Aleksandar Radujkovic

Stefan Fruehauf

W Jens Zeller

Anthony D Ho

Julian Topaly
email: julian.topaly@pk-mx.de

Dr Med Julian Topaly

Synergistic activity of nilotinib and established chemotherapeutic drugs in imatinib-sensitive and -resistant BCR-ABL-positive cells Running title: Combination therapy of CML employing nilotinib

Keywords: BCR-ABL, nilotinib, combination treatment, imatinib resistance, synergism

We investigated various combination treatment regimens employing nilotinib with established chemotherapeutic agents (daunorubicin, mitoxantrone, etoposide and cytarabine) in imatinibsensitive and -resistant BCR-ABL-positive cells. Mitoxantrone or cytarabine showed synergism (CI<1) in combination with nilotinib in imatinib-sensitive LAMA84 cells, whereas in imatinib-resistant LAMA84-R cells synergistic effects could be assessed for daunorubicine, mitoxantrone and etoposide when combined with nilotinib. In both imatinib-sensitive andresistant K562 cells daunorubicin, mitoxantrone and etoposide demonstrated synergism in combination with nilotinib. Moreover, both daunorubicin and mitoxantrone led to synergistic antiproliferative effects when combined with nilotinib in imatinib-resistant Ba/F3 cells carrying point mutations in the ABL TK domain (E255K, E255V and T315I). Annexin V / propidium iodide staining revealed a significant enhancement of nilotinib-induced apoptosis in imatinib-resistant Ba/F3T315I and LAMA84-R cells upon combination with daunorubicine and mitoxantrone, respectively. Our results demonstrate the efficacy of combination treatment regimens employing nilotinib and established chemotherapeutic agents in improving antileukemic effects in imatinib-sensitive and imatinib-resistant cells. This may be the foundation for further study on the potential of the applied combinations in a clinical setting.

INTRODUCTION

Chronic myelogenous leukemia (CML) is a malignant myeloproliferative disease characterized by the chromosomal translocation t(9;22)(q34;q11) known as the Philadelphia (Ph 1 ) chromosome [START_REF] Nowell | A minute chromosome in human chronic granulocytic leukemia[END_REF][START_REF] Rowley | A new consistent chromosomal abnormality in chronic myelogenous leukaemia identified by quinacrine fluorescence and Giemsa staining[END_REF][START_REF] Goldman | Chronic myeloid leukemia--advances in biology and new approaches to treatment[END_REF]. On the molecular level this aberration leads to the expression of the BCR-ABL fusion proto-oncogene with transforming potency due to deregulated ABL tyrosine kinase (TK) activity [START_REF] Goldman | Chronic myeloid leukemia--advances in biology and new approaches to treatment[END_REF][START_REF] Lugo | Tyrosine kinase activity and transformation potency of bcr-abl oncogene products[END_REF]. The development and application of imatinib targeting the ABL TK has revolutionized the treatment of CML making imatinib the therapeutic gold standard for this disease [START_REF] Peggs | Imatinib mesylate--the new gold standard for treatment of chronic myeloid leukemia[END_REF][START_REF] Deininger | The development of imatinib as a therapeutic agent for chronic myeloid leukemia[END_REF]. However, resistance to imatinib may evolve in some patients. Up to now various molecular mechanisms of resistance have been described, most notably mutations in the BCR-ABL TK domain and an increased BCR-ABL activity resulting from gene amplification, up-regulation, or multiple Ph chromosomes [START_REF] Mahon | Selection and characterization of BCR-ABL positive cell lines with differential sensitivity to the tyrosine kinase inhibitor ST571: diverse mechanisms of resistance[END_REF][START_REF] Gorre | Clinical resistance to STI-571 cancer therapy caused by BCR-ABL gene mutation or amplification[END_REF][START_REF] Von Bubnoff | BCR-ABL gene mutations in relation to clinical resistance of Philadelphia-chromosome-positive leukaemia to STI571: a prospective study[END_REF][START_REF] Hochhaus | Resistance to targeted therapy in chronic myelogenous leukemia[END_REF]. Consequently, the effort to overcome imatinib resistance within current treatment strategies lead to the development of second-generation TK inhibitors (TKI).

Nilotinib is a potent, second generation aminopyrimidine ATP-competitive inhibitor of BCR-ABL. It has been shown not only to be at least 20-fold more potent in killing cells with nonmutated BCR-ABL in vitro than imatinib but also to inhibit the activity of the majority of mutant forms of BCR-ABL occurring in imatinib-resistant patients. [START_REF] Weisberg | Characterization of AMN107, a selective inhibitor of native and mutant Bcr-Abl[END_REF][START_REF] Golemovic | AMN107, a novel aminopyrimidine inhibitor of Bcr-Abl, has in vitro activity against imatinib-resistant chronic myeloid leukemia[END_REF]. Furthermore, recent clinical trials found nilotinib effective and safe in patients with CML post imatinib failure or intolerance [START_REF] Kantarjian | Nilotinib in imatinib-resistant CML and Philadelphia chromosome-positive ALL[END_REF][START_REF] Kantarjian | Nilotinib (formerly AMN107), a highly selective BCR-ABL tyrosine kinase inhibitor, is effective in patients with Philadelphia chromosome-positive chronic myelogenous leukemia in chronic phase following imatinib resistance and intolerance[END_REF]. However, the efficacy of nilotinib and other newer TK inhibitors is best in the chronic phase of CML and decreases with disease evolution to further stages. In particular, the blast crisis remains a serious clinical challenge [START_REF] Giles | Nilotinib in Patients (pts) with Philadelphia Chromosome-Positive (Ph+) Chronic Myelogenous Leukemia in Blast Crisis (CML-BC) Who Are Resistant or Intolerant to Imatinib[END_REF][START_REF] Cortes | Dasatinib induces complete hematologic and cytogenetic responses in patients with imatinib-resistant or -intolerant chronic myeloid leukemia in blast crisis[END_REF]. With either conventional intensive chemotherapy or the newer targeted therapies applied as single treatment long-term survival in myeloid blast crisis is rare reaching a plateau at under 10% if the patients are not offered a subsequent allogeneic stem cell transplantation upon achievement of response. On the other hand, an induction therapy leading to remission of blast crisis or to restoration of chronic phase was shown to be associated with improved outcome in patients receiving an allogeneic transplant [START_REF] Gratwohl | Bone marrow transplantation for chronic myeloid leukemia: long-term results. Chronic Leukemia Working Party of the European Group for Bone Marrow Transplantation[END_REF].

