Karima Alim

Amélie Moreau

Arnaud Bruyère

Elodie Jouan

Claire Denizot

Anne T Nies

Yannick Parmentier

Olivier Fardel
email: olivier.fardel@univ-rennes1.fr

Dr Olivier Fardel

Inhibition of organic cation transporter 3 activity by tyrosine kinase inhibitors

Keywords: Drug-drug interaction, molecular descriptors, organic cation transporter, pharmacokinetics, tyrosine kinase inhibitor ALK, anaplastic lymphoma kinase, ALOGP, Ghose-Crippen octanol-water partition, DDI, drug-drug interaction, DiASP, EMT, extraneuronal monoamine transporter, FAU, fluorescence arbitrary unit, OCT, organic cation transporter, PBS, phosphate-buffered saline, TKI, tyrosine kinase inhibitor, TPSA, topological polar surface area

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INTRODUCTION

Organic cation transporter (OCT) 3 (SLC22A3, also known as the extraneuronal monoamine transporter (EMT)), is a membrane drug transporter belonging to the solute carrier (SLC) family [START_REF] Koepsell | Organic Cation Transporters in Health and Disease[END_REF]. OCT3 handles various drugs, including cisplatin, metformin and quinidine, as well as endogenous compounds like neurotransmitters, histamine and creatinine [START_REF] Wagner | Polyspecific organic cation transporters and their impact on drug intracellular levels and pharmacodynamics[END_REF][START_REF] Wu | Identity of the organic cation transporter OCT3 as the extraneuronal monoamine transporter (uptake2) and evidence for the expression of the transporter in the brain[END_REF][START_REF] Nakada | Estimation of changes in serum creatinine and creatinine clearance caused by renal transporter inhibition in healthy subjects[END_REF][START_REF] Schneider | Organic cation transporter 3 modulates murine basophil functions by controlling intracellular histamine levels[END_REF]. Many of its substrates are shared with the two other OCTs, i.e., OCT1 and OCT2. OCT3, unlike OCT1 and OCT2, is rather widely expressed [START_REF] Nies | Organic cation transporters (OCTs, MATEs), in vitro and in vivo evidence for the importance in drug therapy[END_REF]. OCT3 is notably found in tissues relevant for pharmacokinetics, such as the liver and kidney [START_REF] Jonker | Pharmacological and physiological functions of the polyspecific organic cation transporters: OCT1, 2, and 3 (SLC22A1-3)[END_REF], where it is localized at the sinusoidal membrane of hepatocytes and the basolateral membrane of renal proximal tubule epithelial cells, respectively [START_REF] Koepsell | Role of organic cation transporters in drug-drug interaction[END_REF]. The transporter is also present at the luminal membrane of small intestinal enterocytes, where it contributes to oral absorption of metformin [START_REF] Shirasaka | Involvement of organic cation transporter 3 (Oct3/Slc22a3) in the bioavailability and pharmacokinetics of antidiabetic metformin in mice[END_REF]. OCT3 is additionally found in the heart and may consequently play a role in cardiac tissue drug exposure [START_REF] Solbach | Organic cation transporter 3: expression in failing and nonfailing human heart and functional characterization[END_REF], as well as in the placenta, where it facilitates foetal exposure to metformin during pregnancy [START_REF] Lee | Organic Cation Transporter 3 Facilitates Fetal Exposure to Metformin during Pregnancy[END_REF]. Taken together, these data suggest that OCT3 may be a drug transporter to consider according to a pharmacokinetical, pharmacological and/or toxicological point of view, even if it is presently not included in the list of drug transporters that have to be studied during the development of new molecular entities according to regulatory authorities for drug approval in the US and Europe [START_REF]Vitro Drug Interaction Studies -Cytochrome P450 Enzyme-and Transporter-Mediated Drug Interactions Guidance for Industry, US Food and Drug Administration[END_REF][START_REF] Sudsakorn | FDA Drug-drug Interaction Guidance: A Comparison Analysis and Plan by Pharmaceutical Industrial Scientists[END_REF].

Various drugs block OCT3 activity and may consequently theoretically cause drug-drug interactions (DDIs) and/or OCT3 inhibition-related adverse effects [START_REF] Koepsell | Role of organic cation transporters in drug-drug interaction[END_REF]. In particular, some tyrosine kinase inhibitors (TKIs), like the Bcr-Abl inhibitors imatinib, dasatinib and nilotinib, the epidermal growth factor receptor (EGFR) inhibitors gefitinib and erlotinib, and the multi-kinase inhibitors sunitinib and sorafenib, inhibit in vitro OCT3 activity, at concentrations thought to be reached in vivo for at least nilotinib, thus supporting a potential clinical relevance of these effects [START_REF] Minematsu | Interactions of tyrosine kinase inhibitors with organic cation transporters and multidrug and toxic compound extrusion proteins[END_REF]. Whether other TKIs can also behave as inhibitors and/or substrates for OCT3 is however unknown, but likely deserves interest owing to the current development of TKIs as a leading pharmacological class for various therapeutic indications, to the role that transporters play in TKI pharmacokinetics and activity [START_REF] Neul | Impact of Membrane Drug Transporters on Resistance to Small-Molecule Tyrosine Kinase Inhibitors[END_REF][START_REF] Garrison | Role of OATP1B1 and OATP1B3 in Drug-Drug Interactions Mediated by Tyrosine Kinase Inhibitors[END_REF] and to the fact that some TKIs like abemaciclib have been clinically shown to increase exposure to the OCT3 substrate metformin [START_REF] Chappell | Abemaciclib Inhibits Renal Tubular Secretion Without Changing Glomerular Filtration Rate[END_REF]. To get insights about

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this point in the present study, we have analyzed the interactions of OCT3 with 25 TKIs, targeting various tyrosine kinases and regulatory-approved (n=23) or under clinical development (n=2).

MATERIAL AND METHODS

Chemicals

The 23 marketed TKIs abemaciclib, acalabrutinib, afatinib, alectinib, brigatinib, cabozantinib, ceritinib, crizotinib, dacomitinib, entrectinib, ibrutinib, lapatinib, lenvatinib, lorlatinib, neratinib, nintedanib, osimertinib, ponatinib, regorafenib, ribociclib, ruxolitinib, tofacitinib and vemurafenib as well as itacitinib and pacritinib, 2 TKIs under clinical phase 3 development, were provided by MedChemtronica (Sollentuna, Sweden). These TKIs target various kinases, as indicated in Table 1. Corticosterone and unlabeled 1-methyl-4-phenylpyridinium (MPP + ) were from Sigma-Aldrich (Saint Quentin Fallavier, France) and 4-(4-(dimethylamino)styryl)-N-methylpyridiniumiodide (DiASP) from Thermo Fisher Scientific (Waltham, MA, USA). N-[methyl-3 H]-MPP + (specific activity = 81.8 mCi/mmol) was purchased from PerkinElmer (Villebon-sur-Yvette, France).

Cell culture

HEK293 cells overexpressing OCT3 (HEK-OCT3 cells) as well as control HEK293-MOCK cells (HEK-CTR cells), obtained by stable transfection [START_REF] Nies | Proton pump inhibitors inhibit metformin uptake by organic cation transporters (OCTs)[END_REF], were cultured in DMEM medium, supplemented with 10% (vol/vol) fetal calf serum, 1 µg/mL bovine insulin, 1% (vol/vol) nonessential amino acid solution, 800 µg/mL G418, 20 UI/mL penicillin and 20 µg/mL streptomycin.

OCT3 activity inhibition assay

Inhibition of cellular OCT3 activity was investigated using the fluorescent OCT probe DiASP, which is transported into HEK-OCT3 cells in a saturable manner (K m =24.8 ± 5.1 µM) [START_REF] Sayyed | Inhibition of organic cation transporter (OCT) activities by carcinogenic heterocyclic aromatic amines[END_REF].

Briefly, cells were incubated with 10 µM DiASP for 5 min at 37°C, in the absence or presence of the reference OCT3 inhibitor corticosterone or of TKIs, in the transport assay medium previously described [START_REF] Moreau | Drug transporter expression in human macrophages[END_REF]. After washing with phosphate-buffered saline (PBS), intracellular accumulation of the dye was determined by spectrofluorimetry, using a SpectraMax Gemini SX spectrofluorometer

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(Molecular Devices, Sunnyvale, CA, USA); excitation and emission wavelengths were 485 nm and 607 nm, respectively. Data were finally normalized to total protein content, determined by the Bradford's method [START_REF] Bradford | A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[END_REF]. They were expressed as fluorescence arbitrary unit (FAU)/mg protein or as percentages of OCT3 activity or of OCT3 activity inhibition according to the equation (A) or (B), respectively:

Reversibility of OCT3 activity inhibition by TKIs

HEK-OCT3 cells were first incubated in the absence (control) or presence of 10 µM TKIs for 5 min at 37°C. After washing, cells were either used directly for OCT3 activity measurement, performed as described above, or were re-incubated in TKI-free medium for 3 h before OCT3 activity measurement. Data were finally expressed as percentages of OCT3 activity inhibition.

Prediction of in vivo inhibition of OCT3 by TKIs

From in vitro IC 50 values, in vivo inhibition of OCT3 activity by TKIs was predicted through applying the criteria defined by the US Food and Drug Administration (FDA) guidance on in vitro drug interaction studies [START_REF]Vitro Drug Interaction Studies -Cytochrome P450 Enzyme-and Transporter-Mediated Drug Interactions Guidance for Industry, US Food and Drug Administration[END_REF] to OCT3, i.e., a transporter-related DDI can be expected if :

Accepted Article IC50 Imax, u > 0.1 (D)
with I max,u = maximum unbound plasma concentration of the TKI.

Trans-stimulation assay

Trans-stimulation assays were performed in HEK-OCT3 cells using the OCT3 substrate MPP + [START_REF] Gründemann | Agmatine is efficiently transported by non-neuronal monoamine transporters extraneuronal monoamine transporter (EMT) and organic cation transporter 2 (OCT2)[END_REF]; such trans-stimulation assays for OCTs are based on the fact that substrates of these transporters can both cis-inhibit and trans-stimulate them [START_REF] Zhang | The interaction of n-tetraalkylammonium compounds with a human organic cation transporter, hOCT1[END_REF]. Briefly, HEK-OCT3 cells were first incubated with 1 mM unlabeled MPP + or 30 μM TKI for 30 min at 37 °C. After washing with PBS, cells were next re-incubated with radiolabeled [ 3 H]-MPP + (used at 6.0 nM) for 5 min at

TKI molecular descriptor generation

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83 molecular descriptors belonging to the blocks "constitutional indices" (n = 47), "charge descriptors" (n = 15) and "molecular properties" (n = 20), were determined using the Dragon 7.0 software (Talete, Milano, Italy) (See http://www.talete.mi.it/products/dragon_molecular_descriptor_list.pdf for a complete list of these descriptors). TKIs, initially expressed in SMILES format, were converted to 3D format using MarvinView 20.19.0 software (ChemAxon, Budapest, Hungary) before processing by Dragon 7.0 software to obtain molecular descriptors, as previously described [START_REF] Sayyed | Inhibition of organic cation transporter (OCT) activities by carcinogenic heterocyclic aromatic amines[END_REF]. An additional charge descriptor, i.e., the percentage of cationic form of TKIs at pH = 7.4, was determined using MarvinSketch 20.19.0 software; the total number of molecular descriptors analyzed for each TKI was therefore 84.

