addition, in its ability to visually translate the senses created, especially through the proof of concept". As a result, the notion of PoC is increasingly reflected in the world of design thinking and design sprint. Even if this term is very weakly present or even absent from the original texts (see for instance Brown 2008; 2009) and seems to appear from nowhere, it has a real stake and interest in the contracting between design agencies and their clients. For instance, for 'the Sismo', an independent design agency, PoC has become a service that can be sold to clients through a quote. Therefore, a remuneration is ensured while the final object, in the broad sense of the term, has not yet seen the light of day. Generativity and robustness: In terms of robustness, it was usually the client or the sponsor who had to be convinced by the designers. As in a criminal trial in France (where a part of the jury consists in citizens), the clients are themselves the sole judges of "the fullness and sufficiency of the evidence" (Article 353 of the French Code of Criminal Procedure). What we see more and more, especially at the Sismo, the central point is to convince the manager or the management of the client. Indeed, the methods of proof generation and the levels of evidence expected by the client are increasingly demanding. Before going into more detail about the generativity power of the Sismo's PoCs, it is enriching to look at the case of 'Lille 2020, World Design Capital'. For this edition, it is not only about a global event, but the purpose is also to infuse, integrate and transform the metropolis territory through design. The goal of the organising committee is clear: "to make the territory a laboratory for large-scale experimentation [to] imagine and experiment new ways of designing, developing and deciding" through PoCs. While most of the initiatives are still in their infancy, the initial exchanges among committee members are promising. For instance, they stressed that (1) the general public and media character of the event has attracted project promoters of all kinds, (2) PoC has allowed people from diverse institutions to sit together whereas in another context this could have never happen, and (3) for organisations that have already embarked on experimentations, there has been a certain awareness that the transformation to which they aspire has an echo in their own organisation.

Case study of PoC

Introduction of the Sismo

Founded 22 years ago, the Sismo is an independent design studio, renowned for its strategic approaches to support innovation, for French and international large groups, to smaller companies, through public and cultural institutions. Often awarded for its iconic creations, methodological tools and its public exhibitions, the studio has gradually made the service and experience design the point of convergence of its various expertise. The studio is increasingly recognised for its expertise in PoC as evidenced by its involvement in 'Lille 2020, World Design Capital'. PoC describes one step of their project approach which consists of upstream steps (ethnographic study, co-creative workshop and scriptwriting) and downstream steps (valorisation and deployment).