A number of preclinical studies by several investigators including our group could comprehensively show that combination treatment regimens, employing a TK inhibitor (imatinib) and substances with a differing mode of action, lead to synergistic antiproliferative effects presenting a feasible strategy to increase the antileukemic efficacy and to circumvent imatinib resistance [START_REF] Topaly | Synergistic activity of the new ABL-specific tyrosine kinase inhibitor STI571 and chemotherapeutic drugs on BCR-ABL-positive chronic myelogenous leukemia cells[END_REF][START_REF] Topaly | Rationale for combination therapy of chronic myelogenous leukaemia with imatinib and irradiation or alkylating agents: implications for pretransplant conditioning[END_REF][START_REF] Topaly | Combination therapy with imatinib mesylate (STI571): synopsis of in vitro studies[END_REF][21][START_REF] Radujkovic | Combination treatment of imatinibsensitive and -resistant BCR-ABL-positive CML cells with imatinib and farnesyltransferase inhibitors[END_REF][START_REF] Thiesing | Efficacy of STI571, an abl tyrosine kinase inhibitor, in conjunction with other antileukemic agents against bcrabl-positive cells[END_REF][START_REF] Liu | The in vitro activity of the tyrosine kinase inhibitor STI571 in BCR-ABL positive chronic myeloid leukaemia cells: synergistic interactions with anti-leukaemic agents[END_REF][START_REF] Marley | Effects of combinations of therapeutic agents on the proliferation of progenitor cells in chronic myeloid leukaemia[END_REF][START_REF] Yerushalmi | Combined antiproliferative activity of imatinib mesylate (STI-571) with radiation or cisplatin in vitro[END_REF][START_REF] Kano | In vitro cytotoxic effects of a tyrosine kinase inhibitor STI571 in combination with commonly used antileukemic agents[END_REF]. In order to elucidate whether these observations can be translated to nilotinib, in the present study we investigated several combinations applying nilotinib and established chemotherapeutic agents (daunorubicin, mitoxantrone, etoposide and cytarabine) in different imatinib-sensitive and in particular imatinib-resistant BCR-ABL-positive leukemia cell lines.

MATERIALS AND METHODS

Cell lines and culture conditions

K562 and LAMA84 cell lines were obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) and were all sensitive to submicromolar concentrations of imatinib [21,[START_REF] Radujkovic | Combination treatment of imatinibsensitive and -resistant BCR-ABL-positive CML cells with imatinib and farnesyltransferase inhibitors[END_REF]. The imatinib-resistant cell lines LAMA84-R and K562-R were provided by Professor Junia Melo (London; UK) and were characterized in detail previously [21]. BCR-ABL-positive wild type (wt) Ba/F3 wt cells expressing p185 BCR-ABL and the corresponding imatinib-resistant lines carrying mutations in the ABL TK domain (E255K, E255V and T315I) were established in the lab of Professor Justus Duyster (Munich, Germany) [START_REF] Von Bubnoff | Bcr-Abl resistance screening predicts a limited spectrum of point mutations to be associated with clinical resistance to the Abl kinase inhibitor nilotinib (AMN107)[END_REF].

All cells were grown in RPMI-1640 medium supplemented with 10% heat inactivated fetal calf serum (FCS), 2 mM L-glutamine and penicillin / streptomycin (Invitrogen, Karlsruhe, Germany) at standard conditions (37°C, fully humidified atmosphere of 95% air and 5% CO2). In addition, LAMA84-R and K562-R cells were cultured in medium containing 1 µM imatinib.

Drugs

Nilotinib was kindly provided by Novartis Pharma (Basel, Switzerland). Daunorubicin (DNR; Sigma, Steinheim, Germany), mitoxantrone (Sigma), etoposide (Bristol Arzneimittel, Munich) and cytarabine (Ara-C; Cell Pharm, Hannover, Germany) were purchased from the manufacturers. Nilotinib was initially dissolved in sterile 10% dimethyl sulfoxide solution (DMSO; Merck, Darmstadt, Germany). Other applied cytostatic drugs were dissolved in phosphate-buffered saline (Dulbecco's PBS; Invitrogen). Stock solutions at a concentration of 10 mM of each drug were prepared and stored at -20°C until use. For experiments, serial working dilutions were prepared.

Cell proliferation assay and analysis of drug effects and interactions

MTT assays were used for the assessment of cell proliferation with minor modifications as described previously [START_REF] Topaly | Synergistic activity of the new ABL-specific tyrosine kinase inhibitor STI571 and chemotherapeutic drugs on BCR-ABL-positive chronic myelogenous leukemia cells[END_REF]21]. After 48 h of incubation with tested drugs means of four replicate wells for each drug dilution and the control (cells grown in absence of the drug) were used to calculate the extent of relative cell proliferation and growth inhibition (defined as fraction affected (Fa) [START_REF] Topaly | Synergistic activity of the new ABL-specific tyrosine kinase inhibitor STI571 and chemotherapeutic drugs on BCR-ABL-positive chronic myelogenous leukemia cells[END_REF]). Each cell line was treated with five increasing concentrations (doubling with each increment) of nilotinib and chemotherapeutic drug alone or their combination, respectively. Since nilotinib, in contrast to the other agents, was dissolved in 10% DMSO the serial drug dilutions and controls were prepared to finally achieve 0.05% DMSO in all wells (control, single agent and drug combinations; total volume 100 µl). At this concentration no anti-proliferative effects of the applied DMSO solution could be observed in the investigated cell lines (data not shown).

Dose-effect relationships and drug interactions upon combination of nilotinib and established drugs were analyzed according to the median-effect method of Chou and Talalay [START_REF] Chou | Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors[END_REF] employing the CalcuSyn Software (Biosoft, Cambridge, UK). For single agent treatment mean IC50 values for each drug and cell line were calculated; for combinations mean combination index (CI) values based on equitoxic drug ratios were assessed with CI < 1, CI = 1 or CI > 1 representing synergistic, additive. or antagonistic effects of the substances, respectively and combination data were depicted as CI vs. Fa plots [START_REF] Topaly | Synergistic activity of the new ABL-specific tyrosine kinase inhibitor STI571 and chemotherapeutic drugs on BCR-ABL-positive chronic myelogenous leukemia cells[END_REF][START_REF] Topaly | Combination therapy with imatinib mesylate (STI571): synopsis of in vitro studies[END_REF]. In cell lines where no antiproliferative activity upon single agent treatment was assessable, an analysis in terms of sensitization (potentiation) or inhibition of nilotinib activity was performed instead with relative IC50 > 1 indicating inhibition and relative IC50 < 1 indicating potentiation of nilotinib activity, respectively (for details see Topaly et al [START_REF] Topaly | Combination therapy with imatinib mesylate (STI571): synopsis of in vitro studies[END_REF] and Radujkovic et al [START_REF] Radujkovic | Combination treatment of imatinibsensitive and -resistant BCR-ABL-positive CML cells with imatinib and farnesyltransferase inhibitors[END_REF]).

Apoptosis assay

Assessment of apoptosis rates was done by Annexin V / propidium iodide (PI) staining using the Annexin V-FITC Kit (Immunotech, Marseille, France) 48 h after start of incubation as described previously [21,[START_REF] Radujkovic | Combination treatment of imatinibsensitive and -resistant BCR-ABL-positive CML cells with imatinib and farnesyltransferase inhibitors[END_REF]. Data collection and analysis were performed with a FACScalibur flow cytometer (Becton Dickinson, Heidelberg, Germany) applying the CellQuest Software (Becton Dickinson).