Statistical analysis

Experimental data were routinely expressed as means ± SD of at least three independent assays,

RESULTS

Inhibition of in vitro OCT3 activity by TKIs

The effects of 10 µM TKIs towards OCT3-mediated transporter of DiASP were investigated in HEK-OCT3 cells. As shown in nM (for acalabrutinib) (Figure 1). We next investigated whether TKI-mediated inhibition of OCT3 may be reversible. For this purpose, cells were initially exposed to 10 µM TKI for 5 min, washed and re-incubated in TKI-free medium for 3 h before measurement of OCT3 activity. As indicated in Figure 2, inhibition of OCT3 activity by acalabrutinib was fully suppressed by the 3 h TKI-free post-incubation period, whereas those due to brigatinib or pacritinib were partly reversed. This indicates that the inhibitory effects of these TKIs towards OCT3 were fully (acalabrutinib) or partly (brigatinib and pacritinib) reversible after a 3 h TKI-free post-incubation period. By contrast, inhibition of OCT3-mediated transport by ceritinib and crizotinib was not significantly impaired by a 3 h TKI-free post-incubation when compared to the control condition without a TKI-free post-incubation (Figure 2).

In order to investigate physico-chemical structural requirements for OCT3 inhibition by TKIs, percentages of inhibition of OCT3 activity by the 25 TKIs (each used at 10 µM) were next confronted to values of 84 TKI molecular descriptors, through Pearson correlation analysis. 7/84 molecular descriptors were found to be significantly either positively (n=2) or negatively (n=5) correlated with OCT3 inhibition (Table 2). This notably concerns the percentage of H atoms (H%)

and the percentage of cationic forms. The 77/84 molecular descriptors not correlated with OCT3 activity inhibition, including lipophilicity-related parameters such as Moriguchi octanol-water partition coefficient (MLOGP) and Ghose-Crippen octanol-water partition (ALOGP) as well as topological polar surface area (TPSA), are listed in Table S1.

Prediction of in vivo OCT3 inhibition by TKIs

The prediction of in vivo OCT3 inhibition was performed for TKIs defined above as strong inhibitors, i.e., acalabrutinib, brigatinib, ceritinib, crizotinib and pacritinib. It was done using the criteria defined by the U.S. FDA and based on the ratio IC 50 /I max,u . As indicated in Table 3, brigatinib, ceritinib, crizotinib and pacritinib, unlike acalabrutinib, were predicted to inhibit in vivo OCT3 activity at clinically achievable concentrations.

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Transport of TKIs by OCT3

To determine whether TKIs potently interacting with OCT3 activity, i.e., acalabrutinib, brigatinib, ceritinib, crizotinib and pacritinib, may be subtrates for OCT3, trans-stimulation assays were performed, because substrates for OCTs, including OCT3, are postulated to trans-stimulate OCT activities [START_REF] Gründemann | Agmatine is efficiently transported by non-neuronal monoamine transporters extraneuronal monoamine transporter (EMT) and organic cation transporter 2 (OCT2)[END_REF][START_REF] Zhang | The interaction of n-tetraalkylammonium compounds with a human organic cation transporter, hOCT1[END_REF]. As shown in Figure 3

DISCUSSION

The data of the present study fully confirm that TKIs can interact with OCT3 activity. Indeed, only 5/25 TKIs were devoid of any inhibitory effect towards OCT3 activity, whereas 13/25 TKIs were characterized as moderate or strong inhibitors of OCT3 and 7/25 TKIs as weak inhibitors. TKIs acting as moderate and strong inhibitors block more than 50% of OCT3 activity when used at 10 µM, thus supporting the conclusion that their IC 50 values towards OCT3 are less than 10 µM. Such compounds can therefore be unambiguously added to the list of TKIs blocking OCT3, which already comprises 6 other TKIs, including imatinib, nilotinib and erlotinib [START_REF] Minematsu | Interactions of tyrosine kinase inhibitors with organic cation transporters and multidrug and toxic compound extrusion proteins[END_REF]. The kinases targeted by these TKIs blocking OCT3 activity are diverse, including the anaplastic lymphoma kinase (ALK) inhibited by brigatinib, ceritinib and crizotinib, the Janus kinase (JAK) 2 and the Fms-like tyrosine kinase 3 (Flt3) inhibited by pacritinib, the kinase Bcr-Abl inhibited by imatinib and nilotinib and the EGFR inhibited by erlotinib. Inhibition of OCT3 activity by TKIs is therefore not linked to the targeting of a specific tyrosine kinase. For some strong inhibitors of OCT3 such

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as brigatinib, ceritinib and crizotinib, IC 50 values are comprised in the 28-106 nM range, whereas IC 50 values for OCT3 inhibitors are commonly in the 1-100 µM range [START_REF] Nies | Proton pump inhibitors inhibit metformin uptake by organic cation transporters (OCTs)[END_REF][START_REF] Wittwer | Discovery of potent, selective multidrug and toxin extrusion transporter 1 (MATE1, SLC47A1) inhibitors through prescription drug profiling and computational modeling[END_REF][START_REF] Hu | Novel compound, organic cation transporter 3 detection agent and organic cation transporter 3 activity inhibitor, WO2015002150 A1: a patent evaluation[END_REF][START_REF] Li | Interaction of six protoberberine alkaloids with human organic cation transporters 1, 2 and 3[END_REF][START_REF] Zhu | Evaluation of organic cation transporter 3 (SLC22A3) inhibition as a potential mechanism of antidepressant action[END_REF]; this illustrates the rather potent in vitro inhibitory effect of these TKIs towards OCT3, similar to that of reference OCT3 inhibitors such as corticosterone or decynium 22, for which IC 50 values are in the 100-300 nM range [START_REF] Hayer-Zillgen | Expression and pharmacological profile of the human organic cation transporters hOCT1, hOCT2 and hOCT3[END_REF]. Moreover, the inhibition of OCT3 activity by 10 µM ceritinib or 10 µM crizotinib was not impaired by a 3 h TKI-free post-incubation period, suggesting a long-lasting action of these two TKIs. Beyond OCT3, other SLCs handling organic cations such as OCT1, OCT2, multidrug and toxin extrusion protein (MATE) 1 and MATE2-K have been shown to be inhibited by various TKIs [START_REF] Minematsu | Interactions of tyrosine kinase inhibitors with organic cation transporters and multidrug and toxic compound extrusion proteins[END_REF], thus indicating that transporters of organic cations may represent privileged targets of TKIs. In this context, it is noteworthy that one TKI may inhibit multiple SLCs transporting organic cations. As an example, in addition to OCT3, the transporters OCT1, MATE1

and MATE2-K are inhibited by brigatinib [START_REF] Brigatinib | Multi-discipline review[END_REF], whereas clinically relevant concentrations of crizotinib block OCT1 and OCT2 activities [30], therefore supporting the hypothesis that these TKIs may act as powerful inhibitors of organic cation transport.

Among the five strong OCT3 inhibitors reported in the present study, 4, i.e., the ALK inhibitors brigatinib, ceritinib and crizotinib as well as the JAK2/Flt3 inhibitor pacritinib are predicted to in vivo inhibit OCT3 according to the criteria defined by the U.S. FDA. This point may merit consideration in terms of possible DDIs and/or adverse effects. Indeed, even if OCT3 is presently not considered as a priority transporter by drug agencies, it is present at various sites implicated in pharmacokinetics and handles various marketed drugs. It is notably the case for metformin, for which OCT3 is involved in pharmacokinetics and pharmacological response in humans [START_REF] Kwon | OCT3 promoter haplotype is associated with metformin pharmacokinetics in Koreans[END_REF][START_REF] Zaharenko | Single nucleotide polymorphisms in the intergenic region between metformin transporter OCT2 and Accepted Article OCT3 coding genes are associated with short-term response to metformin monotherapy in type 2 diabetes mellitus patients[END_REF][START_REF] Hosseyni-Talei | Association between the synonymous variant organic cation transporter 3 (OCT3)-1233G>A and the glycemic response following metformin therapy in patients with type 2 diabetes[END_REF]. OCT3 may notably be implicated in intestinal absorption of metformin [START_REF] Shirasaka | Involvement of organic cation transporter 3 (Oct3/Slc22a3) in the bioavailability and pharmacokinetics of antidiabetic metformin in mice[END_REF], as well as in fetal exposure to this hypoglycemic agent [START_REF] Lee | Organic Cation Transporter 3 Facilitates Fetal Exposure to Metformin during Pregnancy[END_REF]; it may also govern its antiproliferative effects in OCT3-expressing cancer cells [START_REF] Cai | Cation-selective transporters are critical to the AMPK-mediated antiproliferative effects of metformin in human breast cancer cells[END_REF][START_REF] Cai | Efficacious dose of metformin for breast cancer therapy is determined by cation transporter expression in tumours[END_REF]. TKIs inhibiting in vivo OCT3 activity may therefore be suspected to alter pharmacokinetics and/or pharmacological activity of metformin.

Further studies are likely required to investigate these points. The possible inhibition by TKIs of the OCT3-mediated transport of endogenous compounds, like monoamine neurotransmitters, histamine or creatinine, and its clinical consequences may also deserve interest. This likely concerns the placental OCT3, known to be an essential component of foeto-placental homeostasis of serotonin [START_REF] Karahoda | Serotonin homeostasis in the materno-foetal interface at term: Role of transporters (SERT/SLC6A4 and OCT3/SLC22A3) and monoamine oxidase A (MAO-A) in uptake and degradation of serotonin by human and rat term placenta[END_REF].

