Silvia La Monica

Maricla Galetti

Roberta R Alfieri
email: roberta.alfieri@unipr.it

Andrea Cavazzoni

Andrea Ardizzoni

Marcello Tiseo

Marzia Capelletti

Matteo Goldoni

Sara Tagliaferri

Antonio Mutti

Claudia Fumarola

Mara Bonelli

Daniele Generali

Pier Giorgio Petronini

Everolimus restores gefitinib sensitivity in resistant non-small cell lung cancer cell lines

Keywords: Lung cancer, EGFR, gefitinib, everolimus

A c c e p t e d M a n u s c r i p t

Introduction

Lung cancer is the main cause of cancer deaths in the developed world in both men and women, and non-small cell lung cancer (NSCLC) accounts for about 80-85% of all lung cancers [START_REF] Jemal | Cancer statistics[END_REF]. Current treatments, including chemotherapy, radiotherapy and surgery, have provided only limited improvement in the natural history of the disease. New treatments are thus needed to improve survival of NSCLC patients [START_REF] Carney | Lung cancer--time to move on from chemotherapy[END_REF].

Inhibition of the Epidermal Growth Factor Receptor (EGFR) pathway has been proposed as a novel molecular targeted approach for NSCLC. Two main strategies targeting EGFR have been developed: small-molecule inhibitors of the tyrosine kinase domain (tyrosine kinase inhibitors [TKIs], as erlotinib and gefitinib), and monoclonal antibody (cetuximab), directed against the extracellular domain of EGFR, that inhibits its phosphorylation/activation and stimulates internalization [START_REF] Bunn | Current therapeutic approaches in lung cancer[END_REF].

Clinical data have shown that 10-20% of patients with refractory advanced NSCLC responded to gefitinib or erlotinib [START_REF] Giaccone | Epidermal growth factor receptor inhibitors in the treatment of nonsmall-cell lung cancer[END_REF]. A higher probability of response appears to be associated with certain biological and clinical characteristics (such as adenocarcinoma histotype, female sex, never smoking status and Asian ethnic origin) and with biological features of the tumor [START_REF] Ciardiello | EGFR antagonists in cancer treatment[END_REF].

In particular, molecular analysis showed that in most cases, responders harbored specific mutations in the region that encodes the catalytic domain of EGFR [START_REF] Sharma | Epidermal growth factor receptor mutations in lung cancer[END_REF].

By contrast, acquired resistance to EGFR TKIs involves in same cases the recurrent T790M mutation, which affects the catalytic domain of the kinase weakening the interaction of the inhibitor with its target [START_REF] Pao | Acquired resistance of lung adenocarcinomas to gefitinib or erlotinib is associated with a second mutation in the EGFR kinase domain[END_REF]. Tumor cells can develop other mechanisms of resistance to EGFR TKIs such as the activation of alternative tyrosine kinase receptors (IGF-1R), amplification of the MET gene and constitutive activation of signaling pathways downstream of EGFR (i.e. K-RAS mutations) [START_REF] Camp | Molecular mechanisms of resistance to therapies targeting the epidermal growth factor receptor[END_REF][START_REF] Engelman | Acquired resistance to tyrosine kinase inhibitors during cancer therapy[END_REF].

A c c e p t e d M a n u s c r i p t 4 Two major intracellular signaling pathways activated by EGFR, the PI3K/AKT/mTOR and the RAS/RAF/MAPK cascades, have a central role in controlling cell survival and modulating cell growth and proliferation [START_REF] Jorissen | Epidermal growth factor receptor: mechanisms of activation and signalling[END_REF]. These pathways represent potential novel therapeutic targets.

NSCLC cell lines responsive to EGFR TKIs with growth arrest or apoptosis show a downregulation of the PI3K/AKT/mTOR pathway [START_REF] Engelman | ErbB-3 mediates phosphoinositide 3-kinase activity in gefitinib-sensitive non-small cell lung cancer cell lines[END_REF]. However, deregulation of the AKTdependent pathway has been well documented in a variety of human tumors [START_REF] Hay | The Akt-mTOR tango and its relevance to cancer[END_REF] and has been associated with resistance to EGFR TKIs in NSCLC cell lines [START_REF] Morgillo | Resistance to epidermal growth factor receptor-targeted therapy[END_REF]. Deregulation of the PI3K/AKT/mTOR pathway could result from several alterations, including PI3K isoform gene amplification, activating mutations of PI3K subunits, AKT gene amplification and overexpression, as well as loss of function of PTEN [START_REF] Camp | Molecular mechanisms of resistance to therapies targeting the epidermal growth factor receptor[END_REF][START_REF] Marinov | Targeting mTOR signaling in lung cancer[END_REF]. Frequent AKT activation and mTOR phosphorylation were found in 51% of NSCLC patients and in 74% of NSCLC cell lines [START_REF] Balsara | Frequent activation of AKT in non-small cell lung carcinomas and preneoplastic bronchial lesions[END_REF].

The inhibition of mammalian target of rapamycin mTOR signaling pathway could be a promising therapeutic option. Everolimus (RAD001) is an orally bioavailable derivative of rapamycin that is in current use as a post-transplant immunosuppressive agent and has been shown to inhibit the proliferation of tumor cell growth in preclinical studies, and phase I/II clinical trials in lung cancer are under way. In particular, at a daily dose of 10 mg, Everolimus was reported to be active and safe in pretreated advanced NSCLC patients [START_REF] Johnson | Rationale for a phase I trial of erlotinib and the mammalian target of rapamycin inhibitor everolimus (RAD001) for patients with relapsed non small cell lung cancer[END_REF][START_REF] Papadimitrakopoulou | A phase II study of RAD001 (R) (everolimus) monotherapy in patients with advanced non-small cell lung cancer (NSCLC) failing prior platinum-based chemotherapy (C) or prior C and EGFR inhibitors (EGFR-I)[END_REF]. The crucial role of the constitutive activation of the PI3K/AKT pathway in the development and maintenance of an EGFR-resistant phenotype supports the hypothesis that a combination of EGFR TKIs and mTOR inhibitors may be the best choice for treatment. Clinical trials testing this strategy in advanced NSCLC are ongoing and only preliminary results are available [START_REF] Gridelli | The potential role of mTOR inhibitors in non-small cell lung cancer[END_REF][START_REF] Milton | Phase 1 trial of everolimus and gefitinib in patients with advanced nonsmall-cell lung cancer[END_REF][START_REF] Riely | Prospective assessment of discontinuation and reinitiation of erlotinib or gefitinib in patients with acquired resistance to erlotinib or gefitinib followed by the addition of everolimus[END_REF].

Materials and methods

Cell culture

The human NSCLC cell lines H292, H322, H460, H1299, H1975, H596, H1650, Calu-1, Calu-3, SKMES-1 and SKLU-1 were cultured as recommended. All media were supplemented with 2 mM glutamine, 10% fetal bovine serum (FBS Gibco, Life Technologies). Cell lines were from the American Type Culture Collection (Manassas, VA, USA) and were maintained under standard cell culture conditions at 37°C in a water-saturated atmosphere of 5% CO 2 in air.

Drug treatment

Gefitinib (ZD1839/ Iressa) was synthesized as described elsewhere [START_REF] Carmi | 5-benzylidenehydantoins as new EGFR inhibitors with antiproliferative activity[END_REF]. Everolimus (RAD001) was provided by Novartis International AG (Basel, Switzerland). In all assays, the drugs were dissolved in DMSO immediately before the addition to cell cultures. The concentration of DMSO never exceeded 0.1% (v/v) and equal amounts of the solvent were added to control cells.

Antibodies and reagents

Monoclonal anti-EGFR, polyclonal anti-phospho-EGFR (tyr1068), polyclonal anti-phospho-mTOR (ser2248), polyclonal anti-phospho-AKT (ser273), polyclonal anti-AKT, polyclonal anti-phospho-S6K (thr389), polyclonal anti-S6K, monoclonal anti-phospho-p44/42 MAPK, monoclonal anti-p44/42 MAPK, polyclonal anti-PI3K, polyclonal anti-PTEN antibodies were from Cell Signaling Technology (Beverly, MA, USA). Monoclonal anti-actin (AC-40) antibody was from Sigma-Aldrich (St. Louis, MO, USA). Horseradish peroxidase-conjugated (HRP) secondary antibodies and the enhanced chemiluminescence system (ECL) were from A c c e p t e d M a n u s c r i p t 7 Millipore (Millipore, MA, USA). Reagents for electrophoresis and blotting analysis were obtained from BIO-RAD Laboratories.

Western blot analysis

Procedures for protein extraction, solubilization, and protein analysis by 1-D PAGE are described elsewhere [START_REF] Cavazzoni | Dosedependent effect of FHIT-inducible expression in Calu-1 lung cancer cell line[END_REF]. Briefly, 30-50 µg proteins from lysates were resolved by 5-15% SDS-PAGE and transferred to PDVF membranes. The membranes were then incubated with primary antibody, washed and then incubated with HRP-anti-mouse or HRP-anti-rabbit antibodies. Immunoreactive bands were visualized using an enhanced chemiluminescence system.