Statistics

Three to five independent experiments were performed and the results were depicted as mean value ± standard deviation (s.d.). Statistical significant differences of the data were calculated using the Student t test with significance levels of p < 0.05 and p < 0.01.

RESULTS

Single agent treatment

Imatinib-sensitive LAMA84 and K562 cells were all sensitive to nanomolar concentrations of single agent nilotinib (Table I). In the imatinib-resistant counterparts LAMA84-R and K562-R substantially higher nilotinib concentrations for corresponding antiproliferative effects were required. Imatinib-resistant K562-R cells displayed a remarkable resistance towards nilotinib with an IC50 value of 21,972 nM, i.e. approximately 600-fold higher than in the respective imatinib-sensitive K562 cells (34 nM); in the LAMA84-R line the IC50 value (124 nM) was 10-fold higher as compared to the parental line (11 nM). In imatinib-resistant Ba/F3 cells exhibiting point mutations in the BCR-ABL TK domain Ba/F3 E255K , Ba/F3 E255V and Ba/F3 T315I the IC50 values of nilotinib were 365 nM, 791 nM and 23,582 nM, respectively, i.e. about 16-, 34-and 1,000-fold higher than in the corresponding parental imatinib-sensitive Ba/F3 wt cell line (Table I).

Daunorubicin, mitoxantrone and etoposide displayed a concentration dependent inhibition of proliferation after a 48 h treatment. Both intercalating drugs, the anthracycline daunorubicin and the anthracendione mitoxantrone, showed similar antiproliferative effects in LAMA84 cells (Table I). In imatinib-resistant LAMA84-R cells considerably higher concentrations of both agents were necessary for growth inhibition as compared to the imatinib-sensitive LAMA84 line with mitoxantrone and daunorubicin IC50 values being increased by 10-and 20-fold, respectively, implying cross-resistance towards both compounds (Table I). Regarding imatinib sensitivity, no significant differences in the cytotoxicity of the substances could be observed in K562 cells. The activity of etoposide was substantially higher in the LAMA84 cells as compared to K562 with both imatinib-resistant cell lines (LAMA84-R and K562-R) showing a reduced sensitivity (not significant) towards etoposide (Table I). For cytarabine, only in imatinib-sensitive LAMA84 cells an IC50 value of 175 nM was calculable; in LAMA84-R cells and in the K562 pair no cytarabine activity could be detected. Daunorubicin displayed similar effects on the growth of all investigated Ba/F3 lines; the same applies to mitoxantrone (Table I).

Combination treatment

In the imatinib-sensitive LAMA84 cell line combination treatment with nilotinib plus daunorubicin and nilotinib plus etoposide led to antagonistic (CI > 1; Figure 1a) and additive (CI ~ 1; Figure 1e) effects, respectively. In contrast, the combinations employing mitoxantrone and cytarabine were both clearly synergistic denoted by CI values < 1 above the growth inhibition level > 60% (Fa>0.6) (Figure 1c andg). Combination treatment of imatinibresistant LAMA84-R cells resulted in enhanced antiproliferative effects for every regimen.

Analysis of combined effects revealed synergism with CI values of 0.36±0.35, 0.59±0.24 and 0.91±0.07 for nilotinib plus daunorubicin, nilotinib plus mitoxantrone and nilotinib plus etoposide, respectively, at the 60% growth inhibition level (Fa=0.6) (Figure 1b,d,f). Since cytarabine was inactive in LAMA84-R cells (Table I), the median-effect method was not applicable for investigating combination effects. However, non-cytotoxic doses of cytarabine (≥1,250 nM) led to a significant reduction of nilotinib IC50 values (relative IC50<1) signifying potentiating of nilotinib activity (Figure 1h).

In imatinib-sensitive K562 cells daunorubicin and mitoxantrone showed synergistic effects when combined with nilotinib at Fa≥0.4 and Fa>0.3, respectively (Figure 2a andc); nilotinib plus etoposide acted synergistically at Fa<0.8 (Figure 2e). In the imatinib-resistant K562-R line combination treatment employing daunorubicin and etoposide displayed synergism (Figure 2b andf) whereas the combination nilotinib plus mitoxantrone was at best additive at Fa=0.6 (Figure 2d). In both imatinib-sensitive and -resistant K562 cells no significant potentiation of nilotinib activity could be observed by cytarabine (Figure 2g andh).

Combination treatment of imatinib-sensitive Ba/F3 wt cells with nilotinib plus daunorubicin and nilotinib plus mitoxantrone resulted in improved antiproliferative effects as compared to the respective monotherapies with CI values indicating synergism above affected fractions ≥0.45 and Fa>0.3, respectively (Figure 3a,b). Depending on the level of growth inhibition the combination nilotinib plus daunorubicin showed synergistic cytotoxic effects also in the imatinib-resistant Ba/F3 E255K (Fa≥0.65), Ba/F3 E255V (Fa>0.15) and Ba/F3 T315I (Fa>0.7) cell lines (Figure 3c, e andg). For the combination nilotinib plus mitoxantrone synergistic effects were discernible with CI values < 1 above Fa>0.7 in Ba/F3 E255K and above Fa≥0.2 in both Ba/F3 E255V and Ba/F3 T315I cells (Figure 3d, f andh).

Induction of apoptosis

Monotherapy of the imatinib-resistant cell lines K562-R and LAMA84-R with low dose nilotinib, daunorubicin or mitoxantrone increased the early apoptotic cell fractions (Annexin V+ / PI-cells) as compared to untreated controls. For 1,000 nM daunorubicin this effect was more obvious with apoptotic fractions of 18% (K562-R) and 15% (LAMA84-R) as compared to nilotinib-(13% and 11%) and mitoxantrone-induced apoptosis (10% and 8%) in the respective lines. Compared to the corresponding monotherapies drug combinations led to enhanced apoptosis levels (16% -23%) with a significant increase for nilotinib plus mitoxantrone in the LAMA84-R cell line (Figure 4a).

In the investigated imatinib-resistant Ba/F3 E255K and Ba/F3 T315I cells, respectively, only a marginal increase in the proportion of apoptotic cells upon administration of low dose single agent nilotinib (9% and 4%), daunorubicin (6% for both lines) and mitoxantrone (5% and 6%) could be observed as compared to untreated controls (4% and 3%, Figure 4b). Co-treatment of Ba/F3 E255K cells increased the early apoptotic cell fractions for both combinations (34% for nilotinib plus daunorubicin and 21% for nilotinib plus mitoxantrone). However, only in the Ba/F3 T315I cell line a significant enhancement of apoptosis could be observed after treatment with the combination nilotinib plus daunorubicin vs. monotherapy (Figure 4b).