Various molecular descriptors have been associated with inhibition of SLC transporter activities, notably for OCTs and MATEs [START_REF] Wittwer | Discovery of potent, selective multidrug and toxin extrusion transporter 1 (MATE1, SLC47A1) inhibitors through prescription drug profiling and computational modeling[END_REF][START_REF] Ahlin | Structural requirements for drug inhibition of the liver specific human organic cation transport protein 1[END_REF][START_REF] Kido | Profiling of a prescription drug library for potential renal drug-drug interactions mediated by the organic cation transporter 2[END_REF]. Inhibition of OCT3 by heterocyclic

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aromatic amines is thus positively correlated with percentage of cationic form at pH = 7.4 and percentage of H atoms [START_REF] Sayyed | Inhibition of organic cation transporter (OCT) activities by carcinogenic heterocyclic aromatic amines[END_REF]. Such molecular descriptors are similarly correlated with OCT3 inhibition by TKIs (Table 1), thus supporting the idea that they correspond to robust molecular descriptors to be considered for OCT3 inhibition. Cationic positive fraction at pH = 7.4 has also a high value for OCT1, OCT2 or MATE1 inhibition [START_REF] Wittwer | Discovery of potent, selective multidrug and toxin extrusion transporter 1 (MATE1, SLC47A1) inhibitors through prescription drug profiling and computational modeling[END_REF][START_REF] Ahlin | Structural requirements for drug inhibition of the liver specific human organic cation transport protein 1[END_REF][START_REF] Kido | Profiling of a prescription drug library for potential renal drug-drug interactions mediated by the organic cation transporter 2[END_REF], therefore suggesting that this descriptor is a key-parameter associated with impairment of OCT/MATE activities by drugs. By contrast, parameters such as lipophilicity (LogP) or TPSA are correlated with inhibition of OCT1 and OCT2, but not with blockage of OCT3 by heterocyclic aromatic amines [START_REF] Sayyed | Inhibition of organic cation transporter (OCT) activities by carcinogenic heterocyclic aromatic amines[END_REF] or by TKIs [START_REF] Minematsu | Interactions of tyrosine kinase inhibitors with organic cation transporters and multidrug and toxic compound extrusion proteins[END_REF] (Table S1). This lack of contribution of lipophilicity to OCT3 inhibition however likely merits complementary investigations because it is challenged by the enhanced inhibitory potency of phenylguanidines with increased lipophilic character towards OCT3 activity [START_REF] Pan | A new chemotype inhibitor for the human organic cation transporter 3 (hOCT3)[END_REF]. Excessive lipophilicity may nevertheless make it difficult for a TKI to access the substrate binding region of OCT3 in contact with the aqueous phase, as already proposed for OCT1 [START_REF] Minematsu | Interactions of tyrosine kinase inhibitors with organic cation transporters and multidrug and toxic compound extrusion proteins[END_REF].

Acalabrutinib, brigatinib, ceritinib, crizotinib and pacritinib failed to trans-stimulate OCT3 activity, in contrast to the reference substrate MPP + . Moreover, similar accumulation of these TKIs were found in HEK-CTR and HEK-OCT3 cells, without impairment by the OCT3 inhibitor corticosterone. Taken together, these data suggest that these TKIs are not substrates for OCT3.

OCT3 is therefore unlikely to play a major role in their pharmacokinetic properties. In the same way, the TKI imatinib blocks OCT3, but is not handled by this transporter [START_REF] Minematsu | Interactions of tyrosine kinase inhibitors with organic cation transporters and multidrug and toxic compound extrusion proteins[END_REF][START_REF] Mettral | Imatinib Uptake into Cells is Not Mediated by Organic Cation Transporters OCT1, OCT2, or OCT3, But is Influenced by Extracellular pH[END_REF]. Such data consequently suggest that TKIs likely interact with OCT3 through cis-inhibiting its activity, without being transported.

In summary, various TKIs (13/25) were demonstrated to behave as moderate or strong inhibitors of OCT3. For some of them like brigatinib, ceritinib, crizotinib and pacritinib, realistic clinical concentrations were predicted to in vivo inhibit OCT3 activity, which may have consequences in terms of DDIs or toxicity. OCT3 may therefore appear as a drug transporter worthy of interest when considering the use of TKIs.

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LEGENDS TO FIGURES

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a n = total number of molecular descriptors/block included in the study; these molecular descriptors were given by Dragon 7.0 software, excepted the % of cationic form at pH = 7.4 for the block "Charges descriptors", which was determined using MarvinSketch 20.19.0 software. C max , maximum serum concentration; F u , unbound fraction; I max, u , maximum unbound serum concentration.

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An in vivo inhibition can be predicted if the ratio I max,u /IC 50 >0.1 according to the US FDA [START_REF]Vitro Drug Interaction Studies -Cytochrome P450 Enzyme-and Transporter-Mediated Drug Interactions Guidance for Industry, US Food and Drug Administration[END_REF].

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fcp_12657_f4.pptx

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%

  OCT3 activity = ([DiASP TKI ] -[DiASP Corticosterone ]) × 100 ([DiASP Control ] -[DiASP Corticosterone ]) (A) % OCT3 activity inhibition = 100% -% OCT3 activity (B) with [DiASP TKI ] = DiASP concentration in the presence of a defined concentration of TKI, [DiASP Corticosterone ] = DiASP concentration in the presence of 100 µM corticosterone and [DiASP Control ] = DiASP concentration in control cells not exposed to TKI or corticosterone. Half maximal inhibitory concentration (IC 50 ) for TKIs towards OCT3 activity was calculated using Prism 8.4.2 software (GraphPad Software, La Jolla, CA, USA), through nonlinear regression based on the following four-parameter logistic equation: 𝐴 = 100 1 + 10 ([I] -Log(IC50))x Hill slope (C) with A = percentage of transporter activity for a given concentration of TKI determined as described in equation (A), [I] is the TKI concentration in the medium, and Hill slope is a coefficient describing the steepness of the curve.

  37 °C. Intracellular accumulation of radiolabeled MPP + was next determined by liquid scintillation counting using a Tri-Carb 4810TR counter (Perkin-Elmer), after addition of 2 ml/sample of the liquid scintillation cocktail Ultima Gold™ (Perkin Elmer). Data were normalized to protein content and finally expressed as percentages of [ 3 H]-MPP + accumulation found in control cells not pre-treated by unlabeled MPP + or TKIs.TKI accumulation assayHEK-CTR and HEK-OCT3 cells were incubated with 10 µM TKI, in the absence or presence of 100 µM corticosterone, for 5 min at 37°C. Cells were then washed twice in ice-cold PBS, lysed in distilled water and acetonitrile-based extraction of cell lysates was performed. TKI quantification was next performed through liquid chromatography-tandem mass spectrometry (LC-MS/MS), based on a high-performance liquid chromatography Aria system (Agilent, Les Ulis, France), equipped with a Poroshell 120 C18 (4.6 × 100 mm) column (Agilent, Les Ulis, France) and coupled to a tandem mass spectrometry TSQ Quantum (Thermo Fisher Scientific, Villebon sur Yvette, France), fitted with an electrospray ionization source (ESI+). Monitored ion transitions were at 466.1 > 372.1 m/z (for acalabrutinib), 584.0 >484.2 m/z (for brigatinib), 558.0>433.0 (for ceritinib), 449.9>260.1 (for crizotinib) and 437.1>316.1 (for pacritinib). Data were finally normalized to protein content.

  each being usually performed in triplicate. Normality distribution of percentages of OCT3 activity inhibition by TKIs was checked by D'Agostino and Pearson omnibus normality test. Data were statistically analyzed through analysis of variance (ANOVA) followed by the Dunnett's or Tukey's post-hoc test. Correlation between molecular descriptor indexes and percentages of OCT3 activity inhibition by TKIs was performed through Pearson correlation. The criterion of significance was p < 0.05. All the statistical analyses were done using Prism 8.4.2 software.

  , acalabrutinib, brigatinib, ceritinib, crizotinib and pacritinib failed to trans-stimulate OCT3-mediated radiolabeled MPP + accumulation in HEK-OCT3 cells. By contrast, unlabeled MPP + trans-stimulated OCT3 activity (by a 4.7-fold-factor) (Figure3). Intracellular accumulation of TKIs was next determined in HEK-CTR and HEK-OCT3 cells by LC-MS/MS assay, in the absence or presence of the OCT3 inhibitor corticosterone. As shown in Figure4, accumulations in HEK-CTR and HEK-OCT3 cells were similar for each analyzed TKI and corticosterone failed to reduce TKI concentrations in HEK-OCT3 cells. By contrast, accumulation of the reference OCT3 substrate DiASP was significantly increased in HEK-OCT3 cells when compared to HEK-CTR cells (by a 4.2-fold factor). This enhanced accumulation of DiASP in HEK-OCT3 cells was nevertheless markedly reduced in the presence of corticosterone (Figure4).

Figure 1 .

 1 Figure 1. Concentration-dependent inhibition of OCT3 activity by TKIs. HEK-OCT3 cells were incubated with 10 μM DiASP for 5 min at 37 °C, in the absence or presence of various concentrations of TKIs (from 1 nM to 30 µM) or of 100 µM corticosterone, used here as a reference OCT3 inhibitor. After washing with PBS, intracellular accumulation of DiASP was determined by spectrofluorimetry and normalized to protein content. Data are expressed as percentages of OCT3 activity found in control cells exposed only to DiASP, arbitrarily set at 100%; they are the means ± SD of at least three independent experiments. TKI IC 50 values as well as 95 % confidence intervals (in brackets) are indicated on the top of graphs.

Figure 2 .

 2 Figure 2. Reversibility of TKI-mediated OCT3 activity inhibition.HEK-OCT3 cells were first incubated in the absence or presence of 10 µM TKIs for 5 min at 37°C. After washing, cells were either used directly for OCT3 activity measurement, performed as described in Materials and Methods, or were re-incubated in TKI-free medium for 3 h before OCT3 activity measurement. Data are expressed as percentages of OCT3 activity inhibition and are the means ± SD of at least three independent experiments. ***, p<0.05 when compared to counterparts not subjected to a 3 h TKI-free post-incubation.

Figure 3 .

 3 Figure 3. Trans-stimulation of OCT3 activity by TKIs. HEK-OCT3 cells were first incubated in the absence (untreated) or presence of 1 mM unlabeled MPP + or 30 μM TKIs for 30 min at 37 °C. After washing with PBS, cells were next re-incubated with 6 nM [ 3 H]-MPP + for 5 min at 37 °C. Intracellular accumulation of [ 3 H]-MPP + was finally determined by scintillation counting. Data were expressed as percentages of [ 3 H]-MPP + accumulation found in control untreated cells, arbitrarily set at 100% and indicated by a dashed line on the graph; they are the means ± SD of three independent assays. ***, p < 0.001 when compared to untreated cells.

Figure 4 .