Determination of cell growth

Cell number was evaluated by cell counting in a Burker hemocytometer by trypan blue exclusion and by crystal violet staining as described [START_REF] Gillies | Determination of cell number in monolayer cultures[END_REF]. In brief, cells were fixed with 1% formaldehyde and stained with 0.1% crystal violet in PBS. The unbound dye was removed by washing with water. Bound crystal violet was solubilized with 0.2% TritonX-100 in PBS.

Light extinction which increases linearly with the cell number was analyzed at 570 nm. Proliferation rates were estimated by the growth rate quotient as calculated by the equation: GRQ= (N-N 0 )/ t*1/N 0 ; doubling time (hr) was calculated according to the formula t*ln(2)/ln(N/N 0 ), where N is the final cell number; N 0 is the initial cell number and t is the time elapsed between the two counting.

MTT and colony formation by viable cells were determined as described elsewhere [START_REF] Cavazzoni | Dual mechanisms of action of the 5-benzylidene-hydantoin UPR1024 on lung cancer cell lines[END_REF]. (detection of cleavage products), by Western Blotting procedure as previously described [START_REF] Cavazzoni | Dual mechanisms of action of the 5-benzylidene-hydantoin UPR1024 on lung cancer cell lines[END_REF].

Detection of apoptosis

Statistical Analysis

Using a non-linear regression fit method (Levemberg-Marquardt algorithm), we calculated the dose-response curves of single compounds from the Hill function [START_REF] Jemal | Cancer statistics[END_REF]: [START_REF] Jemal | Cancer statistics[END_REF] where %IN is the % growth inhibition, %IN 0 the amplitude of the signal (e.g. the saturation growth inhibition value), k is the value where %IN was the half of %IN 0 , and n the cooperativity index, which takes into account of possible affinity changes of target sites [START_REF] Goldoni | Models of neurotoxicity: extrapolation of benchmark doses in vitro[END_REF].

In the case that %IN 0 did not reach 100%, we calculated from the fitting parameters the value of IC 50 as that point in which 50% growth inhibition was expected by fitting curve.

Under the hypothesis that everolimus and gefitinib bind at different target sites and can be considered as independent, to study the combined effects of drugs we used the Bliss independence criterion for in vitro co-exposure.

The Bliss criterion is expressed by the following equation: [START_REF] Carney | Lung cancer--time to move on from chemotherapy[END_REF] where E(x) is the effect of the concentration x of the first compound (between 0 and 1), E(y)

E(x,y) = E(x) + E(y) -E(x)*E(y),

the same for the second compound at concentration y, and E(x,y) is the combined effect .

) ( % % 0 n n n x k x IN IN   A c c e p t e d M a n u s c r i p t 9
If the combination effect is higher than the expected value from [START_REF] Carney | Lung cancer--time to move on from chemotherapy[END_REF], the interaction is synergistic, while if this effect is lower, the interaction is antagonistic. Otherwise, the effect is additive and there is no interaction between drugs.

To test the differences between experimental and theoretical points, we applied the method suggested by Goldoni and Johansson [START_REF] Goldoni | A mathematical approach to study combined effects of toxicants in vitro: evaluation of the Bliss independence criterion and the Loewe additivity model[END_REF]. Briefly, the theoretical value was calculated by the experimental points of single dose-response curves [E(x) and E(y), respectively], and its SD was estimated by error propagation of experimental SD calculated separately on E(x) and E(y). Finally, the significance of the differences between theoretical and experimental values was assessed by Student t tests [START_REF] Goldoni | A mathematical approach to study combined effects of toxicants in vitro: evaluation of the Bliss independence criterion and the Loewe additivity model[END_REF]. For all non-linear fits, Origin 6.0 (Originlab, Northampton, USA) was used. For all the statistical tests, SPSS 15.0 (SPSS, Chicago, USA) was used. A p value of 0.05 was always considered as significant.

A c c e p t e d M a n u s c r i p t 10

Results

The IC 50 values for gefitinib in the 11 NSCLC cell lines were determined by MTT assay as previously reported [START_REF] Cavazzoni | Dual mechanisms of action of the 5-benzylidene-hydantoin UPR1024 on lung cancer cell lines[END_REF][START_REF] Tiseo | Epidermal growth factor receptor intron-1 polymorphism predicts gefitinib outcome in advanced non-small cell lung cancer[END_REF]; cells showing IC 50 <1 M were considered sensitive (H322, H292, Calu-3) and cell lines with IC 50 >7 M (SKLU-1, Calu-1, H1650, H596, H1975, H1299, SKMES-1, H460) were considered resistant (Table 1).

Cell lines were also characterized for EGFR, K-RAS, PI3K gene and PTEN protein status. The H1975 cell line carrying the L858R and T790M mutations was resistant to gefitinib [START_REF] Pao | Acquired resistance of lung adenocarcinomas to gefitinib or erlotinib is associated with a second mutation in the EGFR kinase domain[END_REF]; the H1650 cell line, despite harboring a delE746_A750 activating mutation in the exon 19 of the EGFR gene, was resistant to gefitinib and erlotinib as previously reported [START_REF] Van Schaeybroeck | Chemotherapy-induced epidermal growth factor receptor activation determines response to combined gefitinib/chemotherapy treatment in non-small cell lung cancer cells[END_REF][START_REF] Deng | Proapoptotic BH3-only BCL-2 family protein BIM connects death signaling from epidermal growth factor receptor inhibition to the mitochondrion[END_REF][START_REF] Guo | Signaling networks assembled by oncogenic EGFR and c-Met[END_REF]; K-RAS mutations were found in four of the tested resistant cell lines.

We did not observe any basal expression of PTEN protein in the H1650 cell line as also reported by Janmaat et al [START_REF] Janmaat | Enhanced cytotoxicity induced by gefitinib and specific inhibitors of the Ras or phosphatidyl inositol-3 kinase pathways in non-small cell lung cancer cells[END_REF].

We first evaluated the effect of gefitinib on EGFR autophosphorylation on Calu-3 (sensitive) and H1650 and SKLU-1 (both resistant) cell lines. As shown in Fig. 1A, in all the cell lines tested, EGF stimulation induced a significant phosphorylation of tyr1068 as evaluated by Western blot analysis. Gefitinib inhibited EGFR autophosphorylation starting from 0.01 M in Calu-3 and H1650 and from 0.1 M in SKLU-1 cells. At 1 M the inhibition was almost complete in both sensitive and resistant cell lines indicating that other mechanisms were involved in the different sensitivity to gefitinib. Moreover, EGFR levels were not related to gefitinib responsiveness, for example Calu-3, a sensitive cell line showed similar EGFR levels to those of SKMES-1, a highly resistant cell line (see Fig. 1B).

We then analyzed (Fig. 1B) the AKT/mTOR and MAPK pathways in cells exposed to 1 M gefitinib, a concentration that completely inhibits EGFR autophosphorylation. In the sensitive cell lines Calu-3, H292, H322, gefitinib inhibited the phosphorylation of p44/42 MAPK, AKT A c c e p t e d M a n u s c r i p t 11 and S6K; in contrast, in the H460, SKMES-1, H1299, H596 and H1650 cell lines, gefitinib inhibited the phosphorylation of p44/42 MAPK but had little influence on S6K phosphorylation. The T790M mutation in the H1975 cell line conferred resistance to gefitinib enhancing the receptor catalytic activity [START_REF] Yun | The T790M mutation in EGFR kinase causes drug resistance by increasing the affinity for ATP[END_REF], indeed in this cell line no difference was observed between untreated and gefitinib-treated cells. Surprisingly, in both Calu-1 and SKLU-1 (resistant cell lines) gefitinib was effective in reducing the activity of the two signaling pathways analyzed.

We selected the five cell lines (H460, H596, H1299, H1650, SKMES-1) characterized by maintaining S6K phosphorylation after gefitinib treatment, and we evaluated the effect of everolimus on the AKT/mTOR pathway and on cancer cell growth.

The effect of everolimus on the mTOR signaling pathway is shown in Fig. 2A. In cells exposed for 24 hr to 100 nM everolimus, S6K phosphorylation was completely inhibited, whereas p-p44/42 MAPK were not affected. A concomitant increase in AKT phosphorylation was observed in H460, H596 and SKMES-1 cells. We then determined cell number by crystal violet staining on cells treated for 6 days with increasing concentrations of drug from 0.01 to 100 nM. As shown in Fig. 2B, everolimus inhibited in a dose-dependent manner cell proliferation, although to different extents in the different cell models. No apoptotic morphology was observed even when the concentration of everolimus was increased to 1 M (result not shown). It is of note that SKMES-1 and H1650, showing higher basal level of p-AKT, were highly sensitive to everolimus.