DISCUSSION

Our preclinical studies with combinations of imatinib and classic chemotherapeutic drugs demonstrated significant synergistic action that translated in a better efficacy in a clinical setting in myeloid blast crisis of CML [START_REF] Fruehauf | Imatinib combined with mitoxantrone/etoposide and cytarabine is an effective induction therapy for patients with chronic myeloid leukemia in myeloid blast crisis[END_REF]. To present, the majority of patients who progress to a blast crisis of CML have been pre-exposed to imatinib treatment at earlier disease stages and harbor a cell clone resistant to this drug. Several studies demonstrated that BCR-ABL signaling remains one of the principal mechanisms of disease pathogenesis in imatinib resistance. In cases of BCR-ABL TK mutations that confer imatinib-resistance or in cases of up-regulated TK activity as compared to wild type BCR-ABL the newer more potent ABL kinase inhibitors are the rational treatment following imatinib failure.

However, in the course of disease evolution additional pathophysiological mechanisms may gain an increasing influence on cell proliferation thus diminishing the efficacy of single agent TK inhibitors in blast crisis. The logical action to overcome drug resistance is the use of drug combinations with different mechanisms of action. In the present study we investigated the effects of classical chemotherapeutic drugs cytarabine, daunorubicin, etoposide, and mitoxantrone that are typically used in the treatment of acute leukemia. The drugs were used as single agents and as combination partners for nilotinib in imatinib-sensitive CML cells and in BCR-ABL-positive leukemia cells with defined BCR-ABL TK mutations or other mechanisms of imatinib-resistance.

In our previous published reports the effects of imatinib treatment as a single agent and in combination with various compounds were studied extensively both in imatinib-sensitive and -resistant cells [START_REF] Topaly | Synergistic activity of the new ABL-specific tyrosine kinase inhibitor STI571 and chemotherapeutic drugs on BCR-ABL-positive chronic myelogenous leukemia cells[END_REF][START_REF] Topaly | Rationale for combination therapy of chronic myelogenous leukaemia with imatinib and irradiation or alkylating agents: implications for pretransplant conditioning[END_REF]21,[START_REF] Radujkovic | Combination treatment of imatinibsensitive and -resistant BCR-ABL-positive CML cells with imatinib and farnesyltransferase inhibitors[END_REF]. Compared to imatinib treatment, the single agent nilotinib showed an improved efficacy in the investigated imatinib-sensitive cell lines, reflected by IC50 values in the nanomolar range (Table I). In the imatinib-resistant LAMA84-R cell line nilotinib was also more effective than imatinib underlining nilotinib's high potency [START_REF] Weisberg | Characterization of AMN107, a selective inhibitor of native and mutant Bcr-Abl[END_REF][START_REF] Golemovic | AMN107, a novel aminopyrimidine inhibitor of Bcr-Abl, has in vitro activity against imatinib-resistant chronic myeloid leukemia[END_REF]21] (Table I). Only the K562-R cell line, in which several different resistance mechanisms are probably involved [START_REF] Mahon | Selection and characterization of BCR-ABL positive cell lines with differential sensitivity to the tyrosine kinase inhibitor ST571: diverse mechanisms of resistance[END_REF]21,[START_REF] Puttini | In vitro and in vivo activity of SKI-606, a novel Src-Abl inhibitor, against imatinib-resistant Bcr-Abl+ neoplastic cells[END_REF] showed a high resistance to both imatinib and nilotinib (IC50 values >20,000 nM) (Table I). With the exception of Ba/F3 T315I single agent nilotinib was also active in the highly imatinib-resistant Ba/F3 E255K and Ba/F3 E255V cell lines, which is consistent with previous findings [START_REF] Weisberg | Characterization of AMN107, a selective inhibitor of native and mutant Bcr-Abl[END_REF][START_REF] Von Bubnoff | Bcr-Abl resistance screening predicts a limited spectrum of point mutations to be associated with clinical resistance to the Abl kinase inhibitor nilotinib (AMN107)[END_REF]. Monotherapy with established chemotherapeutic agents resulted in variable antiproliferative effects in the investigated cell lines. Interestingly, in both the K562 and LAMA84 pair higher concentrations of the applied intercalating drugs and etoposide were required for growth inhibition of the imatinib-resistant cells implying crossresistance (Table I). Since daunorubicin, etoposide and mitoxantrone are substrates for the Pglycoprotein (Pgp) mediated drug efflux [START_REF] Michieli | Amifostine does not inhibit the toxic effects of anthracycline derivates or mitoxantrone on MDR tumor cell lines[END_REF][START_REF] Rago | Safety and efficacy of the MDR inhibitor Incel (biricodar, VX-710) in combination with mitoxantrone and prednisone in hormone-refractory prostate cancer[END_REF][START_REF] Takara | An update on overcoming MDR1-mediated multidrug resistance in cancer chemotherapy[END_REF], the known increased Pgp activity of these imatinib-resistant cell lines is one possible explanation [START_REF] Mahon | Selection and characterization of BCR-ABL positive cell lines with differential sensitivity to the tyrosine kinase inhibitor ST571: diverse mechanisms of resistance[END_REF]21]. Cytarabine which displayed no antiproliferative effects in the tested dose range in imatinib-resistant LAMA84-R and K562-R cells (Table I), however, has not been demonstrated to be a Pgp substrate [START_REF] Bhalla | Characterization of a human myeloid leukemia cell line highly resistant to taxol[END_REF].

Combination therapy employing nilotinib and classical chemotherapeutic drugs led to improved antileukemic effects. In imatinib-resistant LAMA84-R cells these effects resulted either from synergism (CI values <1) or potentiation of nilotinib activity (Figure 1). In the imatinib-resistant K562-R cells synergistic interactions could be observed at a lesser extent as compared to the imatinib-sensitive counterpart (Figure 2). In addition, the applied drug combinations acted synergistically also in Ba/F3 cells expressing defined imatinib-resistant BCR-ABL mutants. However, in T315I carrying cells, conferring complete resistance to imatinib [START_REF] Weisberg | Characterization of AMN107, a selective inhibitor of native and mutant Bcr-Abl[END_REF], high doses of nilotinib were required to assess the synergistic interaction (>25,000 nM) limiting the clinical significance (Figure 3).

The antiproliferative effects of nilotinib are associated with induction of apoptosis [START_REF] Weisberg | Characterization of AMN107, a selective inhibitor of native and mutant Bcr-Abl[END_REF][START_REF] Weisberg | Beneficial effects of combining nilotinib and imatinib in preclinical models of BCR-ABL+ leukemias[END_REF]. In our study we investigated the pro-apoptotic effects in imatinib-resistant cells. Since clinically isolated BCR-ABL kinase domain mutants show a varying incidence and resistance to imatinib [START_REF] Deininger | The development of imatinib as a therapeutic agent for chronic myeloid leukemia[END_REF][START_REF] Weisberg | Characterization of AMN107, a selective inhibitor of native and mutant Bcr-Abl[END_REF][START_REF] Hochhaus | Imatinib therapy in chronic myelogenous leukemia: strategies to avoid and overcome resistance[END_REF], we gave preference to the imatinib-resistant Ba/F3 T315I and Ba/F3 E255K cell lines harboring the more common BCR-ABL mutants with different levels of imatinib resistance in our experiments. Regarding combination treatment the apoptosis results in Figure 4 appear to be inconsistent with the synergistic effects observed in the cell proliferation assays (Figure 123). In our experiments the extent of apoptosis was defined as the proportion of early apoptotic cells (Annexin V+/PI-) which in contrast to the MTT assays shows no direct correlation to the viable cell number [START_REF] Pieters | Adaptation of the rapid automated tetrazolium dye based (MTT) assay for chemosensitivity testing in childhood leukemia[END_REF]. The corresponding fractions of dead (necrotic) cells (annexin V+/PI+) were considerably higher and displayed a stronger correlation to the cell proliferation experiments (data not shown). In our experiments a significant increase in the number of early apoptotic cells as compared to the respective monotherapies could only be observed in imatinib-resistant Ba/F3 T315I and LAMA84-R cells for the combinations nilotinib plus daunorubicin and nilotinib plus mitoxantrone, respectively, signifying the potential of the applied intercalating drugs to enhance nilotinib-induced apoptosis (Figure 4).