 4 Figure 4. Accumulation of TKIs in HEK-CTR and HEK-OCT3 cells. HEK-CTR and HEK-OCT3 cells were incubated with 10 µM TKIs or 10 µM DiASP, in the absence or presence of 100 µM corticosterone, for 5 min at 37 °C. After washing with PBS, intracellular accumulations of TKIs and DiASP were determined by LC-MS/MS and spectrofluorimetry, respectively, and normalized to protein content. Data are the means ± SD of Accepted Article

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  Figure 3

Table 1

 1 OCT3 activity by more than 50% but less than 80% (Table1) and thus appear as moderate inhibitors of the transporter. 5/25 TKIs, i.e., acalabrutinib, brigatinib, ceritinib, crizotinib and pacritinib were found to inhibit OCT3 by more than 80% (Table1) and are consequently potent inhibitors of OCT3 activity. These five strong OCT3 inhibitors were next found to inhibit OCT3 in a concentration-dependent manner, with IC 50 values ranging from 28.2 nM (for ceritinib) to 1841

	, 5/25 TKIs, i.e., alectinib, cabozantinib, itacitinib,

afatinib, dacomitinib, entrectinib, ibrutinib, lenvatinib, ponatinib and ribociclib, were found to Accepted Article inhibit

Table 1 .

 1 Effect of 10 µM TKIs on OCT3 activity in HEK-OCT3 cells. , p<0.05 and ***, p<0.001, when compared to control cells not exposed to TKIs.

	Accepted Article	TKI Abemaciclib Acalabrutinib Afatinib Alectinib Brigatinib Cabozantinib Ceritinib Crizotinib Dacomitinib Entrectinib Ibrutinib Itacitinib Lapatinib Lenvatinib Lorlatinib Neratinib Nintedanib Osimertinib Pacritinib Ponatinib Regorafenib Ribociclib Ruxolitinib Tofacitinib Vemurafenib	Main targeted kinase CDK4/6 BTK EGFR ALK ALK VEGFR2, c-Met, c-Kit, Axl and Flt3 ALK ALK EGFR Pan-TRK, ROS1, and ALK BTK JAK1 ErbB-2 and EGFR VEGFR1-3, FGFR1-4, PDGFR, Kit, and Ret ROS1 and ALK ErbB-2 and EGFR VEGFR, FGFR and PDGFR EGFR JAK2 and Flt3 CDK4/6 VEGFR1/2/3, PDGFRβ, Kit, Ret and Raf-1 CDK4/6 JAKs JAKs B-Raf	% inhibition of OCT3 activity (Mean ± SD) 69.4 ± 7.1 *** 88.6 ± 6.3 *** 76.3 ± 9.3 *** 11.3 ± 16.7 103.3 ± 7.7 *** 22.8 ± 29.8 99.9 ± 7.9 *** 99.8 ± 4.3 *** 71.4 ± 13.0 *** 77.4 ± 10.8 *** 75.1 ± 14.2 *** 0.0 ± 10.1 1.7 ± 7.3 67.0 ± 11.3 *** 41.0 ± 3.6 *** 39.5 ± 7.6 *** 47.6 ± 12.4 *** 48.5 ± 19.8 *** 90.0 ± 5.8 *** 79.2 ± 7.1 *** 29.0 ± 24.9 * 74.3 ± 7.3 *** 47.6 ± 11.4 *** 46.2 ± 15.0 *** 16.0 ± 3.1

ALK, anaplastic lymphoma kinase; BTK, Bruton's tyrosine kinase; c-Met, hepatocyte growth factor receptor (HGFR); CDK, cyclin-dependent kinase; EFGR, epidermal growth factor receptor (also known as ErbB-1 or HER1); ErbB-2, human epidermal growth factor receptor-2 (also known as HER2); FGFR, fibroblast growth factor receptor; Flt3, Fms-like tyrosine kinase 3; JAK, Janus kinase ; PDGFR, platelet-derived growth factor; *

Table 2 .

 2 List of TKI molecular descriptors correlated with OCT3 activity inhibition by 10 μM TKIs.

		Molecular descriptor	Correlation with OCT3 inhibition %
	Block	Name	Pearson coefficient (r)	Significance (p value)
		Mean atomic van der Waals volume (scaled on Carbon atom) (Mv)	-0.4682	0.018
	Constitutional indices	mean atomic Sanderson electronegativity (scaled on Carbon atom) (Me)	-0.4255	0.034
	(n=47) a	Percentage of H atoms (H%)	0.4886	0.013
		Percentage of C atoms (C%)	-0.4501	0.024
		mean absolute charge (charge polarization) (Qmean)	-0.4135	0.040
	Charge descriptors	percentage of cationic forms at pH=7.4	0.4946	0.012
	(n=16)	Unsaturation index (Ui)	-0.4184	0.037
	Molecular properties

Table 3 :

 3 Prediction of in vivo OCT3 inhibition by TKIs.

	Drug	C max	F u	I max,u (nM)	IC 50 (nM)	Ratio I max,u /IC 50	Predicted in vivo inhibition	Reference
	Acalabrutinib	1209 nM	0.025 30.2 1841.0	0.02	No	[41]
	Brigatinib	2482 nM	0.340 844.0	41.0	20.59	Yes	[29]
	Ceritinib	1800 nM	0.028 50.4	28.2	1.79	Yes	[42]
	Crizotinib	1061 nM	0.093 98.7	106.1	0.93	Yes	[43]
	Pacritinib	22500 nM	0.011 247.5 854.9	0.26	Yes	[44]

ACKNOWLEDGMENTS A.T. Nies was in part funded by the Robert Bosch Stiftung (Stuttgart, Germany) and the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany's Excellence Strategy -EXC 2180 -390900677.

Accepted Article


Sébastien Villotte
email: s.villotte@pacea.u-bordeaux1.fr

Sandra Assis
email: sandraassis78@gmail.com

Francisca Alves Cardoso
email: francicard@fcsh.unl.pt

Charlotte Yvette Henderson
email: c.y.henderson@uc.pt

Valentina Mariotti
email: valentina.mariotti@unibo.it

Marco Milella
email: marco.milella@aim.uzh.ch

Doris Pany-Kucera

Nivien Speith
email: nspeith@bournemouth.ac.uk

Cynthia A Wilczak
email: cwilczak@sfsu.edu

Robert Jurmain
email: robert.jurmain@sjsu.edu

In Search of Consensus: Terminology for Entheseal Changes (EC)

Keywords: Enthesis, terminology, Fibrocartilaginous enthesis, spondyloarthropathies, diffuse idiopathic skeletal hyperostosis (DISH)

This article presents a consensus terminology for entheseal changes that was developed in English by an international team of scholars and then translated into French, Italian, Portuguese, Spanish and German. Use of a standard, neutral terminology to describe entheseal morphology will reduce misunderstandings between researchers, improve the reliability of comparisons between studies, and eliminate unwarranted etiological assumptions inherent in some of the descriptive terms presently used in the literature.

Introduction

"Musculoskeletal stress markers", renamed "entheseal changes" (ECs) by [START_REF] Jurmain | Terminology. Entheses in medical literature and physical anthropology: a brief review[END_REF], have been widely studied in past populations as indicators of activity and social divisions of labor (e.g. [START_REF] Dutour | Enthesopathies (lesions of muscular insertions) as indicators of the activities of Neolithic Saharan populations[END_REF][START_REF] Hawkey | Activity-induced musculoskeletal stress markers (MSM) and subsistence strategy changes among ancient Hudson Bay Eskimos[END_REF][START_REF] Robb | The interpretation of skeletal muscle sites: a statistical approach[END_REF]Villotte et al., 2010a;[START_REF] Havelková | Enthesopathies and activity patterns in the Early Medieval Great Moravian population: Evidence of division of labour[END_REF][START_REF] Havelková | Entheseal changes: do they reflect socioeconomic status in the Early Medieval Central European population? (Mikulčice -Klášteřisko, Great Moravian Empire, 9th -10th century)[END_REF][START_REF] Lieverse | Lower limb activity in the Cis-Baikal: Entheseal changes among middle Holocene Siberian foragers[END_REF][START_REF] Villotte | I sing of arms and of a man…": medial epicondylosis and the sexual division of labour in prehistoric Europe[END_REF]. However, the problem of oversimplified etiological interpretations that primarily equated EC with increased muscle use became widely apparent at a workshop held in 2009 in Coimbra, Portugal [START_REF] Santos | The Coimbra Workshop in Musculoskeletal Stress Markers (MSM): an annotated review[END_REF]. Discussions at this meeting led to the establishment of three working groups aimed at standardizing key EC research aspects, namely terminology, recording methodology, and the understanding of definitions of occupation. The three working groups met again in Coimbra at a new workshop in 2013.

This technical note focuses on the results from the terminology working group (SV, RJ). A survey of researchers taken prior to the 2013 workshop clearly showed the need for standardized descriptions of ECs. Only one third of respondents described the EC in Figure 1c using the same term (Table 1), and a similar lack of consensus was found for other types of ECs. During the 2013 workshop, the terminology working group compiled an initial list of neutral terms and descriptions of the most common EC types. The terms proposed by this group have been discussed with the members of the other working groups (SA, FAC, CYH, VM, MM, DPK, NS, CAW), and a consensus was finally reached. The terminology proposed here includes neutral (i.e. without inherent etiological concepts), descriptive terms with figures (Fig. 1 and 2, see also Table 2 for other illustrations of the EC defined here in previous publications by the authors), which researchers can use to describe ECs arising from any etiology, including those associated with pathological conditions, e.g. the seronegative spondyloarthropathies or diffuse idiopathic skeletal hyperostosis. It should be noted that similar changes can occur at other anatomical locations (e.g. synovial joints), but this is outside the scope of this paper. The proposed terminology (Fig. 3), alongside its translation into five major European languages (Table 2), should reduce communication barriers as well as lead to improved data sharing amongst researchers if widely adopted.

TABLE 1

Proposed terminology

Two groups of entheses can be distinguished according to the tissue type present at the skeletal attachment site [START_REF] Benjamin | The histology of tendon attachments to bone in man[END_REF][START_REF] Benjamin | The skeletal attachment of tendons -tendon "entheses[END_REF][START_REF] Cooper | Tendon and ligament insertion. A light and electron microscopic study[END_REF]see Villotte et al., 2010b for a list of the main post-cranial fibrous and fibrocartilaginous entheses). A normal fibrocartilaginous enthesis (FCE) is smooth, well circumscribed and devoid of vascular foramina [START_REF] Benjamin | The skeletal attachment of tendons -tendon "entheses[END_REF][START_REF] Henderson | Musculo-skeletal stress markers in bioarchaeology: indicators of activity levels or human variation? A re-analysis and interpretation[END_REF][START_REF] Henderson | The new "Coimbra method": a biologically appropriate method for recording specific features of fibrocartilaginous entheseal changes[END_REF][START_REF] Villotte | Connaissances médicales actuelles, cotation des enthésopathies: nouvelle méthode[END_REF][START_REF] Villotte | Enthésopathies et activités des Hommes préhistoriques -Recherche méthodologique et application aux fossiles européens du Paléolithique supérieur et du Mésolithique[END_REF]Villotte et al., 2010b); thus any alteration from this definition is considered an EC. For fibrous entheses, for which there is no clear definition of a "normal" aspect, we consider a theoretical smooth cortical surface as a base line [START_REF] Henderson | Musculo-skeletal stress markers in bioarchaeology: indicators of activity levels or human variation? A re-analysis and interpretation[END_REF][START_REF] Villotte | Connaissances médicales actuelles, cotation des enthésopathies: nouvelle méthode[END_REF][START_REF] Villotte | Enthésopathies et activités des Hommes préhistoriques -Recherche méthodologique et application aux fossiles européens du Paléolithique supérieur et du Mésolithique[END_REF]. In the following text "fibrous entheses" refers only to fibrous tendon attachment sites, and not to sites where fleshy muscle fibers attach to the periosteum (e.g. the origin of tibialis anterior muscle).