To further explore the relationship between the inhibition of AKT/mTOR signaling and the different sensitivity to everolimus, we examined p-AKT, p-S6K, and p-p44/42 MAPK in H1299 (IC 50 : 2.3 nM) and in SKMES-1 (IC 50 : 0.14 nM) exposed to different concentrations of everolimus, ranging from 0.01 to 100 nM (Fig. 2C). The phosphorylation of S6K was inhibited by everolimus in a dose-dependent manner with an almost complete inhibition at 1 In an attempt to restore sensitivity to gefitinib of resistant cell lines we evaluated the effect of a combined treatment with gefitinib with everolimus. As shown in Fig. 3, gefitinib inhibited MAPK signaling, but failed to prevent the activation of S6K, whereas everolimus caused a complete suppression of S6K phosphorylation without influencing MAPK activation.

Combined treatment suppressed both signaling pathways in all the cell models tested.

The pure dose-response curves of growth inhibition of H460, SKMES-1, H1299, H596 and H1650 in the presence of gefitinib or everolimus are illustrated in Fig. 4. We also show in this figure, the dose-response curves of gefitinib in the presence of a fixed concentration of everolimus for all cell lines. The fixed concentration of everolimus was 0.01-1 nM depending on the cell line. However, different concentrations of everolimus combined with a fixed gefitinib concentration gave similar results in term of synergism or additivity (results not shown).

Comparing the experimental combination points with that expected by the Bliss criterion, only additive effects were observed with the H596 and H1650 cell lines. In fact, no significant differences between experimental and theoretical points were observed. By contrast, synergistic effects on growth inhibition were observed for H1299 at gefitinib 1 M (p<0.05), 5 M (p<0.01) and 10 M (p<0.01), for H460 with all tested gefitinib concentrations (1 M p<0.05, 2.5-5-10-20 M, p<0.01) and finally for SKMES-1 at gefitinib concentrations of 2.5-5 M (p<0.05). Clonogenic assay performed on H460 cells confirmed the results obtained by crystal violet staining (Fig. 5).

A c c e p t e d M a n u s c r i p t 13

In the three cell lines sensitive to gefitinib (H322, H292 and Calu-3) as well as in the resistant Calu-1 and SKLU-1 cell lines (not maintaining S6K phosphorylation following gefitinib treatment) Bliss analysis showed that gefitinib is additive with everolimus (data not shown).

The effect of the combination of gefitinib and everolimus as a function of the protocol was then analyzed. The efficacy of the two drugs in combination was measured after six days of treatment adopting different schedules of administration ("simultaneous", "gefitinib before", "gefitinib after", "gefitinib before simultaneous" and "everolimus before simultaneous"). In SKMES-1 and H1650 cells, the "simultaneous" schedule was the only one producing synergistic or additive effects on cell proliferation (data not shown).

Considering that the cell lines analyzed showed a different growth rate quotient with significantly different doubling times (Fig. 6A), we checked whether different proliferation rates of the cells affected the growth inhibition induced by the combined treatment. Growth rate was varied by modifying the number of SKMES-1 cells initially plated. SKMES-1 were seeded at different cell densities, treated with everolimus and gefitinib for 3 days and growth inhibition was then evaluated and plotted as a function of growth rate quotient. As shown in Fig. 6B, cells having a higher value of GRQ were more responsive to the combined treatment. This result is consistent with the lack of apoptosis (evaluated by caspase-3 activation and morphological analysis) under any of the tested treatments at all time points analyzed suggesting that the combined everolimus-gefitinib treatment exerts, at least until ten days, a cytostatic and not a cytotoxic effect. Taken together, these results indicate that in selected high proliferating non-small cell lung cancer cell lines the combination of gefitinib with everolimus increases the growth-inhibitory effect.

A c c e p t e d M a n u s c r i p t 14

Discussion

Intrinsic and acquired resistance to EGFR-targeting agents is an important issue in clinical practice. Clear evidence exists for the involvement of constitutive activation of the PI3K/AKT signaling pathway in lung carcinogenesis and in resistance to tyrosine-kinase inhibitors [START_REF] Balsara | Frequent activation of AKT in non-small cell lung carcinomas and preneoplastic bronchial lesions[END_REF].

Of the eleven NSCLC cell lines tested, three (H322, H292, Calu-3) having an IC 50 <1 M were considered highly sensitive to gefitinib, while the others, with an IC 50 >7 M were considered resistant. The gefitinib concentration of 1 M was used as cutoff for sensitivity because this concentration approaches levels observed in serum from patients under treatment with 250 mg/day [START_REF] Sharma | Epidermal growth factor receptor mutations in lung cancer[END_REF]. In line with other reports [START_REF] Van Schaeybroeck | Chemotherapy-induced epidermal growth factor receptor activation determines response to combined gefitinib/chemotherapy treatment in non-small cell lung cancer cells[END_REF][START_REF] Parra | Analysis of epidermal growth factor receptor expression as a predictive factor for response to gefitinib ('Iressa', ZD1839) in non-small-cell lung cancer[END_REF][START_REF] Ono | Sensitivity to gefitinib (Iressa, ZD1839) in non-small cell lung cancer cell lines correlates with dependence on the epidermal growth factor (EGF) receptor/extracellular signalregulated kinase 1/2 and EGF receptor/Akt pathway for proliferation[END_REF] no association between responsiveness to gefitinib and EGFR level was observed.

The response of sensitive cell lines to gefitinib was previously correlated with dependence on AKT and MAPK activation in response to EGFR signaling [START_REF] Ono | Sensitivity to gefitinib (Iressa, ZD1839) in non-small cell lung cancer cell lines correlates with dependence on the epidermal growth factor (EGF) receptor/extracellular signalregulated kinase 1/2 and EGF receptor/Akt pathway for proliferation[END_REF] and on inhibition of both PI3K/AKT/mTOR and MAPK pathways following gefitinib treatment [START_REF] Van Schaeybroeck | Chemotherapy-induced epidermal growth factor receptor activation determines response to combined gefitinib/chemotherapy treatment in non-small cell lung cancer cells[END_REF][START_REF] Paez | EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy[END_REF][START_REF] Sordella | Gefitinib-sensitizing EGFR mutations in lung cancer activate anti-apoptotic pathways[END_REF]. In the three highly sensitive cell lines we observed a marked reduction on p-p44/42 MAPK, on p-AKT, and on p-S6K following gefitinib treatment (Fig. 1B). In contrast, in five resistant cell lines (H460, SKMES-1, H1299, H596, H1650) there was no reduction in the phosphorylation of S6K suggesting that the maintenance of this survival pathway is related to resistance to gefitinib.

The presence of a mutation in the PI3K gene in H596 and the deletion of PTEN in the H1650 cells could justify the persistent phosphorylation of AKT after treatment with gefitinib (Fig. 1B and Fig. 3). However in H460, SKMES-1 and H1299 p-AKT was significantly reduced. This result is in contrast to that reported by Engelman J.A. et al [START_REF] Engelman | ErbB-3 mediates phosphoinositide 3-kinase activity in gefitinib-sensitive non-small cell lung cancer cell lines[END_REF] on the reduction of p-AKT levels solely in sensitive NSCLC cell lines in which gefitinib inhibited growth. gefitinib was able to reduce the phosphorylation of p44/42 MAPK as previously reported for the H460 cell line [START_REF] Janmaat | Enhanced cytotoxicity induced by gefitinib and specific inhibitors of the Ras or phosphatidyl inositol-3 kinase pathways in non-small cell lung cancer cells[END_REF] indicating that K-RAS mutations cannot sustain an EGFR-independent MAPK pathway activity.

The rapamycin derivative everolimus is a potent inhibitor of tumor cell proliferation in vitro and in animal models of cancer [START_REF] Boulay | Antitumor efficacy of intermittent treatment schedules with the rapamycin derivative RAD001 correlates with prolonged inactivation of ribosomal protein S6 kinase 1 in peripheral blood mononuclear cells[END_REF]. In general, rapamycin and everolimus are cytostatic. In KLN-205 and A549 NSCLC cells, rapamycin induced cell cycle arrest and blocked proliferation but did not induce apoptosis [START_REF] Boffa | Rapamycin inhibits the growth and metastatic progression of non-small cell lung cancer[END_REF]. In H460, H596, H1299, H1650, SKMES-1 characterized by maintaining S6K phosphorylation after gefitinib treatment, everolimus inhibited phosphorylation of S6K and slowed proliferation, albeit to different extents especially at the lower doses. This result is in agreement with that reported by Sun et al on H460 and H1299 cell lines treated with rapamycin [START_REF] Sun | Activation of Akt and eIF4E survival pathways by rapamycin-mediated mammalian target of rapamycin inhibition[END_REF]. The high basal level of p-AKT observed in H1650 and SKMES-1 may be associated with the higher sensitivity to everolimus as reported in ovarian cancer cells [START_REF] Mabuchi | RAD001 inhibits human ovarian cancer cell proliferation, enhances cisplatin-induced apoptosis, and prolongs survival in an ovarian cancer model[END_REF] and in breast cancer cells [START_REF] Noh | Determinants of rapamycin sensitivity in breast cancer cells[END_REF].