The underlying molecular mechanisms of the synergistic antiproliferative and pro-apoptotic effects of tyrosine kinase inhibitors in combination with classic antineoplastic agents are poorly investigated. Chow et al. [START_REF] Chow | The tyrosine kinase inhibitor AMN107 (Nilotinib) exhibits off-target effects in lymphoblastic cell lines[END_REF] could show that the pro-apoptotic activity of nilotinib is not restricted to BCR-ABL, c-Kit, or PDGFR-positive cells. Nilotinib-induced apoptosis in cell lines of lymphatic origin was shown to critically rely on activation of caspase-6 and caspase-9 and differential expression of Src-kinases [START_REF] Chow | The tyrosine kinase inhibitor AMN107 (Nilotinib) exhibits off-target effects in lymphoblastic cell lines[END_REF]. With regard to our results in Ba/F3 T315I cells, originally derived from a murine pro B cell line [START_REF] Palacios | Il-3-dependent mouse clones that express B-220 surface antigen, contain Ig genes in germ-line configuration, and generate B lymphocytes in vivo[END_REF] and showing no or only negligible expression of c-Kit or PDGFR, one could speculate that nilotinib is able to exert BCR-ABL independent pro-apoptotic effects which are only evident after sensitization by daunorubicin. Furthermore, the possibility of a physically altered interaction between nilotinib and the kinase domain of BCR-ABL after sensitization by standard chemotherapy allowing nilotinib to bind to the mutated T315I target cannot be ruled out either. In a recent report, Tiwari et al. [START_REF] Tiwari | Nilotinib (AMN107, Tasigna) reverses multidrug resistance by inhibiting the activity of the ABCB1/Pgp and ABCG2/BCRP/MXR transporters[END_REF] could demonstrate that nilotinib is able to reverse multidrug resistance by inhibiting the activity of ABCB1/Pgp and ABCG2/BCRP/MXR transporters. In one of our previous studies comprising functional rhodamine 123 efflux assays we could show that imatinib-sensitive LAMA84 cells displayed a lower Pgp activity than -resistant LAMA84-R cells, whereas K562 exhibited negligible and K562-R cells low Pgp activity [21].

Consequently, in these cell lines the observed synergy between nilotinib and the investigated agents, which with the exception of cytarabine are substrates for the Pgp mediated drug efflux [START_REF] Michieli | Amifostine does not inhibit the toxic effects of anthracycline derivates or mitoxantrone on MDR tumor cell lines[END_REF][START_REF] Rago | Safety and efficacy of the MDR inhibitor Incel (biricodar, VX-710) in combination with mitoxantrone and prednisone in hormone-refractory prostate cancer[END_REF][START_REF] Takara | An update on overcoming MDR1-mediated multidrug resistance in cancer chemotherapy[END_REF][START_REF] Bhalla | Characterization of a human myeloid leukemia cell line highly resistant to taxol[END_REF], may be attributed to the inhibition of Pgp by nilotinib.

The drug concentrations applied in our in vitro synergy experiments were primarily selected to enable a reliable analysis according to the Chou and Talalay method, i.e. around the IC50 in vitro. Of course, it is of great relevance, whether these concentrations are also achievable in vivo and the respective synergistic effects can be expected in a clinical setting. Zhou et al. reported a steady state C max of 2,160.7 ng/mL (corresponding to 4,081 nM) in CML patients treated with the standard dose of nilotinib 400 mg twice daily [START_REF] Zhou | Nilotinib for imatinib-resistant or -intolerant chronic myeloid leukemia in chronic phase, accelerated phase, or blast crisis: a single-and multiple-dose, open-label pharmacokinetic study in Chinese patients[END_REF]. For daunorubicin, depending on the drug formulation varying plasma concentration can be observed. Feldman et al. [START_REF] Feldman | Pharmacology of CPX-351: a nano-scale liposomal fixed molar ratio cytarabine-daunorubicin for patients with advanced leukemia[END_REF] demonstrated a C max of 30,185±6,198 ng/mL (57,221±11,749 nM) in patients treated with a liposomal daunorubicin formulation. In the work of Canal et al. [START_REF] Canal | Plasma and cellular pharmacokinetics of mitoxantrone in highdose chemotherapeutic regimen for refractory lymphomas[END_REF] the mean maximum plasma concentration of mitoxantrone reached 6,429±2,427 µg/L (14,464±5,460 nM) at maximum tolerated dose (90 mg/m 2 ) versus 789±158 µg/L (1,775±355 nM) after administration of 15 mg/m 2 . In 14 patients, who received the standard dose of 100 mg/m 2 etoposide, peak and trough serum concentrations of 18.5 µg/mL (31,433 nM) and 0.2 µg/mL (340 nM), respectively, could be observed [START_REF] Kato | Pharmacokinetics of etoposide with intravenous drug administration in children and adolescents[END_REF]. In patients with relapsed AML or a blast crisis of CML steady-state plasma levels of cytarabine ranged between 7,000 and 24,000 nM after infusion at a dose rate of 250 mg/m 2 /h for 36 to 72 h [START_REF] Spriggs | Prolonged high dose ARA-C infusions in acute leukemia[END_REF]. Thus, with the exception of K562-R and Ba/F3 T315I cells, the concentrations used in our in vitro studies are achievable in a clinical setting (Table I).