We distinguished three main categories of EC: mineralized tissue formation, surface discontinuity, and complete loss of original morphology with subcategories where appropriate (Fig. 3). Note that the definition of EC does not include architectural variation (e.g. a more or less developed deltoid tuberosity, or "waving" of the surface itself).

Mineralized tissue formation

The morphological change exceeds the level of the original surface, from roughness of the surface to a clear structure of any size or shape.

Diffuse and non-protruding formation

Not clearly distinct from the surface of the enthesis. Two morphological subtypes are identified according to the enthesis type.

Grained surface

A textural change of a diffuse granular nature (similar to fine grained sandpaper, [START_REF] Henderson | The new "Coimbra method": a biologically appropriate method for recording specific features of fibrocartilaginous entheseal changes[END_REF] at FCEs (Fig. 1a). The surface does not look as smooth as it is in unaltered FCEs, and it can feel roughened to the touch.

Based on a picture of a histological section of such a change (Fig. 6-11 and 6-63 in Schultz, 2003), this granular texture is likely to be related to the mineralization of the uncalcified fibrocartilage. 1 This type of change is mainly seen at the ischial and radial tuberosities.

Diffuse cortical irregularity

The area is rough to the touch due to diffuse tissue formation (Fig. 1b). The surface looks well remodeled (i.e. likely to be long standing). This is an extremely common feature at fibrous entheses and may be related to the direct attachment of tendon fibers to bone [START_REF] Villotte | Understanding entheseal changes: definition and life course changes[END_REF]. It is also seen at the margin (Zone 1 in [START_REF] Henderson | Recording specific entheseal changes of fibrocartilaginous entheses: initial tests using the Coimbra Method[END_REF], see also [START_REF] Villotte | Connaissances médicales actuelles, cotation des enthésopathies: nouvelle méthode[END_REF] of FCEs, a region with little fibrocartilage.

Isolated protrusion

Identified as a clearly distinct protrusion from the surface of the enthesis.

Enthesophyte

A clear bony projection (Fig. 1c) mostly seen at the margin of FCEs. The height of the projection can be easily measured, tends to be greater in length than in width, and tapers towards the end. In histological studies, these spurs have been found to contain (and be surrounded by) calcified fibrocartilage from the original entheses (Abreu, et al.2003;Moriggl et al.2001;[START_REF] Rufai | Structure and histopathology of the insertional region of the human Achilles tendon[END_REF]. Enthesophytes form through endochondral ossification in the Achilles tendon and subscapular ligament [START_REF] Moriggl | Fibrocartilage at the entheses of the suprascapular (superior transverse scapular) ligament of man -a ligament spanning two regions of a single bone[END_REF][START_REF] Rufai | Structure and histopathology of the insertional region of the human Achilles tendon[END_REF], but they form through intramembranous and chondroidal ossification in the plantar fascia [START_REF] Kumai | Heel spur formation and the subcalcaneal enthesis of the plantar fascia[END_REF].

Raised margin

A raised rim at the margin of a FCEs (Fig. 1d) that is not as developed as an enthesophyte (i.e. it is difficult to measure the height of the bony projection).

Longitudinal protrusion 1

Or, as stated by [START_REF] Schultz | Light microscopic analysis in skeletal paleopathology[END_REF], related to the ossification of the tendon. However, this second hypothesis seems less likely. In the caption of the figure 6.63, [START_REF] Schultz | Light microscopic analysis in skeletal paleopathology[END_REF] stated that the two layers of calcified tissue (green structures) correspond to mineralized tendon and that the process occurred in two waves. Actually, it seems to correspond to the layers of uncalcified and calcified fibrocartilage separated by the tidemark (see [START_REF] Benjamin | The skeletal attachment of tendons -tendon "entheses[END_REF], the first layer being mineralized in that pathological case.

A longitudinal bony protrusion at a fibrous enthesis normally affects nearly the entire length of the enthesis (Fig. 1e), either continuously or discontinuously. The protrusion is clearly distinct from the surrounding cortical surface, but it is not as developed and distinctive as an enthesophyte (i.e. it is difficult to measure the height of the bony projection). It can occur in conjunction with a smooth or a coarse surface.

Shapeless protrusion

A relatively flat or mound-like protruding mineralized tissue formation (Fig. 1f). It is highly variable in size, affecting a very limited part of the enthesis (a small raised area, i.e. nodule) to the entire attachment site. It occurs at both fibrocartilaginous (typically the central portion) and fibrous entheses.

FIGURE 1

Surface discontinuity

Depressions and other mineralized tissue loss of the surface [START_REF] Rothschild | Nondestructive, epi-illumination surface microscopic characterization of surface discontinuity in bone: A new approach offers a descriptive vocabulary and new insights[END_REF]. In fibrous entheses, cortical surface discontinuity can also involve the trabecular bone. In FCEs, the discontinuity involves the calcified cartilage and the subchondral bone (i.e. the subchondral bone plate and the trabecular bone). Possible origins include a resorptive process, a resorptive and hypertrophic process, hypervascularisation or avulsions (e.g. [START_REF] Flemming | Osseous involvement in calcific tendinitis: a retrospective review of 50 cases[END_REF][START_REF] Miller | Sonography of injury of the ulnar collateral ligament of the elbowinitial experience[END_REF][START_REF] Milz | Molecular composition and pathology of entheses on the medial and lateral epicondyles of the humerus: a structural basis for epicondylitis[END_REF].

Porosity

Pores are macroscopically observable small rounded openings in the surface 2 .

Fine porosity

Very small pinholes with a diameter of less than 1 mm, usually occurring in a cluster [START_REF] Henderson | The new "Coimbra method": a biologically appropriate method for recording specific features of fibrocartilaginous entheseal changes[END_REF].

The opening is too small to see the subsurface morphology. These occur at both fibrocartilaginous and fibrous entheses (Fig. 2a).

Macro porosity

Pores ≥ 1mm in diameter [START_REF] Henderson | The new "Coimbra method": a biologically appropriate method for recording specific features of fibrocartilaginous entheseal changes[END_REF], which occur singularly or in small clusters at fibrocartilaginous or fibrous entheses (Fig. 2b). The subsurface morphology is variable and, depending on the size of the opening, can sometimes be seen. When the subsurface morphology has the appearance of a channel, the pore is very likely a vascular foramen (often seen during skeletal development in FCEs). A macro pore can also be the entrance to a cavitation (see 2.2.3).

Cortical defect 2

We avoided the term "pitting", very often used as a descriptive term in paleopathology, but usually not defined (e.g. [START_REF] Ortner | Identification of Pathological conditions in human skeletal remains[END_REF]Roger and Waldron, 2015). However, it should be noted that our definition of "porosity" is similar to the definition of "pitting" given by Waldron (2008:27) in the context of changes taking place in articulating bones: "pitting on the joint surface manifested as a series of holes on the joint surface, some of which may communicate with sub-chondral cysts."

Focal bone loss, usually longitudinal in shape, often remodeled, and of variable depth (Fig. 2c). The floor of the depression often exhibits numerous pores. Cortical defects are mostly seen at the metaphyseal region of long bones, especially on the humerus at the insertions of the MM. pectoralis major and teres major, and at the attachment of the costoclavicular ligament on the clavicle. Possible origins include an avulsion, or an erosion from the periosteal or endosteal surface [START_REF] Villotte | Ostéoarchéologie et techniques médico-légales: tendances et perspectives. Pour un "Manuel pratique de paléopathologie humaine[END_REF]. In immature skeletons, cortical defects at metaphyseal attachment sites are likely related to the unbalanced activity of osteoclasts and osteoblasts when the attachments of tendons and ligaments migrate as long bones grow in length [START_REF] Villotte | Understanding entheseal changes: definition and life course changes[END_REF].

Erosive lesion

Destruction of the mineralized cartilage and subchondral bone [START_REF] Henderson | The new "Coimbra method": a biologically appropriate method for recording specific features of fibrocartilaginous entheseal changes[END_REF]. The erosive lesion is generally greater in width than depth. Its edges are often irregular and can be minimally or significantly remodeled. The floor of the depression exhibits exposed trabeculae or numerous pores (Fig. 2d). The morphology is very similar to erosions at synovial joints (see Rogers and Waldron, 1995:12). It can be observed at virtually all the appendicular FCEs.

Cavitation

Spherical subchondral lacuna with an opening at the surface that is smaller than the diameter of the cavitation and a clear floor that is not a channel [START_REF] Henderson | The new "Coimbra method": a biologically appropriate method for recording specific features of fibrocartilaginous entheseal changes[END_REF]) (Fig. 2e). Usually cavitations have remodeled trabecular bone walls. They may correspond to the cysts described in the biomedical literature [START_REF] Milz | Molecular composition and pathology of entheses on the medial and lateral epicondyles of the humerus: a structural basis for epicondylitis[END_REF][START_REF] Fritz | Cystic changes at supraspinatus and infraspinatus tendon insertion sites: association with age and rotator cuff disorders in 238 patients[END_REF]. Cavitations are only seen at FCEs, especially at the proximal humerus.

Furrowed surface (fibrous entheses)

This feature seems to occur only at fibrous entheses in immature skeletons. The cortical surface is covered by very thin, straight furrows of variable length (usually no more than a 2-3 millimeters) that are generally oriented along the long axis of the attachment (Fig. 2f). The appearance of numerous areas of bone resorption interspersed with hypertrophic bone formation suggests the furrows are related to the active process of bone growth [START_REF] Villotte | Understanding entheseal changes: definition and life course changes[END_REF].

Complete loss of original morphology

Associated with bone formation, and / or erosion, and porosity (Fig. 2g). Loss of morphology is most often seen at the greater tuberosity of the humerus.