Generativity power of the Sismo's PoCs

For designer teams: Due to the nature of the subjects of work related to service and experience design, they must deal with "intangible, immaterial things that it is sometimes difficult [for the Sismo's designers] to represent" and to anticipate. Then, the PoC helps to reduce some uncertainties. The interviewed project manager took the example of project A., "we had worked with advisors on a way to separate the flow of arrival [of job seekers] in three: administrative reception, express reception and second level reception, with the hope of decreasing and removing the long tail, but we were not sure at all [that the expected effect was going to happen]". One another objective of the PoC for the Sismo is to gather evidence necessary to convince stakeholders and then ensure massive deployment and adoption. Thus, as the project manager pointed out, it is not always easy to find the right arguments for both the management teams and the field teams. These gathered elements will feed a "deployment booklet where we put [...] photos and verbatim to prove what we were trying". The PoC is also an opportunity to continue the design, as the project manager said, "it's a playground, it's a board game, you put the cards, the pawns, […] you have the conditions to continue to design, that's why I say it's a design phase [and not just a test]". For field teams: As mentioned above, the Sismo often works on things are immaterial which require "a strong projection capacity of people". Then, the PoC has a role of conviction through an experiential scenario. Thus, as the project manager emphasised, "it is by making them feel that we convinced them". To support this, she talked about the project P.: "There are some advisers for whom we only managed to convince and board them through the PoC, if I take an example, when we said to some of them, you are going to work on a narrower table, they saw only security problems, while in situation they told us "ah but we are closer [with the job seeker] so we speak less loud and so there is less aggression!"". Moreover, the PoC is an opportunity for the field teams to have the means and framework to do and dare to do otherwise. As emphasised by the project manager, "it is rare that they [the field team] have the means to do that!", and thus, "it's important to change their outfit to become [this new character]". More than desired, this appropriation is, almost in all cases, necessary because the Sismo are not often experts in the field of the client organisation or subject of work. Indeed, the challenge is to put the right bases or good cards, pawns on the board game to use the expression of the project manager and "after that it is up to [the field teams] to play!". For example, if we take again the example of project A. with the partition of the reception flow in three, "it is to the advisers to define which questions relate to which reception and to determine if express reception is related to an answer that takes 5, 10 or 15 minutes? etc." For management teams: As the project manager pointed out, "in all cases, it [the PoC] makes communicate the headquarters and the field where you have two realities". Thus, the PoC allows a certain awareness of management teams by reconnection with the field. However, certain conditions are necessary to generate this effect. Management teams must come to the PoC in the field and adopt a position of observer with empathy or even take the posture of the field teams, and this on a time long enough. For example, in project B., the project manager recounted the client's comments: "She said that it had been such a transformation for her, it was good, ..., it had been a long time since she had not had the opportunity to spend 6 weeks at the same store, [...] which allowed her to realise that sometimes agents were asked very complicated things". The PoC also allows to "give an account that some things need to be improved" and thus to open new (transformation) programs and projects and position these as priorities. Furthermore, just like the field teams, the PoC is an opportunity to dare and test new things. As the project manager commented in the context of project B., "what amused me was that the client [...] was offering iterations herself, she was proposing things that were not always very interesting […] but we let them do so that they see by themselves that it does not work [...]. I felt that she was in this dynamic of saying there is a problem and I am looking for solutions but that it has its limits … Fortunately we were there for the mediation and hopefully, they still let us do the restitution because they had made certain shortcuts, taken certain things at face value, they did not have the overall view as we can have".