Nilotinib is approved for imatinib-resistant and -intolerant CML patients in the chronic and accelerated phase of the disease [START_REF] Kantarjian | Nilotinib in imatinib-resistant CML and Philadelphia chromosome-positive ALL[END_REF][START_REF] Kantarjian | Nilotinib (formerly AMN107), a highly selective BCR-ABL tyrosine kinase inhibitor, is effective in patients with Philadelphia chromosome-positive chronic myelogenous leukemia in chronic phase following imatinib resistance and intolerance[END_REF]. The newer studies report hematologic and cytogenetic responses to nilotinib in blast crisis, although the response rates are as expected lower [START_REF] Giles | Nilotinib in Patients (pts) with Philadelphia Chromosome-Positive (Ph+) Chronic Myelogenous Leukemia in Blast Crisis (CML-BC) Who Are Resistant or Intolerant to Imatinib[END_REF][START_REF] Giles | Nilotinib Therapy after Dasatinib Failure in Patients with Imatinib-Resistant or -Intolerant Chronic Myeloid Leukemia (CML) in Chronic Phase (CP), Accelerated Phase (AP) or Blast Crisis (BC)[END_REF]. With nilotinib, as a representative of second-generation TKI, displaying synergistic or additive drug interactions with a number of established chemotherapeutics in CML cells, the preclinical data reported in this work support combination treatment regimens for clinical testing. Based on our previous clinical experience with imatinib-based combination treatment in myeloid blast crisis patients [START_REF] Fruehauf | Imatinib combined with mitoxantrone/etoposide and cytarabine is an effective induction therapy for patients with chronic myeloid leukemia in myeloid blast crisis[END_REF] we propose a modern treatment concept for this patient group beginning with an induction treatment with a well tolerated chemotherapy regimen, e.g. mitoxantrone/etoposide (NOVE regimen) given along with nilotinib. Upon response to induction treatment, eligible patients should be assigned to a consolidation with allogeneic stem cell transplantation. Patients not eligible for allogeneic transplantation but fit enough to receive an intermediate-intensive chemotherapy treatment can be offered an alternative consolidation with e.g. intermediate-dose cytarabine combined with nilotinib. After completion of consolidation, a life-long maintenance treatment with nilotinib can be expected to be advantageous for patients not receiving an allogeneic transplant and therefore still harboring a residual CML clone. Regarding the low incidence of CML blast crisis under efficient imatinib treatment nowadays a concerted multicenter effort is needed to implement such a treatment concept in order to yield patient recruitment sufficient for further analysis. [a] Imatinib-sensitive LAMA84 cells displayed antagonistic (CI > 1) effects upon combination treatment with nilotinib and daunorubicin.

[c, g] Treatment of imatinib-sensitive LAMA84 cells with combinations of nilotinib plus mitoxantrone and nilotinib plus cytarabine showed synergistic effects (CI<1) above affected fractions approximately > 0.6.

[e] For the combination nilotinib plus etoposide additive (CI~1) antiproliferative effects were discernible in LAMA84 cells.

[b, d, f] In imatinib-resistant LAMA84-R cells synergistic effects were observable for the combination nilotinib plus daunorubicin (Fa≤0.7), nilotinib plus mitoxantrone (0.4≤Fa<0.8) and nilotinib plus etoposide (Fa≥0.65).

[h] Non-cytotoxic doses of cytarabine potentiated nilotinib activity in LAMA84-R cells (cf. [a, c, e] In imatinib-sensitive K562 cells synergistic effects were detectable for the combination nilotinib plus daunorubicin (Fa≥0.4), nilotinib plus mitoxantrone (Fa>0.3) and nilotinib plus etoposide (Fa<0.8).

[b, f] The combinations nilotinib plus daunorubicin and nilotinib plus etoposide acted synergistically in imatinib-resistant K562 cells at 0.35<Fa<0.9 and 0.45<Fa<0.75, respectively.

[d] Treatment of K562-R cells with nilotinib plus mitoxantrone displayed at best additive effects (CI~1 at 0.4<Fa<0.9).

[g, h] Cytarabine showed no significant potentiation of nilotinib activity in both imatinibsensitive and -resistant K562 cells. [a] Combination treatment of imatinib-resistant K562-R and LAMA84-R cell lines increased early apoptotic cell fractions (annexin V+ / PI-cells) as compared to the respective monotherapies. A significant increase of the early apoptotic cell fraction could be demostrated for the combination nilotinib plus mitoxantrone in the LAMA84-R cell line.

[b] In imatinib-resistant Ba/F3 E255K and Ba/F3 T315I cells similar effects were discernible.

Figure 1 .

 1 Figure 1. Analysis of drug interactions in imatinib-sensitive and -resistant LAMA84 cells.

  Materials and Methods) CI: combination index; Fa: fraction affected. The dotted line at CI = 1 in Figure 1 a-g represents additive, > 1 represents antagonistic, < 1 represents synergistic effects of the applied drug combinations. The dashed line in a-g represents the standard deviation (s.d.). The dotted line at 1 in h represents the relative nilotinib IC50 value; > 1 indicates inhibition, < 1 indicates potentiation of nilotinib activity. The error bars in h represent the standard deviation (s.d.). n = 5 for each experiment. *p<0.05, **p<0.01.

Figure 2 .

 2 Figure 2. Analysis of drug interactions in imatinib-sensitive and -resistant K562 cells.

  CI: combination index; Fa: fraction affected. The dotted line at CI = 1 in Figure 1 a-g represents additive, > 1 represents antagonistic, < 1 represents synergistic effects of the applied drug combinations. The dashed line in a-f represents the standard deviation (s.d.). The dotted line at 1 in g and h represents the relative nilotinib IC50 value; > 1 indicates inhibition, < 1 indicates potentiation of nilotinib activity. The error bars in g and h represent the standard deviation (s.d.). n = 5 for each experiment.

Figure 3 .

 3 Figure 3. Analysis of drug interactions in imatinib-sensitive Ba/F3 wt and imatinibresistant Ba/F3 E255K , Ba/F3 E255V and Ba/F3 T315I cell lines. [a, b] Both investigated combinations nilotinib plus daunorubicin and nilotinib plus mitoxantrone showed synergism in Ba/F3 wt above affected fractions ≥0.45 and > 0.3, respectively.[c, e, g] Co-treatment with nilotinib and daunorubicin resulted in synergistic effects in all investigated imatinib-resistant Ba/F3 E255K , Ba/F3 E255V and Ba/F3 T315I cell lines above Fa≥0.65, Fa>0.15 and Fa>0.7, respectively.[d, f, h] Synergism could also be observed for the combination nilotinib plus mitoxantrone above affected fractions >0.7 and ≥0.2 in Ba/F3 E255K and both Ba/F3 E255V and Ba/F3 T315I cells, respectively. CI: combination index; Fa: fraction affected. The dotted line at CI = 1 represents additive, > 1 represents antagonistic, < 1 represents synergistic effects of the applied drug combinations.The dashed line represents the standard deviation (s.d.). n = 5 for each experiment.

Figure 4 .

 4 Figure 4. Induction of apoptosis in imatinib-resistant cell lines after treatment with different concentrations of single agent nilotinib, daunorubicin and mitoxantrone or with combinations.

  Significant enhancement of apoptosis could only be observed in the Ba/F3 T315I cell line upon treatment with the combination nilotinib plus daunorubicin. (*p<0.05) The error bars represent the standard deviation (s.d.). n = 3 for each experiment.

Table I .

 . Effects of single agent nilotinib and established chemotherapeutic drugs on the cell growth of different BC R-ABL-positive imatinibsensitive and -resistant cell lines after 48 h treatment.