An increase in AKT phosphorylation after everolimus treatment was observed in H460, H596 and SKMES-1 cells. It is recognized that mTOR inhibition induces insulin receptor substrate-1 expression and inhibits a normally negative feedback loop resulting in AKT activation in some cancer cell lines [START_REF] Sun | Activation of Akt and eIF4E survival pathways by rapamycin-mediated mammalian target of rapamycin inhibition[END_REF][START_REF] O'reilly | mTOR inhibition induces upstream receptor tyrosine kinase signaling and activates Akt[END_REF].

It is noteworthy that up-regulation of pAKT induced by everolimus in H596 and SKMES-1 cells was reduced in the presence of gefitinib, but to a lesser extent in H460 cells.

In vitro studies with rapamycin and erlotinib in the gefitinib-resistant H460, Calu-6 and SW1573 NSCLC cell lines [START_REF] Buck | Rapamycin synergizes with the epidermal growth factor receptor inhibitor erlotinib in non-smallcell lung, pancreatic, colon, and breast tumors[END_REF] showed a synergistic growth inhibition associated with a down-modulation of rapamycin-stimulated AKT activity.

A c c e p t e d M a n u s c r i p t 16 However, in H460 treated with rapamycin and gefitinib, Janmaat et al. [START_REF] Janmaat | Enhanced cytotoxicity induced by gefitinib and specific inhibitors of the Ras or phosphatidyl inositol-3 kinase pathways in non-small cell lung cancer cells[END_REF] did not observe an inhibition of AKT phosphorylation, and an enhanced cytotoxicity was obtained only when gefitinib was associated with the PI3 kinase inhibitor LY294002.

The association between mTOR inhibitors (rapamycin, everolimus or temsirolimus) and anti-EGFR drugs (gefitinib, erlotinib or cetuximab) has also been tested in human cancer cell lines derived from different types of tumors [START_REF] Buck | Rapamycin synergizes with the epidermal growth factor receptor inhibitor erlotinib in non-smallcell lung, pancreatic, colon, and breast tumors[END_REF][START_REF] Bianco | Inhibition of mTOR pathway by everolimus cooperates with EGFR inhibitors in human tumours sensitive and resistant to anti-EGFR drugs[END_REF][START_REF] Azzariti | Synergic antiproliferative and antiangiogenic effects of EGFR and mTor inhibitors on pancreatic cancer cells[END_REF][START_REF] Jimeno | Dual EGFR and mTOR targeting in squamous cell carcinoma models, and development of early markers of efficacy[END_REF][START_REF] Johansson | Effective in vivo targeting of the mammalian target of rapamycin pathway in malignant peripheral nerve sheath tumors[END_REF].

In particular Bianco et al. [START_REF] Bianco | Inhibition of mTOR pathway by everolimus cooperates with EGFR inhibitors in human tumours sensitive and resistant to anti-EGFR drugs[END_REF] showed that in resistant human GEO colon, PC3 prostate and MDA-MB-468 breast cancer cells the everolimus and gefitinib combined therapy restored the anti-proliferative effect induced by anti-EGFR drugs and induced, in colon cells, a strong reduction of AKT activation. Moreover, the combined treatment potentiated the antiangiogenic effect of everolimus alone. This result was also confirmed on pancreatic cancer cells [START_REF] Azzariti | Synergic antiproliferative and antiangiogenic effects of EGFR and mTor inhibitors on pancreatic cancer cells[END_REF] with a combination of gefitinib and rapamycin.

In our experimental models, we demonstrated in a panel of NSCLC cell lines that the everolimus and gefitinib combination exerted a cytostatic and not a cytotoxic effect independently from inhibition of AKT phosphorylation. The effect of the combination was deeply studied on the basis of a plausible interaction model (e.g. Bliss) and we showed that the combination was exactly additive for H596 and H1650 and synergistic for H460, SKMES-1 and H1299 with suppression of proliferation rather than enhancement of cell death in contrast to the findings of Buck et al [START_REF] Buck | Rapamycin synergizes with the epidermal growth factor receptor inhibitor erlotinib in non-smallcell lung, pancreatic, colon, and breast tumors[END_REF] on a single NSCLC model (Calu-6 cells).

The authors suggested that the synergy for rapamycin with erlotinib was linked to the capability of erlotinib to promote apoptosis by reducing the rapamycin-stimulated AKT activity, although this conclusion was supported by observations on only one of the models responsive to the double treatment. On the other hand, we did not detect cell death, although AKT levels were partially reduced by the combined treatment. promising on NSCLC patients. In a phase I trial, 10 advanced NSCLC patients received the combination of everolimus and gefitinib administered orally and daily and the maximumtolerated dose of everolimus was 5 mg daily when combined with gefitinib at a dose of 250 mg [START_REF] Milton | Phase 1 trial of everolimus and gefitinib in patients with advanced nonsmall-cell lung cancer[END_REF]. To date, only preliminary results from the subsequent phase II trial are available. In that trial, untreated or platinum-based pretreated advanced NSCLC patients, all current or former smokers, were enrolled. The results are promising; in particular, positive responses were observed in male former smokers and in one K-RAS mutated patient, all features negative for response to EGFR-TKIs [START_REF] Kris | Combined inhibition of mTOR and EGFR with everolimus (RAD001) and gefitinib in patients with non-small cell lung cancer who have smoked cigarettes: A phase II trial[END_REF].

In this study we provide new insights regardless a rationale applicability of the combined treatment based on tumor growth rate. Indeed cells showing higher proliferation index are more responsive to treatment than are cells with a low level of proliferation. We propose that proliferation index might be a good marker for selecting those patients who will benefit from the combined treatment.

Tumor proliferative activity, in general evaluated by Ki-67 expression, is a prognostic marker in different type of tumours included NSCLC and it is considered of potential interest in defining populations at high or low risk of recurrence [START_REF] Martin | Ki-67 expression and patients survival in lung cancer: systematic review of the literature with meta-analysis[END_REF]. No data are available about the possible correlation between high tumor proliferation and response to EGFR inhibitors.

Nevertheless in this context, it has been recently reported that 18F-FDG PET, which reflects glucose metabolism and proliferative activity of tumor cells [START_REF] Vesselle | Relationship between non-small cell lung cancer FDG uptake at PET, tumor histology, and Ki-67 proliferation index[END_REF], may predict response to gefitinib in the treatment of advanced NSCLC patient [START_REF] Sunaga | Usefulness of FDG-PET for early prediction of the response to gefitinib in non-small cell lung cancer[END_REF].

In conclusion, our observations on combinations of everolimus and gefitinib on various NSCLC lines in vitro may help devise new therapies for selected high Ki-67 expressing lung cancer patients by targeting the AKT/mTOR pathway in an attempt to overcome gefitinib resistance. Curves of growth inhibitory effects of gefitinib (left), everolimus (middle) and combined treatment versus theoretical Bliss additivity curve (right) are reported. In the combined treatment, everolimus was 0.01 nM in H460 and SKMES-1, 0.1 nM in H1650 and 1 nM in H1299 and H596. Cells were treated with the drugs for six days and then cell number was assessed using crystal violet staining as described in Materials and Methods. Data are A). Proliferation rates were estimated by the growth rate quotient as calculated by the equation: GRQ= (N-N 0 )/ t*1/N 0 . Doubling time (hr) was calculated according to the formula t*ln(2)/ln(N/N 0 ), where N is the final cell number; N 0 is the initial cell number and t is the time elapsed between the two counts. B) SKMES-1 were plated at different density (2500, 5000, 10000, 25000/cm 2 ) and exposed for three days to 5 M gefitinib and 0.01 nM everolimus and then cell number was assessed using crystal violet staining as described in Materials and Methods. Percent inhibition of cell proliferation versus control cells was plotted as function of growth rate quotient.

The experiments, repeated three times, yielded similar results.

A c c e p t e d M a n u s c r i p t 5 To

 5 better support in vivo studies and to further clarify their interaction, we assessed the effects of combining the mTOR inhibitor everolimus with gefitinib on an extended panel of NSCLC cell lines characterized by gefitinib resistance. A c c e p t e d M a n u s c r i p t 6

A c c e p t e d M a n u s c r i p t 8 Apoptosis

 8 was assessed by: (a) morphology on stained (Hoechst 33342, PI) or unstained cells using light-, phase contrast-and fluorescence-microscopy; (b) activation of caspase-3

  significant increase in the level of p-AKT was observed only in the SKMES-1, this cell line also displayed a higher basal level of p-AKT. No modulation on the p-p44/42 MAPK level was detected in both cell models tested.

  SKMES-1, Calu-1 and SKLU-1 harbor mutations in the K-RAS gene,

A c c e p t e d M a n u s c r i p t 17 Preliminary

 17 clinical data on combining mTOR inhibitors with EGFR-TKIs might be

  FIGURE CAPTIONS.