Conclusion

The evaluation of "ECs" in bioarchaeological research became a common practice 30 years ago. As more researchers evaluated these morphological changes it became clear that both the common name for them and the varied descriptive terminology lacked scientific rigor and consistency [START_REF] Jurmain | Terminology. Entheses in medical literature and physical anthropology: a brief review[END_REF], and our goal here is to propose an initial attempt towards greater standardization. In so doing, we hope to encourage further discussion and a more focused intellectual exchange among bioarcheologists and paleopathologists. Table 2. The proposed new terminology alongside translations into French, Italian, Portuguese, Spanish, and German. References to other illustrations in publications by the authors of the EC defined here are also provided.

  FIGURE 2 FIGURE 3 TABLE 2

Figure list Figure 1 .

 list1 Figure list Figure 1. Examples of entheseal changes (mineralized tissue formation) to illustrate the terminology. a) grained surface. b) diffuse cortical irregularity. c) enthesophyte. d) raised margin. e) longitudinal protrusion. f) shapeless protrusion. Scale: 2cm.Photo credits: Villotte, S. (a, c, e, f); Assis, S. (b); Reichmann, W. and Pany-Kucera, D. (d).

Figure 2 .

 2 Figure 2. Examples of entheseal changes (surface discontinuity, and complete loss of original morphology) to illustrate the terminology. a) fine porosity. b) macro porosity. c) cortical defect. d) erosive lesion. e) cavitation. f) furrowed surface. g) complete loss of original morphology. Scale: 2cm. Photo credits: Mariotti, V. (a); Villotte, S. (b, c, f, g); Henderson, C. (d); Reichmann, W. and Pany-Kucera, D. (e).

Figure 3 .

 3 Figure 3. Graphical summary of the proposed terminology. "Fibrous"; fibrous entheses, "FC"; fibrocartilaginous entheses.

  Figure 1 Click here to download high resolution image

Table list Table 1 .

 list1 Results of a survey eliciting responses on the naming of EC, specifically what is defined in this paper as an enthesophyte (Fig 1c white arrrow).

Table 1 .

 1

	Table
	Answers	N
	Enthesophyte(s)
	Osteophyte(s)
	Exostosis / exostoses
	Bone spur(s)
	Bone growth(s) / outgrowth(s)
	EC
	Bone formation
	Enthesitis
	Hyperostotic change(s)
	Osteophytic formation
	Total

Acknowledgements

The authors would like to thank the Wenner-Gren Foundation (grant number Gr. CONF-632) for funding the workshop entitled 'EC and Reconstruction of Human Behavior: Towards Standardization' hosted by CIAS (Research Centre for Anthropology and Health, University of Coimbra, Portugal -UID/ANT/00283/2013) with support from CRIA (Centro em Rede de Investigação em Antropologia, Universidade de Nova, Lisbon, Portugal -UID/ANT/04038/2013). We also thank W. Reichmann, who took the pictures in Figures 1d and2e together with D. Pany-Kucera at the 18th Paleopathology Association meeting in Vienna, Austria, with the permission of M. Teschler-Nicola. The copyright of those pictures is retained by the Natural History Museum Vienna, Austria. Fig. 2a has been taken by V. Mariotti at the Dpt. BiGEA of the Alma Mater Studiorum, University of Bologna, with permission of M. G. Belcastro. We are also grateful to the many colleagues who kindly participated in the survey concerning the terms they use for entheseal changes. We would like to thank Maria Alejandra Acosta (CEF-Centre for Functional Ecology and the Laboratory of Forensic Anthropology, University of Coimbra) for translating the terminology into Spanish. Finally, we would like to thank the three anonymous reviewers for their useful comments. Authors C. Henderson and F. Alves Cardoso are funded by the Portuguese FCT -Fundação para a Ciência e a Tecnologia bursary SFRH/BPD82559/2011 and Investigator Programme IF/00127/2014, respectively and supported by the European Commission ESF and POPH.


Sébastien Q4

Villotte
email: s.villotte@pacea.u-bordeaux1.fr

Christopher J Knüsel

"I sing of arms and of a man.": medial epicondylosis and the sexual division of labour in prehistoric Europe q

Keywords:

Thrower's elbow (medial epicondylosis) is a rare occupational syndrome today. The lateral to medial (L/M) epicondylar ratio is greater than 1 in living populations. The L/M ratio permits comparisons in past populations. European prehistoric males display a specific pattern of lesions for the right elbow. Right upper limb use in throwing motions is a long-term status-linked male proclivity.

Introduction

In prehistory sexual division of labour or gendered activity, a part of gender relations that contributes to the roles that men and women play in society, is often inferred from analyses of grave goods, as a part of the social identity of the deceased (see, for example, Q2 Chapman, 1997; O 'Shea and Zvelebil, 1984;Rega, 1997;Stig-Sorensen, 1997;Treherne, 1995), and artistic representations, both pictographs and figurines [START_REF] Conkey | Contexts of action, contexts for power: material culture and gender in the Magdalenian[END_REF][START_REF] Duhard | Upper Palaeolithic figures as a reflection of human morphology and social organization[END_REF]Joyce 2004;[START_REF] Nelson | Gender in Archaeology[END_REF]. Interpretations depend on analogies drawn from cross-cultural studies that provide evidence for ostensibly universal sex differences in labour [START_REF] Wood | A cross-cultural analysis of the behavior of women and men: implications for the origins of sex differences[END_REF]. Within non-industrial societies, the majority of tasks are strongly gendered, but substantial diversity exists across societies with respect to which sex performs a particular task [START_REF] Gilchrist | Archaeology and the life course: a time and age for gender[END_REF][START_REF] Moore | Understanding sex and gender[END_REF] with regard to the effect of age and social change, respectively). In almost all groups hunting large game and warfare appear to be largely, albeit not exclusively, men's activities (Keeley 1986: 35;[START_REF] Knüsel | The osteology of conflict: what does it all mean[END_REF][START_REF] Murdock | Factors in the division of labor by sex: a crosscultural analysis[END_REF][START_REF] Testart | Essai sur les fondements de la division sexuelle du travail chez les chasseurs-cueilleurs[END_REF][START_REF] Wood | A cross-cultural analysis of the behavior of women and men: implications for the origins of sex differences[END_REF]. With regard to hunting, [START_REF] Testart | Essai sur les fondements de la division sexuelle du travail chez les chasseurs-cueilleurs[END_REF] points out that among modern hunter-gatherers, females can and do hunt, but almost never with weapons that penetrate the flesh. In a sample of 50 worldwide traditional societies from the Human Relations Area Files probability sample, men as opposed to women overwhelmingly engage in games and sports employing projectiles, such as spears, sticks, javelins, darts, and stones [START_REF] Deaner | Sex differences in sports across 50 societies[END_REF]. As a whole, men perform movements involving percussive force, as in the use of an adze or an axe [START_REF] Testart | Essai sur les fondements de la division sexuelle du travail chez les chasseurs-cueilleurs[END_REF]; these require similar movements of the upper limb to those employed in throwing, the biomechanics and injurious effects of which has been studied in detail for some years [START_REF] Bennett | Shoulder and elbow lesions of the professional baseball pitcher[END_REF][START_REF] Bennett | Shoulder and elbow lesions distinctive of baseball players[END_REF][START_REF] Bennett | Elbow and shoulder lesions of baseball players[END_REF][START_REF] Fleisig | Biomechanics of overhand throwing with implications for injuries[END_REF][START_REF] Gainor | The throw: biomechanics and injury[END_REF][START_REF] Jobe | An EMG analysis of the shoulder in throwing and pitching: a preliminary report[END_REF][START_REF] Mccue | Throwing injuries to the shoulder[END_REF][START_REF] Priest | Elbow injuries in highly skilled tennis players[END_REF][START_REF] Tullos | Examination of the throwing elbow[END_REF].

Throwing is among the most dynamic and demanding of physical activities, requiring complex sequential movements of the shoulder, arm, and forearm, as well as each segment of the body from the fingers to the toes in various combinations for the different forms of projectile use. The power of the throw is as much a product of the trunk, hips and lower limbs as it is of the upper q From the opening lines of Virgil's Aeneid. [START_REF] Toyoshima | Contribution of body parts to throwing performance[END_REF][START_REF] Pappas | Biomechanics of baseball pitching: a preliminary report[END_REF], but the shoulder and elbow joints and their associated soft tissue structures are those most often injured [START_REF] Ciccotti | Diagnosis and treatment of medial epicondylitis of the elbow[END_REF]Jobe and Cicciotti, 1984).

The torques generated by dynamic movements (i.e. those in excess of the range of normal joint motions) produce injuries in the shoulder and elbow joints of the dominant limb [START_REF] Mccue | Throwing injuries to the shoulder[END_REF][START_REF] Tullos | Examination of the throwing elbow[END_REF]. The overhead throwing motion places valgus stress on the elbow that is transmitted via the flexorpronator muscles to the medial epicondyle of the humerus, producing "valgus extension overload syndrome", a form of repetitive stress syndrome that is the most common cause of elbow injuries in the throwing athlete (Cain et al., 2003). Medial epicondylosis,1 also called "thrower's elbow" is a condition affecting the site of attachment of the medial collateral ligament and origin of the common flexor tendon at the medial epicondyle [START_REF] Baccarani | Su due casi di epitrocleite in lanciatori di giavellotto[END_REF][START_REF] Bramhall | Injuries in throwing sports[END_REF][START_REF] Ciccotti | Diagnosis and treatment of medial epicondylitis of the elbow[END_REF][START_REF] Jobe | Lateral and medial epicondylitis of the elbow[END_REF][START_REF] Miller | Javelin thrower's elbow[END_REF][START_REF] Ouellette | MR imaging of the elbow in baseball pitchers[END_REF]. It is a rare condition seen mainly in sports involving overhead throwing but also in occupational activities creating similar forces (as in hammering in carpentry) [START_REF] Ciccotti | Diagnosis and treatment of medial epicondylitis of the elbow[END_REF][START_REF] Jobe | Lateral and medial epicondylitis of the elbow[END_REF]. Lateral epicondylosis or "tennis elbow" is more common than medial epicondylosis and described in connection with numerous sports and occupational activities [START_REF] Jobe | Lateral and medial epicondylitis of the elbow[END_REF][START_REF] Shiri | Lateral and medial epicondylitis: role of occupational factors[END_REF]. When medial and lateral epicondylosis are studied together in living populations the frequency of lateral epicondylosis divided by the frequency of medial epicondylosis (the L/M ratio) is almost always greater than 1 (Table 1). Of those groups studied, baseball pitchers are the only group where the occurrence of medial epicondylosis is clearly greater than lateral epicondylosis (Table 1). Unilateral enthesopathy of the medial epicondyle has been recognized previously as a good skeletal marker of throwing and used to discuss the sexual division of labour in prehistoric groups [START_REF] Dutour | Enthesopathies (lesions of muscular insertions) as indicators of the activities of Neolithic Saharan populations[END_REF]Villotte et al., 2010a) but for small samples, and the L/M ratio has never been studied in detail. The goal of this article is to present the frequencies of lateral and medial epicondylosis in several prehistoric and historic samples and use the L/M ratio in order to discuss the sexual division of labour in activities associated with the throwing motion. Because these repeated movements affect the skeleton in a patterned way, their relative distribution in male and female skeletons provides an unrivalled opportunity to assess sexual division of labour directly from the remains of those who performed tasks.