Acknowledgments 12

We thank Bernhard Berkus, Hans-Jürgen Engel and especially Carmen Hoppstock (German Cancer Research Center, Heidelberg, Germany) for their expert technical assistance and Dr. E. Buchdunger (Novartis, Basel, Switzerland) for supplying nilotinib. We are also grateful to Professor Dr. Junia Melo (Hammersmith Hospital, London, UK) and to Professor Dr. Justus Duyster (Department of Internal Medicine III, Technical University of Munich, Munich, Germany) for providing the imatinib-resistant K562-R/LAMA84-R and Ba/F3 cell lines, respectively. We also thank Dr. Christine Volk for critically reading the manuscript.

Authors' contributions:

A.R. designed research, performed research, analyzed data, wrote manuscript. S.F. designed research, approved manuscript. W.J.Z. assisted with interpretation and analysis of data.

A.D.H. assisted with interpretation and analysis of data, approved manuscript. J.T. wrote manuscript and is responsible for conception of research and integrity and analysis of data.

Disclosures: None


Ornella Letari

Catherine Booth

Albino Bonazzi

Paolo Garofalo

Francesco Makovec

Lucio C Rovati

Gianfranco Caselli
email: gianfranco.caselli@rottapharm.com

Efficacy of CR3294, a new benzamidine derivative, in the prevention of 5-fluorouracil-induced gastrointestinal mucositis and diarrhea in mice

Keywords: CR3294, mucositis, chemotherapy, diarrhea, cancer

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

Introduction

Chemotherapy and radiation therapy are useful for treating many forms of cancer. However, they also affect rapidly-dividing cells in healthy tissues, in particular the epithelial cells of the alimentary tract. This is the reason why oral and gastrointestinal (GI) mucositis commonly arises as an adverse effect of anticancer therapy and often causes dose delays or reductions, which can hamper treatment success [START_REF] Duncan | Oral and intestinal mucositis -causes and possible treatments[END_REF][START_REF] Sonis | Perspectives on cancer therapy-induced mucosal injury: pathogenesis, measurement, epidemiology, and consequences for patients[END_REF][START_REF] Rubenstein | Clinical practice guidelines for the prevention and treatment of cancer therapy-induced oral and gastrointestinal mucositis[END_REF].

GI mucositis is associated with many chemotherapy protocols and with radiotherapy directed to any part of the GI tract. These treatments kill progenitor cells in the crypts of Lieberkühn, at the base of the villi, with consequent breakdown of the mucosal barrier [START_REF] Duncan | Oral and intestinal mucositis -causes and possible treatments[END_REF]. Diarrhea is one of the most common manifestations of GI mucositis. The incidence and severity of diarrhea vary greatly, depending on the therapeutic regimen chosen.

Chemotherapy-induced diarrhea (CID) occurs in as many as 80% of cancer patients treated with 5-fluorouracil (5-FU) and irinotecan. Of even greater concern, increased death rates from GI toxicity have been reported in patients receiving irinotecan plus bolus 5-FU/leucovorin [START_REF] Rubenstein | Clinical practice guidelines for the prevention and treatment of cancer therapy-induced oral and gastrointestinal mucositis[END_REF][START_REF] Leichman | Phase II study of fluorouracil and its modulation in advanced colorectal cancer: a Southwest Oncology Group study[END_REF][START_REF] Arnold | Clinical implications of chemotherapy-induced diarrhea in patients with cancer[END_REF][START_REF] Wisinski | Chemotherapy-induced mucositis: focusing on diarrhea[END_REF][START_REF] Rothenberg | Mortality associated with irinotecan plus bolus fluorouracil/leucovorin: summary findings of an independent panel[END_REF]. As the impact of CID rises with the advent of more aggressive anticancer therapies, new therapeutic options for preventing GI mucositis and diarrhea are awaited [START_REF] Arnold | Clinical implications of chemotherapy-induced diarrhea in patients with cancer[END_REF][START_REF] Wisinski | Chemotherapy-induced mucositis: focusing on diarrhea[END_REF][START_REF] Bowen | Prevention and treatment of regimen-related mucosal toxicity[END_REF][START_REF] Keefe | Emerging drugs for chemotherapyinduced mucositis[END_REF]. CR3294 belongs to a novel series of anti-inflammatory agents that inhibit the expression of inducible nitric oxide synthase (iNOS). Notably, this compound protects the epithelium from injury in validated animal models of GI inflammation [START_REF] Tafani | Induction of autophagic cell death by a novel molecule is increased by hypoxia[END_REF][START_REF] Makovec | Benzamidine derivatives having anti-inflammatory and immunosuppressive activity[END_REF].

Mucositis is now regarded as a complex phenomenon in which many cellular and tissue components of the mucosa play a part. In essence, reactive oxygen species (ROS) activate downstream signaling pathways, transcription factors mediate the expression of target genes such as interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α), and these cytokines fuel a vicious loop that harms mucosal integrity [START_REF] Sonis | The pathobiology of mucositis[END_REF][START_REF] Logan | Characterisation of mucosal changes in the alimentary tract following administration of irinotecan: implications for the pathobiology of mucositis[END_REF][START_REF] Leitao | Role of nitric oxide on pathogenesis of 5fluorouracil induced experimental oral mucositis in hamster[END_REF][START_REF] Al-Dasooqi | Matrix metalloproteinases: key regulators in the pathogenesis of chemotherapy-induced mucositis?[END_REF]. On the basis of the evidence available, and because of the unmet medical need, CR3294 was tested as a preventive agent against mucositis.

The aims of this study were to provide in vitro data that support a role for CR3294 in reducing the molecular triggers for mucositis and to assess, in a relevant animal model, whether CR3294 has the potential to prevent GI mucositis and diarrhea. dextran T500 was from Amersham Pharmacia Biotech (Uppsala, Sweden); and multiplex human cytokine kits were from Bio-Rad (Milan, Italy). All general purpose reagents used in animal studies were from Sigma Aldrich (Poole, UK). Animals were purchased from Harlan Laboratories UK Ltd., and tumor cell lines were from the American Type Culture Collection (ATCC). We purchased 5-FU from Roche (Welwyn Garden City, UK). Rat anti-5-bromo-2'-deoxy-uridine (BrdU) clone U1/75 (ICR1) antibody was from AbD SeroTec MorphoSys UK Ltd., with labeling detected using the ABC Elite system from Vector Laboratories UK Ltd.

Materials and Methods

Materials

In vitro studies

ROS production by human polymorphonuclear leukocytes (neutrophils)

Venous blood was collected from healthy human donors in sodium citrate 0.38% and centrifuged at 275 g for 15 minutes. The resulting buffy coat was diluted 3 times (3 mL to 9 mL) with phosphate buffered saline (PBS) and mixed with 2 mL of 6% dextran T500 in 0.9% saline. This mixture was left for 45 minutes at room temperature, then the supernatant was collected and centrifuged at 275 g for 15 minutes. The pellet (leukocytes) was subjected to hypotonic lysis of contaminating erythrocytes by addition of distilled water. The osmolarity was restored with 0.8% NaCl solution. Leukocytes were then centrifuged at 275 g for 15 minutes and washed again in PBS.