Robustness power of the Sismo's PoCs

To begin with, the first statement of the project manager in reaction to the analytical framework of [START_REF] Sekaran | Research methods for business[END_REF] was: "Overall, these criteria well describe robustness". For most of these criteria, we will not only provide quotes from the project manager interview, but also comments to mediate and complete citations to ensure a more comprehensive view of the Sismo's practice and a finer systematic comparison with the analytical framework. Purposiveness: "For the Sismo, it works obviously, […] behind this there is the hypothesis we want to test". Rigour: "There, honestly, we don't take the time to do it well, we rarely have a very extensive theoretical basis, besides, I don't really know where and how to look, I'm bad at doing these things. [...] I have no personal knowledge of ethnology literature." This statement should be moderated; they always start their work with an enrichment phase, but which is more empirical than theoretical. They preferably perform immersions, interviews and basic research on the internet than investigate scientific literature. "At the method level, we don't necessarily have the means to compare in two groups [...] but we try to vary the means to collect the data (interviews, observations, etc.)." Indeed, there are few situations where they will use a control sample to perform an A/B testing analysis. They prefer to mix the way to produce knowledge through different modes of observation on the observed sample. Their sampling does not seek to be random, and this property depends on the level of analysis chosen, i.e., the agency/store was chosen according to certain criteria and was therefore not chosen randomly, on the other hand the people who had frequented these places were random. In addition, people who was selected for further observations or interviews was often chosen because designers had observed unusual behaviours or things that surprised them. Testability: "It's complicated, on project B., we don't control the number of people a day, … it would be distorted the situation to control it, … and then we did [experiment] it over six weeks". In fact, experiments are not carried out in the laboratory or in the chamber where high confinement can be performed but, in the field, and in real life (or almost) where control is more difficult. Moreover, it is often not clear, a priori, what parameters need to be controlled. Replicability: "Yes, concretely, we could do PoC in several situations [...] but there are budgetary issues, […] we had been paid to conduct 3 PoCs [PoC in 3 agencies], we didn't have the means and the time to test everything [all the concepts in each agency], but they [the clients] did [tests] in other agencies." Admittedly, PoC can be replicated, however the observed effects are never only related to the new object alone but to the object/environment pair, so it is important to clarify this interaction so as not to produce abusive inferences. In addition, there is a feedback effect between the observer (the designer) and the observed so the conditions of knowledge production are plural. Precision and confidence: "It is clear that we are always trying to be closed to reality, even if we can found few counter-examples […] we try, during PoC, on the advice of [a middle manager of the Sismo], to be careful not to generalise an observation on the basis of one person." There is a desire in the Sismo's projects to get closer to what they call 'reality', e.g., with tests in the operational environment, for instance, an agency that already hosts jobseekers, an existing store in a shopping centre. However, sometimes the test conditions can be more favourable than the 'reality', for example, by selecting a voluntary agency, supporting the field teams with advice or actions, ...). Objectivity: "Of course, with PoC I have concrete evidence, a verbatim, a behaviour, a film, …, but I think that where there is still a subjective side [...] [it is related to the fact that PoC] is still part of the design phase, we can still remain in the intuition". In view of her statement, the project manager listed the methods of proof that she considered admitted, without asking whether these proofs or their nature were enough for the receiving party, whereas the importance of the way the data was collected, and its related context was mentioned. Generalizability: "For Project A., this is not the same issue that the system works everywhere [with reference to project B.], it is a public service, [...] they have the challenge that the reception works in all types of agencies with different audiences [...]. In Project B., they can be more authoritarian, it's their brand, [...] I think they won't test things anywhere else because the sample is representative everywhere [in all their shops]." To complete, each context of observation is quite different but the question to answer behind is whether these differences have a real impact. The notion of representativeness is not something that is directly sought, even though it is often one of the criteria for selecting the field. Knowing that the size of their sample is limited, there is little discussion about statistical inference. Parsimony: "When [a colleague] came to me for advice for a PoC […], she said she wanted to test a lot of things at the same time, I told her to be careful about that."

Main findings and discussion

To investigate the basic research question about what proofs-of-concept really do, we systematically compare PoC practices through an historical study and a case study regarding an analytical framework based on two performance dimensions that are generativity and robustness [START_REF] Hatchuel | A systematic approach of design theories using generativeness and robustness[END_REF]. Trough the historical review of PoC related to TRLs, medicine, security, start-up and design, we have seen that PoCs are used in different contexts with different objectives and go beyond boundary objects based on a demonstration of feasibility for the purpose of contractualisation. While PoC practices seem to share a common property -federate and structure a collaborative exploration community -, PoCs produce various effects depending on why and how the PoC was designed and carried out. Through the case of the Sismo, we have seen that their PoCs have plural generative dimensions and they are of different natures according to the typology of stakeholders: (1) for designer teams: reduce some uncertainties, gather evidence necessary to convince stakeholders and then ensure massive deployment and adoption, continue the design, (2) for field teams: conviction through an experiential scenario, an opportunity to have the means and framework to do and dare to do otherwise, (3) for management teams: awareness by reconnection with the field, namely the need to open new (transformation) programs and projects, and an opportunity to dare and test new things. These three typologies allow to go beyond the classical client-supplier partition of PoC by opening the way to a more collaborative model. Thus, designer teams often do not reduce themselves to Sismo's designers and can also be composed of people from the client organisation and civil society. In terms of robustness or guarantee of evidence, a further comparative study between proof of concept and scientific proof based on hypothesis testing should be considered. Indeed, it is commonly accepted that PoC constitutes proof but may not be complete, which raises ambiguity. One can then wonder what natures the constraints are set aside also whether this ambiguity is desired or on the contrary should be better controlled. We hypothesise that the more cautious the designers are in identifying uncontrolled variables, the better the scientific evidence, and the more the PoC will be generative according to these variables.