Figure 1 :

 1 Figure 1: Effects of gefitinib on EGFR signalling pathways.

Figure 2 :

 2 Figure 2: Effect of everolimus on PI3K/AKT/mTOR, MAPK signaling pathways and on cell

A

  c c e p t e d M a n u s c r i p t (B) H460, H596, H1299, H1650 and SKMES-1 cells were exposed for six days to different concentrations of everolimus (from 0.01 to 100 nM) and then cell number was assessed using crystal violet staining as described in Materials and Methods. Data are expressed as percent inhibition of cell proliferation versus control cells. IC 50 values are also indicated. The mean values of three independent measurements (± SD) are shown. (C) H1299 and SKMES-1 cellswere treated with different concentrations of everolimus (from 0.01 to 100 nM) for 24 hr and then cells were lysed and Western blot analysis was performed on lysate proteins by using monoclonal antibodies directed against p-AKT (ser273), AKT, p-S6K (thr389), S6K, p-p44/42 MAPK, p44/42 MAPK and actin. Data are from a representative experiment. The experiment, repeated three times, yielded similar results.

Figure 3 :

 3 Figure 3: Effect of gefitinib-everolimus combined treatment on PI3K/AKT/mTOR, MAPK

Figure 4 :

 4 Figure 4: Interaction between gefitinib and everolimus on the growth inhibition dose-

Figure 5 :

 5 Figure 5: Effect of gefitinib-everolimus combined treatment on colony formation ability.

Figure 6 :

 6 Figure 6: Effect of proliferative index on growth inhibition induced by gefitinib-everolimus

  A c c e p t e d M a n u s c r i p t

Figure 1 A

 1 Figure 1

Figure 2 PageFigure 3 A

 23 Figure 2

Figure 4 Page 33 of 35 A

 435 Figure 4

Figure 5 A

 5 Figure 5

Figure 6 Page

 6 Figure 6

Table 1

 1 Page 29 of 35

Acknowledgements

Grant support: Ministero della Salute (Programma straordinario di ricerca oncologica 2006); Regione Emilia Romagna; Associazione Marta Nurizzo, Brugherio MI; Associazione Chiara Tassoni, Parma; A.VO.PRO.RI.T., Parma; Associazione Davide Rodella, Montichiari BS, Lega Italiana per la lotta contro i tumori, Sezione di Parma and CONAD, Bologna.


Florian Eyer

Darren M Roberts

Nicholas A Buckley

Michael Eddleston

Horst Thiermann

Franz Worek

Peter Eyer
email: peter.eyer@lrz.uni-muenchen.de

Extreme variability in the formation of chlorpyrifos oxon (CPO) in patients poisoned by chlorpyrifos (CPF)

Keywords:

EC 3.1.1.7); BChE, butyrylcholinesterase (EC 3.1.1.

patients with intentional CPF poisoning. A wide concentration range of CPO and CPF was observed and the ratio of CPO/CPF varied considerably between individuals and over time.

The ratio increased during the course of poisoning from a mean of 0.005 in the first few hours after ingestion up to an apparent steady state mean of 0.03 between 30 and 72 hrs. There was a hundred fold variation in the ratio between samples and the interquartile range (between individuals) indicated over half the samples had a 5-fold or greater variation from the mean.

The ratio was independent of the CPF concentration and the pralidoxime regimen. CPO was present in sufficient quantities to explain any observed acetylcholinesterase inhibitory activity. The effectiveness of pralidoxime in reactivating the inhibited acetylcholinesterase is strongly dependent on the CPO concentration. Differences in clinical outcomes and the response to antidotes in patients with acute poisoning may occur due to inter-individual variability in metabolism.

Word count 233

Introduction

Chlorpyrifos (CPF) is one of the most commonly used organophosphorus pesticides for domestic, agricultural and industrial purposes. It may be estimated that it causes tens of thousands of deaths per year worldwide [START_REF] Gunnell | The global distribution of fatal pesticide self-poisoning: systematic review[END_REF][START_REF] Eddleston | Differences between organophosphorus insecticides in human self-poisoning: a prospective cohort study[END_REF]. CPF is a pro-poison that requires metabolic activation to become a potent phosphorylating agent, namely chlorpyrifos oxon (CPO) [START_REF] Sams | Biotransformation of chlorpyrifos and diazinon by human liver microsomes and recombinant human cytochrome P450s (CYP)[END_REF]. At low concentrations, the desulfuration of CPF by human liver microsomes is due to the action of the polymorphically expressed CYP2B6 owing to its favorable V max /K m ratio [START_REF] Foxenberg | Human hepatic cytochrome p450-specific metabolism of parathion and chlorpyrifos[END_REF]. At high poison concentration, however, an increasing proportion of CPF desulfuration occurs due to the abundant CYP3A4 [START_REF] Buratti | CYP-specific bioactivation of four organophosphorothioate pesticides by human liver microsomes[END_REF]. CPO is also rapidly detoxified by human liver microsomes via CYP-dependent deethylation and dearylation, and by glutathione-S-transferase [START_REF] Fujioka | Glutathione S-transferase conjugation of organophosphorus pesticides yields S-phospho-, S-aryl-, and S-alkylglutathione derivatives[END_REF]. In addition, reactions with A-esterases such as paraoxonase 1 (PON 1; EC 3.1.8.1) or Besterases such as carboxylesterase (EC 3.1.1.1) and butyrylcholinesterase (BChE; EC 3.1. 1.8) in the liver may rapidly degrade or scavenge CPO [START_REF] Choi | Metabolism of chlorpyrifos and chlorpyrifos oxon by human hepatocytes[END_REF]. These polymorphisms in the activating and detoxifying enzymes [START_REF] Mutch | The relationship between PON1 phenotype and PON1-192 genotype in detoxification of three oxons by human liver[END_REF] are expected to lead to wide inter-individual variability in exposure to CPO for a given CPF load. However, further exploration of the importance of these factors has been limited by the absence of a reliable method of quantifying CPO in vivo.

Until recently [START_REF] Heilmair | Enzyme-based assay for quantification of chlorpyrifos oxon in human plasma[END_REF], CPO had never been detected in the blood of CPF-poisoned patients [START_REF] Drevenkar | Chlorpyrifos metabolites in serum and urine of poisoned persons[END_REF][START_REF] Timchalk | A Physiologically based pharmacokinetic and pharmacodynamic (PBPK/PD) model for the organophosphate insecticide chlorpyrifos in rats and humans[END_REF]]. Yet we repeatedly observed plasma of CPF-poisoned patients was able to inhibit red blood cell (RBC) acetylcholinesterase (AChE; EC 3.1.1.7) from healthy donors in vitro. It had been noted that BChE had extremely high reaction rates with CPO [START_REF] Amitai | Inhibition of acetylcholinesterase and butyrylcholinesterase by chlorpyrifos-oxon[END_REF] and an enzyme-based assay was developed using BChE (described in detail in [START_REF] Heilmair | Enzyme-based assay for quantification of chlorpyrifos oxon in human plasma[END_REF]). In the current study, we determined the ratio of CPO/CPF in patients with moderate to severe self-inflicted poisoning.

A c c e p t e d M a n u s c r i p t informed consent was taken from each patient, or their relatives, in their own language. Study details are reported elsewhere [START_REF] Eddleston | Differences between organophosphorus insecticides in human self-poisoning: a prospective cohort study[END_REF][START_REF] Jayawardane | The spectrum of intermediate syndrome following acute organophosphate poisoning: a prospective cohort study from Sri Lanka[END_REF][START_REF] Eddleston | Multiple-dose activated charcoal in acute self-poisoning: a randomised controlled trial[END_REF]. Treatment consisted of fluid resuscitation and atropine as described [START_REF] Eddleston | Management of acute organophosphorus pesticide poisoning[END_REF] and pralidoxime chloride 1g every 6 h for 48 h (RCT1 and Nuwara Eliya cohort), although there was no strict adherence to this regimen. Patients in RCT2 were randomised to receive pralidoxime (2g loading dose over 20 min, then 0.5g/h until a maximum of 7 days) or an equal volume of normal saline in addition to the supportive therapy mentioned above. The blood samples were collected between 25 May 2004 and 18

October 2006.

Blood samples were collected into tubes containing EDTA prior to pralidoxime, then at 1, 4, 12, and 24 h after administration, then once daily until discharge or death. A 0.2 mL blood sample was diluted at the bedside immediately after collection with 4 mL of ice-cold saline and frozen to -23°C until analysis of AChE activity. The remaining sample was promptly centrifuged and the aspirated plasma frozen at -23°C until analysis of BChE activity, inhibitory activity, and the concentration of CPF and CPO [START_REF] Eddleston | Management of acute organophosphorus pesticide poisoning[END_REF].