Material and methods

The frequencies of lateral and medial epicondyle enthesopathies in several prehistoric and historic groups have been calculated, and the L/M ratio has been determined for three large samples of prehistoric, pre-industrial historic, and modern documented populations (Table 2). Two different analyses were performed: one involving the total number of observed right and left condyles, and a second of humeri with both condyles preserved and recorded. A single observer (S.V.) performed all enthesis examinations employing the same methodological criteria [START_REF] Villotte | Connaissances médicales actuelles, cotation des enthésopathies: nouvelle méthode[END_REF]Villotte et al., 2010b). These samples include adult specimens only. Age at death and sex are known for the identified skeletal collections (the modern documented populations) (see [START_REF] Villotte | Enthésopathies et activités des hommes préhistoriques -Recherche méthodologique et application aux fossiles européens du Paléolithique supérieur et du Mésolithique[END_REF] for a description of these collections). Estimation of age-atdeath and sex of the prehistoric and pre-industrial historic individuals was based on recently published methods with demonstrably good reliability (references in Villotte et al., 2010a).

Results

Fig. 1 displays the frequencies of medial and lateral epicondyle enthesopathies in the modern documented (i.e. identified) sample. Frequencies of enthesopathies increase with age, but for each age category and for both sides the L/M ratio falls into the range of variation seen for living samples (Table 1). For most of the age categories considered, enthesopathies of the lateral epicondyle are two or three times more common than they are for the medial epicondyle, as for living samples. Moreover, there is no apparent correlation between age-at-death and the ratio. For all comparisons except one, enthesopathies are more frequent for the lateral epicondyle. The ratio below 1 for the first age group for the left side, as well as the very high ratio for the 30e39 age-class, can be explained by the very low frequency of cases seen for the medial epicondyle (one in both cases).

Frequencies for the right and left sides for each collection are presented in Table 2. As there is no control for age, frequencies cannot be compared between groups, but this can be done within each sub-sample. In all modern documented and pre-industrial historic collections, enthesopathies are more common at the lateral epicondyle for both sides and both sexes. For the prehistoric groups, the situation is dramatically different. For females, medial epicondylosis is more frequent in two cases (from 12 comparisons): for the site of Stuttgart-Mühlhausen (Early Neolithic) and for the sample dating from the Early/Middle Mesolithic, a minor difference (less than 1%) is observed for the left side. For males and for each of the groups considered, the ratio clearly favours the medial epicondyle of the right side. When groups are pooled (Fig. 2) the L/M ratio is always positive and varies from 1.8 to 3.4, except in the right side of the male prehistoric group (0.6), indicating that, although the lateral epicondyle is affected, it is more often accompanied by medial epicondylar enthesopathies in these males than for their same sex counterparts from more recent times and for females. When only humeri with both medial and lateral epicondyles scored are taken into account and when only one enthesopathy is recorded, there is a significant tendency for the lesion to occur at the lateral epicondyle for all groups and both sides, except for prehistoric males for the right side (Table 3).

Discussion

Although entheseal changes are age-related (e.g. [START_REF] Villotte | Enthésopathies et activités des hommes préhistoriques -Recherche méthodologique et application aux fossiles européens du Paléolithique supérieur et du Mésolithique[END_REF]Villotte et al., 2010b), for the L/M ratio there is no associated age correlation (Fig. 1). We did not attempt to control for age-at-death in the present study due the number of individuals for which it had not been possible to assess it, or for which the assessment provided a very broad age range. Thus, frequencies of medial or lateral epicondylosis cannot be compared across samples, but the L/M ratio can. Moreover, the present study clearly shows that this ratio is almost always greater than 1 in the archaeological and clinical samples. The L/M ratio for the right side in the prehistoric male sample, below the range of variation seen for historic samples presented here, as well as in clinical samples (Table 1), highlights a specific pattern for the right arm. This very low L/M ratio is seen today only in young baseball pitchers [START_REF] Lyman | Longitudinal study of elbow and shoulder pain in youth baseball pitchers[END_REF]. This provides strong evidence for the mechanical origin of these lesions, as the pattern would have been seen for both sides if they had resulted from hormonal or other intrinsic factors. Medial epicondyle enthesopathy frequently co-occurs with lateral epicondyle enthesopathy when it is unrelated to the throwing motion [START_REF] Gold | Specific and non-specific upper extremity musculoskeletal disorder syndromes in automobile manufacturing workers[END_REF][START_REF] Walker-Bone | Prevalence and impact of muskuloskeletal disorders of the upper limb in the general population[END_REF]. When only humeri with both medial and lateral epicondyles scored are taken into account, and when only one enthesopathy (medial or lateral) is recorded, the right medial epicondyle tends to be more often injured in Please cite this article in press as: Villotte, S., Knüsel, C.J., "I sing of arms and of a man.": medial epicondylosis and the sexual division of labour in prehistoric Europe, Journal of Archaeological Science (2014), http://dx.doi.org/10.1016/j.jas.2013.12.009 prehistoric males (Table 3). Considering the high specificity of medial epicondyle injuries today, we suggest that the unilateral pattern of medial epicondylosis indicates that most of these lesions were likely due to the throwing motion. Cross-culturally, humans employ a single hand for tasks demanding skilled use of implements. This is most often the right hand [START_REF] Coren | Fifty centuries of right-handedness: the historical record[END_REF][START_REF] Calvin | A stone's throw and its launch window: timing precision and its implications for language and hominid brains[END_REF][START_REF] Faurie | Handedness frequency over more than ten thousand years[END_REF][START_REF] Steele | When did directional asymmetry enter the record?[END_REF], and our study reveals that this was the same in the past. Today, ballistic skills, as opposed those requiring fine motor control, disproportionately employ the right hand [START_REF] Annett | A classification of hand preference by association analysis[END_REF]. Within each prehistoric sample, the frequency of medial versus lateral epicondylar enthesopathies is higher in males. The consistency across these groups of hunter-gatherers and farmers provides strong support for a universal biocultural phenomenon linked to throwing, rather than being an artefact of sampling bias.

Hunting was essential during the Upper Palaeolithic and Mesolithic, but also appears to have played a considerable role in European Neolithic groups, providing dietary supplements and as a prestige-enhancing means of social distinction [START_REF] Hachem | La conception du monde animal sauvage chez les éleveurs du Rubané[END_REF][START_REF] Hachem | Les faunes du Néolithique moyen dans le nord de la France: bilan et pistes de recherches[END_REF][START_REF] Jeunesse | Changements sociaux et signification de la chasse dans les sociétés du Néolithique circumalpin. Une hypothèse alternative au déterminisme écologique[END_REF][START_REF] Uerpmann | Animaux sauvages et domestiques du Rubané "le plus ancien" (LBK 1) en Allemagne[END_REF]. The use of complex projectile weapons, like the bow and arrow, spear-thrower, and dart, which do not seem to especially stress the medial epicondyle (see Villotte et al., 2010 and references therein), is attested in Europe for the latter part of the Upper Palaeolithic, Mesolithic and Neolithic Fig. 2. L/M ratios for the prehistoric, pre-industrial historic, and modern documented samples. Full line and dashed lines: 25, 50th, and 75th percentiles calculated from L/M ratios from clinical studies (Table 1), excepted the group of pitchers (for which the ratio is presented by a dotted line). Please cite this article in press as: Villotte, S., Knüsel, C.J., "I sing of arms and of a man.": medial epicondylosis and the sexual division of labour in prehistoric Europe, Journal of Archaeological Science (2014), http://dx.doi.org/10.1016/j.jas.2013.12.009 [START_REF] Cattelain | Apparition et évolution de l'arc et des pointes de flèches dans la préhistoire européenne (Paléo-, Méso-, Néolithique)[END_REF][START_REF] Cattelain | La chasse dans la Préhistoire du Paléolithique au Néolithique en Europe[END_REF][START_REF] Clark | Neolithic bows from Somerset, England, and the prehistory of archery in North-Western Europe[END_REF][START_REF] Rozoy | Le mode de vie au Mésolithique[END_REF][START_REF] Shea | The origins of lithic projectile point technology: evidence from Africa, the Levant and Europe[END_REF]. However, this does not exclude the use of more simple technology simultaneously: barbed points, javelins, killing sticks and boomerangs are known for the same period [START_REF] Bahn | Return of the Euro-boomerang[END_REF][START_REF] Burov | The use of vegetable materials in the Mesolithic of Northeast Europe[END_REF][START_REF] Cattelain | La chasse dans la Préhistoire du Paléolithique au Néolithique en Europe[END_REF][START_REF] Valde-Nowak | Upper Palaeolithic boomerang made of a mammoth tusk in south Poland[END_REF]. Even thrown stones are highly effective in hunting and warfare [START_REF] Isaac | Throwing and human evolution[END_REF]. As documented in ethnographic literature (see [START_REF] Churchill | Weapon technology, prey size selection, and hunting methods in modern hunter-gatherers: implications for hunting in the Palaeolithic and Mesolithic[END_REF] for a review), hand-thrown projectiles likely formed a frequent complement to the use of more complex weaponry in the prehistoric groups studied here. Thus, it appears that most cases of medial epicondylosis observed for these individuals are related to the use of overhead throwing during games, warfare, and hunting. Overhead throwing with force and accuracy is a skill uniquely developed in our species that played an important role in human evolution [START_REF] Calvin | A stone's throw and its launch window: timing precision and its implications for language and hominid brains[END_REF][START_REF] Darlington | Group selection, altruism, reinforcement, and throwing in human evolution[END_REF][START_REF] Darwin | The Descent of Man, and Selection in Relation to Sex[END_REF][START_REF] Isaac | Throwing and human evolution[END_REF][START_REF] Knüsel | Throwing and hominid evolution[END_REF]. Effective throwing behaviour (probably of stones initially) may date as far back as 4 million years ago [START_REF] Calvin | A stone's throw and its launch window: timing precision and its implications for language and hominid brains[END_REF][START_REF] Darlington | Group selection, altruism, reinforcement, and throwing in human evolution[END_REF][START_REF] Isaac | Throwing and human evolution[END_REF]. Today, males perform significantly better than females in spatial ability tests, as in gauging movements across the visual field [START_REF] Jardine | Spatial ability and throwing accuracy[END_REF][START_REF] Voyer | Magnitude of sex differences in spatial abilities: a meta-analysis and consideration of critical variables[END_REF], as well as in both throwing velocity and accuracy exercises [START_REF] Jardine | Spatial ability and throwing accuracy[END_REF][START_REF] Tillaar | Effect of body size and gender in overarm throwing performance[END_REF][START_REF] Whittaker | Primitive weapons and modern sport: atlatl capabilities, learning, gender, and age[END_REF]. Significant correlations have been found between throwing accuracy and spatial ability, and these sex differences may reflect the evolutionary adaptations required of male hunters and later as armed warriors [START_REF] Jardine | Spatial ability and throwing accuracy[END_REF][START_REF] Kolakowski | Spatial ability, throwing accuracy and man's hunting heritage[END_REF]. Because this proclivity appears to form a fundamental part of masculine identity, prolonged training and socialization to achieve proficiency may be implicated in the occurrence of observed medial epicondyle enthesopathies.