Analyses

Laboratory analyses were conducted in Munich, Germany. AChE activity was determined in native RBCs (AChE in vivo) and after reactivation with supra-therapeutic obidoxime (AChE in vitro) to estimate the reactivatable fraction. These assays were performed using a modified Ellman assay as described [START_REF] Worek | Improved determination of acetylcholinesterase activity in human whole blood[END_REF]. BChE activity [START_REF] Eddleston | Management of acute organophosphorus pesticide poisoning[END_REF] and inhibitory activity of plasma [START_REF] Thiermann | Cholinesterase status, pharmacokinetics and laboratory findings during obidoxime therapy in organophosphate poisoned patients[END_REF] were assessed as reported. In short, inhibitory activity of patient plasma was tested by A c c e p t e d M a n u s c r i p t CPO was determined by an enzyme-based assay as described recently [START_REF] Heilmair | Enzyme-based assay for quantification of chlorpyrifos oxon in human plasma[END_REF]. In short, CPO was extracted with n-pentane and the reconstituted residue titrated with purified horse BChE, allowing an LOQ of 0.5 nM. The data were corrected for 83% recovery of CPO from plasma [START_REF] Heilmair | Enzyme-based assay for quantification of chlorpyrifos oxon in human plasma[END_REF].

Horse BChE (Cat. No C-1057) and CPF Pestanal® were from Riedel-de Haen, Sigma, Taufkirchen, Germany; CPO was a generous gift from DowAgroSciences, Indianapolis, IN.

Both compounds had purities ≥ 99%. Fenitrothion (95.5% pure) was obtained from Dr.

Ehrenstorfer, Augsburg, Germany. All other reagents were from commercial sources at the purest grade available.

Pralidoxime was determined by HPLC on reversed phase material with an ion-pairing reagent. Specifically, 100 µL plasma was mixed with 30 µL trichloroacetic acid Typically, pralidoxime was eluted after 5.6 min. LOQ was 1 µM pralidoxime in plasma.

Free EDTA in plasma samples was titrated with a standard calcium chloride solution (50 mM) in the presence of the calcium indicator eriochrome black T (CAS 1787-61-7; 10 mg eriochrome black + 1 g sodium chloride triturated and dissolved in 2.5 mL water).

Specifically, 50 µL plasma was diluted with 450 µL distilled water, 5 µL sodium hydroxide (2 M) and 1 µL indicator solution added and titrated with calcium chloride until formation of a pink coloration.

Calculations

Data were included when CPF was ingested (on the basis of history and/or analytical data)

with no other organophosphorus compound being detected, BChE was mostly inhibited (typical for CPO) and AChE not significantly aged (typical for a diethylphosphorylated enzyme). Data were excluded when CPF and/or CPO were below the LOQs or when the time of ingestion or blood sampling was ambiguous. Data were also excluded when plasma pralidoxime was higher than 0.5 mM, indicating that blood was withdrawn proximally from the infusion site. In these cases significant post-sampling reactions had to be suspected.

The relationship between measured and calculated (predicted) CPO concentration was determined using equations as deduced elsewhere [START_REF] Eyer | The role of oximes in the management of organophosphorus pesticide poisoning[END_REF]. This approach has been validated for paraoxon previously [START_REF] Worek | Reappraisal of indications and limitations of oxime therapy in organophosphate poisoning[END_REF]. The basis for the calculation of free CPO is the equation given below: The constants for reactivation of human diethylphosphoryl-AChE by pralidoxime are known from in vitro experiments using human red blood cells [START_REF] Eyer | The role of oximes in the management of organophosphorus pesticide poisoning[END_REF][START_REF] Worek | Reappraisal of indications and limitations of oxime therapy in organophosphate poisoning[END_REF], [E], [E+EPOx] and [Ox] were determined, hence [P] can be calculated. We used k r = 0.3 min -1 ; K D = 330 µM and k i = 6 x 10 6 M -1 min -1 [START_REF] Eyer | The role of oximes in the management of organophosphorus pesticide poisoning[END_REF].

[E] [EP  EPOx]  k r k i  [P]  1 K D [Ox]   E =
The fraction of active test AChE upon incubation with known amounts of CPO in plasma samples was calculated discretely in 1-min increments using the following equation:

E = E 0 -k i *(E -

Results

Time course of CPO formation

Formation of CPO occurred quite early after CPF ingestion. In one patient we detected significant amounts of CPO 70 min after CPF ingestion with BChE and AChE depressed to less than 2% and 10% of normal, respectively. Figure 1 shows the time course of plasma CPF and CPO in a patient who was admitted some 5 h post ingestion and received no pralidoxime.

Here, maximal CPO concentration appeared after a delay of several hours. As expected, the ratio of CPO/CPF increased with time, starting with less than 0.005 in the period between 0 and 4 h after ingestion and approaching 0.03 on average in the period between 24 and 120 hours. Table 1 shows the median plasma concentrations along with the interquartile ranges of CPF and CPO in 72 eligible patients (with and without pralidoxime treatment, no significant difference), where sufficient data were available to determine the CPO/CPF ratio at various time intervals post-ingestion. Fig. 2 shows the time course of the ratio of CPO/CPF and a mono-exponential association curve that was fitted to the median values, without implying a specific mechanism.

The variation of the ratio CPO/CPF was large, and some individuals showed a ratio below 0.01, while others had >0.05 in the period between 24 and 120 h, when a steady state was reached (cf. Fig. 2). The ratio CPO/CPF showed no dependence on the CPF concentration.

When plotting the ratio CPO/CPF in a logarithmic scale vs. the CPF concentration (Fig. 3), the linear regression showed no significant deviation of the slope from zero (p > 0.4; not shown). These data were indicative that in this setting CPO formation and decomposition did not show a saturation (dose-limiting) phenomenon.

Dependence of AChE-inhibitory material in plasma on CPO concentration

A large scattering of data was observed in the ratio of AChE-inhibitory activity of patients' plasma and the CPO concentration, whether or not patients had received pralidoxime. As expected, the inhibitory activity was on average higher in the group who did not receive pralidoxime. Fig. 4 shows the scatter of data from patients who were not administered inhibition rate constant of 6 x 10 6 M -1 min -1 [START_REF] Eyer | The role of oximes in the management of organophosphorus pesticide poisoning[END_REF] allows a rough estimate of the inhibition progress, which was essentially complete after the 60 min incubation time. As shown in Fig. 4, in most cases AChE was inhibited to a lesser extent than predicted from the CPO concentration (dotted line). The ratio of inhibitory activity found/predicted in samples with and without PAM did not depend on the CPO concentration as shown in the semilogarithmic plot of Fig. 5. Linear regression analysis of the data indicated that the slopes were not significantly different from zero (p>0.2 and >0.5, respectively). These results pointed to competing side reactions of CPO during the incubation period.

Since BChE was essentially completely inhibited at even low concentrations of CPO we suspected that OP-hydrolases were degrading CPO in the inhibition assay. PON 1 is notorious for its high activity towards CPO [START_REF] Li | Catalytic efficiency determines the in-vivo efficacy of PON 1 for detoxifying organophosphorus compounds[END_REF]. This enzyme, however, requires free calcium ions, both for stability and activity [START_REF] Kuo | Calcium binding by human and rabbit serum paraoxonases. Structural stability and enzymatic activity[END_REF]. Hence, this enzyme should be inactivated in EDTA-based plasma samples. We measured the free concentration of EDTA and found in 487 out of 490 plasma samples that excess EDTA was higher than 0.5 mM (median 4 mM). This should be sufficient to completely inactivate PON 1 and this implies other mechanisms of CPO inactivation/sequestration may exist.

Influence of CPO and pralidoxime concentrations on AChE activity

Figure 6 shows the effects of CPO and pralidoxime plasma concentrations on the percentage of active AChE in two selected patients with a similar time course of the CPF concentrations.

Patient A received pralidoxime by the continuous infusion regimen while patient B received the intermittent lower dose scheme. In both cases, AChE activity was largely inhibited before pralidoxime. In case A, AChE reactivation was sustained and approached some 90% when pralidoxime was given for 5 days. In contrast, intermittent doses of pralidoxime for less than 48 h resulted in gradual re-inhibition of reactivated AChE, particularly since CPO concentrations were much higher than in patient A. The mutual dependence of the RBC-AChE activity on the pralidoxime and the CPO concentration was analyzed according to the equation given in the Methods section. Forty four patients who received pralidoxime, were eligible for the analysis contrasting the CPO concentration predicted from the RBC-AChE activity with the measured CPO. We excluded all data where CPO was below 0.5 nM (LOQ), pralidoxime given less than 1 hour before sampling and where the pralidoxime concentration was below 13 µM. At this concentration the half-life for reactivation is approximately 1 hr [START_REF] Worek | Reappraisal of indications and limitations of oxime therapy in organophosphate poisoning[END_REF] and steady-state conditions could not be expected at lower concentrations. The expected free CPO concentration could be calculated from the ratio of inhibited, but reactivatable enzyme and the active enzyme, i.e.

[EP +EPOx]/[E]. In doing so, we observed a large scattering of the ratio of CPO found/CPO predicted. Fig. 7 shows a plot of data (from 20 subjects with at least 3 samples that met the above criteria). The ratio of CPO found vs. CPO predicted is given in a logarithmic scale (median, IQR). The predicted CPO concentration is the free (active) fraction. Thus we expected a mean ratio of about 6 (dashed line), corresponding to 85% reversible albumin binding [START_REF] Choi | Metabolism of chlorpyrifos and chlorpyrifos oxon by human hepatocytes[END_REF] as CPO from plasma was extracted by an organic solvent determining both the bound and free fraction of CPO.

Discussion

Since CPO reacts more than 2 orders of magnitude faster with BChE than with AChE [START_REF] Amitai | Inhibition of acetylcholinesterase and butyrylcholinesterase by chlorpyrifos-oxon[END_REF],

CPF-poisoned patients usually arrive at hospital with completely inhibited BChE while RBC-AChE is often less affected. These sequestering reactions along with the effective hydrolysis brought about by PON 1 were thought to keep the CPO steady-state concentration in blood too low for conventional determination (HPLC, GC-MS, LC-MS) [START_REF] Choi | Metabolism of chlorpyrifos and chlorpyrifos oxon by human hepatocytes[END_REF][START_REF] Timchalk | A Physiologically based pharmacokinetic and pharmacodynamic (PBPK/PD) model for the organophosphate insecticide chlorpyrifos in rats and humans[END_REF]. However, in previous studies in CPF-poisoned patients [START_REF] Eddleston | Differences between organophosphorus insecticides in human self-poisoning: a prospective cohort study[END_REF] we observed that the patient's plasma was able to inhibit AChE of added test erythrocytes. The consistent presence of "inhibitory activity" of plasma led us to develop the enzyme-based assay for CPO.

Using the standard assay for CPO determination [START_REF] Heilmair | Enzyme-based assay for quantification of chlorpyrifos oxon in human plasma[END_REF], reliable results were obtained in the concentration range of 2 to 20 nM CPO in plasma (accuracy ± 10%, reproducibility ± 4% (95% CI)). When it turned out that BChE was almost completely inhibited, plasma was diluted 1:10 or more with buffer to arrive at the useful range. Of course, the biological assay lacks specificity since all highly reactive inhibitors that are extracted with n-pentane may mimic CPO. With the exception of the nerve agents soman and cyclosarin no oxons are so reactive as CPO in inhibiting BChE [START_REF] Amitai | Inhibition of acetylcholinesterase and butyrylcholinesterase by chlorpyrifos-oxon[END_REF][START_REF] Schopfer | Reaction kinetics of biotinylated organophosphorus toxicant, FP-biotin, with human acetylcholinesterase and human butyrylcholinesterase[END_REF][START_REF] Aurbek | Suitability of human butyrylcholinesterase as therapeutic marker and pseudo catalytic scavenger in organophosphate poisoning: A kinetic analysis[END_REF]. Of the insecticidal organophosphates diazoxon is next reactive, but still 20-times less than CPO [START_REF] Schopfer | Reaction kinetics of biotinylated organophosphorus toxicant, FP-biotin, with human acetylcholinesterase and human butyrylcholinesterase[END_REF]. We intentionally minimized the reaction time for BChE inhibition to largely exclude the effect of potential cross-reacting oxons. Significant CPF concentrations were verified in all patients included and the presence of the lipophilic organophosphates fenthion and quinalphos were excluded by HPLC analysis.

Next, the presence of major concentrations of dimethylphosphoryl compounds could be ruled out because erythrocyte AChE did not indicate the rapid ageing seen with these compounds [START_REF] Eddleston | Management of acute organophosphorus pesticide poisoning[END_REF]. Earlier studies in Sri Lanka have shown that the alleged poison was confirmed in more than 90% of the patients [START_REF] Eddleston | Differences between organophosphorus insecticides in human self-poisoning: a prospective cohort study[END_REF]. Hence, we are confident that our assay detected in fact CPO and no other BChE inhibitors.

The present study confirms that CPO is present in more than sufficient quantities to be responsible for all the "inhibitory activity" of the plasma of CPF poisoned patients. It also casts doubt on the accuracy of non-specific bioassays such as the ex-vivo "inhibitory activity" and suggests that they may significantly and variably under-estimate the true extent of in-vivo AChE inhibitory activity. As shown in Fig. 4 the correlation of CPO with inhibition of test AChE was not as strong as anticipated. Even if we assume that some 85% of CPO is bound to 12 albumin [START_REF] Heilmair | Enzyme-based assay for quantification of chlorpyrifos oxon in human plasma[END_REF], we would expect somewhat slower but finally complete inhibition of AChE if the molar concentration of CPO exceeded that of AChE (11.3nM). This expectation was usually not met (Figs. 4 and5), so it appears likely that CPO was subject to competing reactions ex vivo. Hydrolysis of CPO by PON 1 in the inhibition assay can be ruled out since EDTA in the plasma samples leads to complete inactivation of PON 1 [START_REF] Kiderlen | The phosphoryl oxime-destroying activity of human plasma[END_REF]. Albumin, however, not only binds CPO reversibly, but also hydrolyses CPO as demonstrated by the liberation of 3,5,6-trichloropyridinol in the absence of free calcium ions [START_REF] Sultatos | The interaction of the phosphorothioate insecticides chlorpyrifos and parathion and their oxygen analogues with bovine serum albumin[END_REF]. This reflects the covalent binding of CPO to tyrosine 411 in human albumin [START_REF] Li | Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry assay for organophosphorus toxicants bound to human albumin at Tyr 411[END_REF]. Based on the assumption of an albumin concentration in human plasma of about 4 g/100 mL, our inhibition assay contained 1% albumin. It is known that bovine serum albumin 'hydrolyses' CPO at V max of 0.9 nmol/min/mg protein with a K m value of 0.4 mM at pH 7.4 and 37°C [START_REF] Sultatos | The interaction of the phosphorothioate insecticides chlorpyrifos and parathion and their oxygen analogues with bovine serum albumin[END_REF]. Given that human serum albumin has similar kinetic constants, we can deduce that 20 nM CPO will be inactivated at a rate of 0.45 nM/min. This may be compared with the more rapid inhibition rate of 1.35 nM/min (11.3 nM AChE, 20 nM CPO, k i = 6 x 10 6 M -1 min -1 , see above). It is clear that the (initial) inhibition rate will be slowed down upon gradual inhibition of AChE, while covalent binding to albumin that has a large molar excess of about 150 µM still proceeds. This latter pseudo-first-order reaction explains the observation that the consumption of CPO was largely independent of the CPO concentration (Fig. 5). If other plasma proteins were also involved in CPO binding the effect of competing reactions might be even greater. In fact, data exist showing that mouse plasma contains at least 11 proteins capable of binding organophosphorus compounds under physiological conditions [START_REF] Peeples | Albumin, a new biomarker of organophosphorus toxicant exposure, identified by mass spectrometry[END_REF].

The influence of pralidoxime on the inhibitory activity of the patent's plasma (Fig. 5 13 capacity, which in the rat was estimated to be 3 orders in magnitude higher than of BChE [START_REF] Timchalk | A Physiologically based pharmacokinetic and pharmacodynamic (PBPK/PD) model for the organophosphate insecticide chlorpyrifos in rats and humans[END_REF]. Circulating carboxylesterase is not found in human plasma [START_REF] Li | Butyrylcholinesterase, paraoxonase, and albumin esterase, but not carboxylesterase, are present in human plasma[END_REF], but located in tissues.

When these B-esterases are saturated, free CPO may escape the major site of production, the liver. Concomitantly, CPO is prone to degradation by A-esterases (true hydrolysis) and by CYP450-mediated inactivation [START_REF] Timchalk | A Physiologically based pharmacokinetic and pharmacodynamic (PBPK/PD) model for the organophosphate insecticide chlorpyrifos in rats and humans[END_REF]. PON 1 has favorable kinetic properties towards CPO hydrolysis, but shows a polymorphism that affects differently the catalytic efficiency of organophosphates. Thus, PON 1 192R is superior in hydrolyzing CPO and provides better protection against CPO exposure in PON 1 null mice than PON 1 192Q [START_REF] Richter | Paraoxonase 1 (PON1) status and substrate hydrolysis[END_REF]. Interethnic variability of the genetic makeup is remarkable in that PON 1 RR was rarely found in Indians (3%) but often in Chinese (32%), the inverse relationship was found with the PON 1 QQ genotype [START_REF] Chia | Distribution of PON1 polymorphisms-PON1Q192R and PON1L55M among Chinese, Malay and Indian males in Singapore and possible susceptibility to organophosphate exposure[END_REF]. In addition, a large variation in enzyme levels are found among individuals even with the same genotype [START_REF] Li | Catalytic efficiency determines the in-vivo efficacy of PON 1 for detoxifying organophosphorus compounds[END_REF]. Hence, steady-state concentrations of CPO (at constant CPF) are expected to vary widely. We observed steady-state ratios of CPO/CPF after some 30 h post-ingestion with a spread of the interquartile range of about 5 (cf. µM, respectively, at comparable V max values. Since the former CYP is prone to polymorphism in protein expression [START_REF] Wang | CYP2B6: new insights into a historically overlooked cytochrome P450 isozyme[END_REF][START_REF] Ramachandran | Association of high T allele frequency of CYP2B6 G516T polymorphism among ethnic south Indian HIV-infected patients with elevated plasma efavirenz and nevirapine[END_REF], large variability in oxon formation can be expected [START_REF] Foxenberg | Human hepatic cytochrome p450-specific metabolism of parathion and chlorpyrifos[END_REF]. In fact, a variability of CPO/CPF of more than 1 order in magnitude was observed (Fig. 5).