Other activities may be associated with some of the medial epicondylosis seen in prehistoric males. The sling, an ancient implement used by herders to tend flocks, farmers to protect crops from scavengers e and as a weapon especially well known in the classical ancient world [START_REF] Paunov | New data on the use of war sling in Thrace (4th-1st century BC)[END_REF][START_REF] Dohrenwend | The sling: forgotten firepower of antiquity[END_REF] e suggests one such activity, with evidence for what may be sling stones from prehistoric sites worldwide [START_REF] Finney | Middle Iron Age Warfare of the Hillfort Dominated Zone c[END_REF][START_REF] Rosenberg | Flying stones e the slingstones of the Wadi Rabah Cuture of the southern Levant[END_REF][START_REF] Vega | New experimental data on the distance of sling projectiles[END_REF], including the European Neolithic [START_REF] Paunov | New data on the use of war sling in Thrace (4th-1st century BC)[END_REF]. Interestingly, as for other throwing activities men have a greater range in sling use [START_REF] Vega | New experimental data on the distance of sling projectiles[END_REF]. The intensive use of axes and adzes, objects which are well known from the Neolithic and throughout the Mesolithic (e.g. [START_REF] Valdeyron | Derniers chasseurs et premiers bûcherons? La question des haches et des herminettes dans le Mésolithique européen[END_REF], may also have produced valgus stress in the medial compartment of the humerus similar to that seen in overhead throwing. Unilateral wood-working is a masculine activity in non-industrial societies (the average percentage of male participation calculated from 185 societies is 98.8%; [START_REF] Murdock | Factors in the division of labor by sex: a crosscultural analysis[END_REF], and it is noteworthy that thick-bladed adzes are found almost exclusively in male burials from the Early Neolithic Linearbandkeramik [START_REF] Bakels | On the adzes of the Northwestern Linearbandkeramik[END_REF], perhaps as a means of signalling masculine identity.

Conclusion

Sexual division in the use of throwing weapons is one of the most striking universal occurrences noted in cross-cultural studies [START_REF] Murdock | Factors in the division of labor by sex: a crosscultural analysis[END_REF][START_REF] Testart | Essai sur les fondements de la division sexuelle du travail chez les chasseurs-cueilleurs[END_REF][START_REF] Wood | A cross-cultural analysis of the behavior of women and men: implications for the origins of sex differences[END_REF]. Our results indicate that sex differences indicative of this proclivity date back at least 30,000 years. Moreover, this sexual division of labour appears to have been a persistent feature of human groups through millennia and across societies with radically different subsistence economies and living a diverse range of environments. An ability to throw with accuracy and at increasing distance may represent one of the fundamental structuring factors in past human societies, with both biological and social implications.

Uncited references

Churchill and Rhodes, 2009;[START_REF] Dunsworth | Throwing and bipedalism: a new look at an old idea[END_REF][START_REF] Knüsel | Bone adaptation and its relationship to physical activity in the past[END_REF][START_REF] Rhodes | Throwing in the Middle and Upper Paleolithic: inferences from an analysis of humeral retroversion[END_REF] Please cite this article in press as: Villotte, S., Knüsel, C.J., "I sing of arms and of a man.": medial epicondylosis and the sexual division of labour in prehistoric Europe, Journal of Archaeological Science (2014), http://dx.doi.org/10.1016/j.jas.2013.12.009

Fig. 1 .

 1 Fig. 1. Modern documented sample (four collections of individuals of known sex and age at death). Frequencies of enthesopathies and L/M ratios (triangles) for each age group, Top: right side, down: left side.

Table 1

 1 Reported prevalence rates of epicondylosis in modern clinical groups performing various trades, professions, and activities. The higher lesion frequencies for lateral or medial epicondyles is indicated in bold.Please

	Population

cite this article in press as: Villotte, S., Knüsel, C.J., "I sing of arms and of a man.": medial epicondylosis and the sexual division of labour in prehistoric Europe, Journal of Archaeological Science (2014), http://dx.doi.org/10.1016/j.jas.2013.12.009

Table 2

 2 Frequencies of lateral and medial epicondylosis for each sub-sample. The higher lesion frequencies for lateral or medial epicondyles is indicated in bold.

		Left epicondyles	N total and % L/M	ratio Lateral Medial	49 36.7% 50 12.0% 3.1	133 11.3% 112 7.1% 1.6	120 10.8% 114 7.0% 1.5	65 29.2% 56 12.5% 2.3	367 17.7% 332 8.7% 2.0		16 56.3% 14 28.6% 2.0	34 47.1% 39 20.5% 2.3	16 18.8% 18 16.7% 1.1	66 42.4% 71 21.1% 2.0		9 22.2% 11 18.2% 1.2	9 22.2% 10 0.0% /	29 24.1% 31 6.5% 3.7	15 6.7% 15 6.7% 1.0	10 20.0% 10 0.0% /		17 11.8% 17 0.0% /	89 18.0% 94 5.3% 3.4
	Males	Right epicondyles	N total and % L/M	ratio Lateral Medial	53 39.6% 46 19.6% 2.0	119 20.2% 121 6.6% 3.1	110 20.9% 110 8.2% 2.5	64 39.1% 56 8.9% 4.4	346 26.9% 333 9.3% 2.9		11 72.7% 16 43.8% 1.7	32 68.8% 36 33.3% 2.1	20 25.0% 17 11.8% 2.1	63 55.6% 69 30.4% 1.8		15 26.7% 15 53.3% 0.5	11 18.2% 11 27.3% 0.7	35 28.6% 36 36.1% 0.8	19 0.0% 24 12.5% 0.0	11 27.3% 9 33.3% 0.8		15 6.7% 16 25.0% 0.3	106 18.9% 111 30.6% 0.6
		Left epicondyles	N total and % L/M	ratio Lateral Medial	60 70.0% 55 34.5% 2.0	124 21.8% 111 14.4% 1.5	/	/	184 37.5% 166 21.1% 1.8		96 53.1% 85 21.2% 2.5	34 58.8% 41 31.7% 1.9	7 28.6% 8 12.5% 2.3	137 53.3% 134 23.9% 2.2		20 25.0% 21 23.8% 1.1	14 35.7% 15 13.3% 2.7	24 8.3% 22 9.1% 0.9	22 22.7% 21 9.5% 2.4	12 8.3% 11 9.1% 0.9		9 11.1% 10 0.0% /	101 18.8% 100 12.0% 1.6
	Collections Periods Females	Right epicondyles	N total and % L/M	ratio Lateral Medial	Spitalfields (England) 18e20th c. AD 67 68.7% 64 39.1% 1.8	Coimbra (Portugal) 131 35.1% 118 16.1% 2.2	Sassari (Italy) /	Bologna (Italy) /	Total Modern Documented 198 46.5% 182 24.2% 1.9	Sample	Post-medieval (France) 16e18th c. AD 106 59.4% 92 33.7% 1.8	Medieval (England) 10e11th c. AD 39 64.1% 40 45.0% 1.4	Antiquity (France) 2nd-3rd c. AD 4 50.0% 6 0.0% /	Total Pre-industrial Historic 149 60.4% 138 35.5% 1.7	Sample	Middle Neolithic (Switzerland) 3,2e2700 BC 21 42.9% 18 22.2% 1.9	Middle Neolithic (France) 4,5e4000 BC 12 41.7% 16 12.5% 3.3	Early Neolithic (Germany) 5000 BC 25 20.0% 28 10.7% 1.9	Late Mesolithic (Portugal) 6,4e5300 BC 27 29.6% 42 11.9% 2.5	Early/Middle Mesolithic 10e7000 BC 14 21.4% 11 9.1% 2.4	(Europe)	Upper Palaeolithic (Europe) 30e10,000 BC 10 10.0% 10 0.0% /	Total Prehistoric Sample 109 28.4% 125 12.0% 2.4
	Samples				Modern Documented	Sample (223 females	and 430 males)				Pre-industrial Historic	Sample (211 females	and 89 males)			Prehistoric Sample	(169 females and	139males)

  Please cite this article in press as: Villotte, S., Knüsel, C.J., "I sing of arms and of a man.": medial epicondylosis and the sexual division of labour in prehistoric Europe, Journal of Archaeological Science (2014), http://dx.doi.org/10.1016/j.jas.2013.12.009Please cite this article in press as: Villotte, S., Knüsel, C.J., "I sing of arms and of a man.": medial epicondylosis and the sexual division of labour in prehistoric Europe, Journal of Archaeological Science (2014), http://dx.doi.org/10.1016/j.jas.2013.12.009

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Please cite this article in press as: Villotte, S., Knüsel, C.J., "I sing of arms and of a man.": medial epicondylosis and the sexual division of labour in prehistoric Europe, Journal of Archaeological Science (2014), http://dx.doi.org/10.1016/j.jas.2013.12.009

Contrary to "epicondylitis", the more neutral term "epicondylosis" does not suggest inflammation and is thus more appropriate (even if less used). As in most of the cases of medial or lateral epicondyle enthesopathies there is no histopathological evidence of either acute or chronic inflammation[START_REF] Khan | Histopathology of common tendinopathies. Update and implications for clinical management[END_REF].

Acknowledgements

The authors thank the two reviewers for their useful comments. S.V. thanks the many curators across Europe who allowed him access to the material.

Grant sponsors: Maison des Sciences de l'Homme d'Aquitaine; European Union (Synthesys GB-TAF-636); CNRS; Ministère de l'Enseignement Supérieur et de la Recherche (PhD research grant, and "Aires Culturelles"); Fondation Fyssen (Post doctoral research fellowship), British Academy (Small Research Grant).