Pralidoxime was able to reactivate inhibited RBC-AChE in CPF-poisoned patients as shown in Fig. 6. Its effectiveness depended both on its plasma concentration and on the CPO concentration. The two examples shown indicate that the variability in CPO production and also that if pralidoxime is ceased while there is measureable CPO present then the RBC-AChE will not remain reactivated. Such considerations are likely to apply to all OP, particularly when highly lipophilic agents are involved [START_REF] Eddleston | Differences between organophosphorus insecticides in human self-poisoning: a prospective cohort study[END_REF][START_REF] Eddleston | Management of acute organophosphorus pesticide poisoning[END_REF]. As deduced recently [START_REF] Eyer | The role of oximes in the management of organophosphorus pesticide poisoning[END_REF], the theoretical plasma concentration of CPO should be able to be calculated from the ratio of inhibited vs. active AChE, the plasma concentration of the oxime and the kinetic constants of the reactions involved (see Methods section). However, we observed a very large variation in the found/predicted CPO concentration ratio. We expected a mean ratio of about 6 (dashed line), corresponding to 85% reversible albumin binding [START_REF] Heilmair | Enzyme-based assay for quantification of chlorpyrifos oxon in human plasma[END_REF] as CPO from plasma was extracted by an organic solvent determining both the bound and free fraction of CPO and the IQRs of 72 eligible patients from Table 1. For illustration a mono-exponential association function was fitted to the median values.

  (1 M) and spun down. Eighty µL of supernatant was mixed with 40 µL of n-heptanesulfonate reagent (PIC B7 low UV, Millipore) and 30 µL of a sodium formate buffer (1 M, pH 3.5) to give a final pH ≥ 3.0. Fifty µL were applied to a Purosphere Star RP-18 column (55 x 4 mm, 3 µm, Merck) and eluted with a linear methanol gradient (0-15 % within 7.5 min) in 5 mM PIC-B7 A c c e p t e d M a n u s c r i p t 22.04.2009 6 low UV reagent in water at a flow rate of 1.4 mL/min (20-25°C) and detection at 293 nm.

  active enzyme EP = phosphorylated, but unaged enzyme EPOx = complex of oxime and phosphorylated, but unaged enzyme [EP + EPOx] is the reactivatable enzyme = AChE in vitro -AChE in vivo k r is the first-order reactivation rate constant K D is the dissociation constant of the complex EPOx k i is the second-order inhibition rate constant [P] is the oxon concentration [Ox] is the oxime concentration.

  A c c e p t e d M a n u s c r i p t 22.04.2009 7

  pralidoxime (absence confirmed by HPLC). A hyperbola fitted by non-linear regression is shown (full line). The normal concentration of AChE in whole blood is about 15 nM [20, 21], hence the concentration of AChE in the inhibition assay was about 11.3 nM. Using the A c c e p t e d M a n u s c r i p t 22.04.2009 9

A c c e p t e d M a n u s c r i p t 22 .

 22 04.2009

Fig 2 )

 2 . The ratio was not dependent on the CPF concentration, indicating that the equilibrium of CPO formation and degradation did not show saturation phenomena. Desulfuration of CPF by human hepatic CYPs is mainly brought about by CYP2B6 and CYP3A4 with K m values of about 1 and 30

Fig. 1

 1 Fig. 1 Time course of CPF and CPO concentrations in a patient with CPF self-poisoning. The patient did not receive pralidoxime. X-axis shows the time post ingestion. Note the different concentration scales.

Fig. 2

 2 Fig. 2 Ratio of CPO/CPF vs time post-ingestion of CPF. Data are median values with their

Fig. 3 Fig. 4

 34 Fig. 3 Ratio of CPO/CPF vs CPF concentration in patient samples 24 to 120 h post ingestion from the data set shown in Fig. 2. Linear regression analysis of the logarithmically transformed ratio versus CPF concentration gave a slope not significantly different from zero.

Fig. 5

 5 Fig. 5 Semilogarithmic plot of the ratio of found and expected inhibitory activity of plasma from CPF-poisoned patients versus the total CPO concentration. (Closed circles and full line from pralidoxime-free samples; Xes and broken line samples containing pralidoxime between 13 and 150 µM).

Fig. 6

 6 Fig. 6 Influence of CPO and pralidoxime concentrations on AChE activity. Two patients, A and B, with a similar time course of plasma CPF had very different CPO concentrations in their plasma. Patient A received the intensified pralidoxime regimen with continuous infusion for >4 days; patient B received the intermittent bolus doses for<2 days, resulting in the re-inhibition of reactivated AChE. (%AChE refers to the fraction of active enzyme from the pool of reactivatable AChE, thus ignoring the aged fraction.)

  Figure legends

Fig. 3

 3 Fig. 3

A c c e p t e d M a n u s c r i p t

Fig. 4

 4 Fig. 4

Fig. 5

 5 Fig. 5

  EP)*0.15*(CPO 0 -EP), with E 0 and CPO 0 being the AChE and total CPO concentration at t=0 (the factor 0.15 corrects for the fraction not bound reversibly to albumin).Calculations were done with Microsoft Excel 2004. Correlation analyses, Mann-Whitney U test and graphical presentations were performed with Prism4, GraphPad San Diego CA.

	22.04.2009	8
		Formatted: Portuguese (Brazil) a n u s c r i p t M
		A c c e p t e d

  ) was only evident at low CPO concentrations, because the bioreactor AChE + pralidoxime has only a limited capacity to degrade CPO, due to the low molar AChE concentration present. The large scattering of the data at low-to-intermediate CPO concentrations (up to 100 nM CPO) in the presence of pralidoxime may be mainly caused by variations of its concentration in the plasma samples. Taken together in the absence of pralidoxime, roughly 2/3 of CPO in EDTA based plasma was capable of inhibiting AChE, the remainder had most probably reacted covalently with other proteins with albumin being the most favorite candidate.

	22.04.2009
	M a n u s c r i p t
	A c c e p t e d
	Appearance of CPO in plasma lags behind CPF. This may be due to delayed formation or,
	more probably, due to sequestering reactions with limited capacity. CPO is rapidly bound to
	B-esterases, such as BChE, AChE and carboxylesterase. The latter enzyme has a high binding

Table 1

 1 Plasma concentrations of CPF and CPO after various time intervals post poisoning Patients were eligible (CPF and CPO >LOQ). The ratio CPO/CPF was calculated from the individual sample values. Data are median values along with the interquartile range in brackets.

	Time interval	CPF	CPO	CPO/CPF x 1000	n
	(h)	(µM)	(nM)
	0-4	2.54 (1.6-4.7) 10.1 (4.5-22.2)	4.2 (1.9-9.5)	52
	4-8	1.65 (0.7-3.2) 11.9 (6.5-21.5)	6.8 (4.3-14.0)	67
	8-16	0.70 (0.4-1.2) 12.7 (5.5-22.5)	17.5 (9.2-31.6)	59
	16-24	0.44 (0.3-1.5) 9.6 (4.1-30.1)	14.2 (10.5-42.6)	30
	24-36	0.47 (0.3-1.1) 14.1 (4.7-34.5)	28.6 (11.3-44.4)	45
	36-60	0.66 (0.2-1.0) 16.7 (3.4-68.4)	29.2 (14.4-70.7)	41
	60-84	0.46 (0.2-0.8) 22.7 (2.2-59,9)	37.3 (12.7-65.3)	27
	84-120	0.39 (0.2-0.7) 7.1 (4.2-40.2)	25.0 (15.4-53.6)	28
	Plasma samples (n=357) of 72

Acknowledgements:

We thank the directors, consultant physicians and medical and nursing staff of the study hospitals for their support and Renate Heilmair, Bodo Pfeiffer and Elisabeth Topoll for technical assistance. The South Asian Clinical Toxicology Research Collaboration is funded by a Wellcome Trust/National Health and Medical Research Council International Collaborative Research Grant GR071669MA.

In conclusion, this newly developed CPO assay gives reliable results that enable a better assessment of the mechanisms behind inter-individual variation in susceptibility in CPF poisoning. The study has shown that the steady-state ratios of CPO/CPF varies widely and probably mirrors the activating and detoxifying capabilities. Thus, a large variation in the susceptibility for CPF-and CPO-mediated toxicities has to be taken into account when debating safety margins of CPF.