Polymorphonuclear leukocytes (PMNL) were isolated by gradient centrifugation (400 g for 30 minutes) on HISTOPAQUE-1077. Purified PMNL were suspended in PBS plus 0.87 mM CaCl 2 and 1 mM MgCl 2 , counted, and diluted to 2.5x10 6 cells/mL. For chemiluminescence assays, the PMNL suspension was premixed with luminol (5 µM final). Aliquots of cell suspension (200 µL) were incubated with CR3294 (1, 3, or 10 µM) or its vehicle (distilled water) for 10 minutes at 37ºC. Next, PMNL were activated with 0.1 µM PMA, and light emission was monitored at 3-minute intervals for 24 minutes in a HTS7000 plus microplate reader. Results are expressed as relative luminescence units (RLU)

Cytokine release from peripheral blood mononuclear cells

Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation on HISTOPAQUE-1077, according to the manufacturer's instructions.

In vivo studies

Ethics and animal care

All studies were conducted in accordance with the UK Home Office Animals (Scientific Procedures) Act 1986, at standards equivalent to the UKCCCR guidelines for the welfare of animals in experimental neoplasia [START_REF]UKCCCR guidelines for the welfare of animals in experimental neoplasia[END_REF]. Animals were held in individually ventilated cages in a specific pathogen-free barrier unit. On arrival from the supplier, mice were housed for 2 weeks to enable acclimatization and stabilize the circadian rhythm. Mice were identified by numbered cages and by ear punches. There was a constant room temperature of 21ºC±2ºC and a mean relative humidity of 55%±10%.

The day-night cycle was constant, with light and dark phases of 12 hours each. Both feed and water (from drinking bottles) were available ad libitum. Animal health was monitored daily and cages were cleaned at regular intervals.

Analysis of intestinal crypt survival

Male BDF1 mice (10-12 weeks of age, n=8 per group) were used in this experiment.

CR3294 was stored at room temperature in the dark: solutions were prepared fresh each day in deionized water, and concentrations were adjusted to give the appropriate dose (2.5, 5, 10, or 20 mg/kg) in 100 µL. Intestinal damage was induced by either 5-FU (2x450 mg/kg intraperitoneally, 6 hours apart) or irradiation (13 Gy) on day 0 of the study, after 3 days of once-daily oral administration of CR3294 or its vehicle.

Irradiation was administered by a Pantak HF320 X-ray unit (Agfa NDT Ltd., Reading, UK). The machine was operated at 300 kV, 10 mA. The X-ray tube was fitted with additional filtration to give a radiation quality of 2.3 mm Cu half-value layer (HVL).

Mice were restrained in a jig positioned at a distance of 700 mm from the focus of the X-ray tube, and irradiation was delivered at a dose rate of 70 cGy/min. An examination of body weight, general condition, behavior, and mortality was performed daily for each mouse. Animals were sacrificed 4 days after the last dose of the cytotoxic insult. The small intestine was removed, fixed in Carnoy's fixative, paraffin embedded, and processed for histology with hematoxylin and eosin (H&E) stain. Ten intestinal circumferences per mice were analyzed. The number of surviving crypts per circumference was scored and the group mean was determined [START_REF] Withers | Microcolony survival assay for cells of mouse intestinal mucosa exposed to radiation[END_REF][START_REF] Potten | The microcolony assay in mouse small intestine[END_REF]. Only crypts containing 10 or more strongly H&E-stained cells (excluding Paneth cells), and only intact circumferences not containing Peyer's patches, were scored. The mean crypt width (at its widest point) was also measured to correct for scoring errors due to crypt size differences [START_REF] Potten | The correction of intestinal microcolony counts for variation in size[END_REF]. The following correction was applied:

Corrected number of crypts per circumference = (mean crypt width in untreated controls/mean crypt width in treatment group) X mean number of surviving crypts in treatment group

The effects of CR3294 were also expressed as Protection Index, i.e., the ratio of surviving crypts in treatment group to surviving crypts in vehicle control group.

Cell proliferation and apoptosis

Male BDF1 mice (10-12 weeks of age, n=6 per group) were randomized into 7 groups and divided into 2 experiments. In the first one we tested the effect of CR3294 (20 mg/kg) on crypt cell proliferation 24 hours after 3 daily doses (i.e., the time at which 5-FU is administered). Animals were dosed with 10 mg of BrdU (0.5 mL intraperitoneally) 40 minutes before sacrifice. In the second experiment we examined the levels of apoptosis 4.5 hours after exposure to single or repeated (2 times, 6 hours apart) cytotoxic doses of 5-FU (450 mg/kg). Mice were sacrificed by cervical dislocation in both studies. The small intestine was removed, fixed in Carnoy's fixative, paraffin embedded, and sectioned to generate 2 non-serial sections per slide. Carnoy's fixed sections were either labeled to reveal BrdU incorporation or H&E stained to measure the incidence of apoptosis. Crypts were analyzed on a cell positional basis to identify drug-induced changes. In all cases, 50 half-crypts per mouse were scored-300 scores per group, from which the frequencies were generated [START_REF] Merritt | Apoptosis in murine intestinal crypts[END_REF]. Quantification of BrdU labeling and apoptosis was performed by the purpose-built WinCrypts software program, which analyzes the statistical significance of any distributions using an extension of the median test. At each cell position the common median for all the animals was calculated, and then the individual values for each animal were classified as being below or above the median [START_REF] Potten | The temporal and spatial changes in cell proliferation within the irradiated crypts of the murine small intestine[END_REF].

Diarrhea assessment

Male BDF1mice (10-12 weeks of age, n=15 per group) were randomized into 2 groups. Dosage regimens for 5-FU and CR3294 (20 mg/kg) were consistent with those used in the experiments described above. Mice were weighed once-daily after the beginning of treatment. Similarly, animal well-being was checked daily, and the incidence and severity of diarrhea were scored. Checks increased to twice a day during days 4-11.

Scores were recorded as 0, 1, 2, or 3 where 0 is normal stool consistency, 1 is loose stools, 2 is overt diarrhea, and 3 is liquid feces with severe perianal/tail soilage (extreme diarrhea). Surviving animals were to be sacrificed 20 days after the cytotoxic insult.

Tumor proliferation models

Two experiments were carried out to rule out adverse effects of CR3294 on tumor growth or 5-FU activity. In the first study, 16 male and 16 female BALB/c nude mice were xenografted with human colorectal (HT29) and breast (MDA-MB-231) cancer cells, respectively. Tumors were measured with calipers 3 times per week and were allowed to grow until the mean tumor volume of the group reached 100-150 mm 3 . Animals were treated with 20 mg/kg CR3294 or vehicle (sterile water) daily for 14 days. In the second study, 32 male BALB/c nude mice were xenografted with the colorectal HT29 cell line and randomized into 4 treatment groups. Groups 2 and 4 were treated with oral CR3294 on days 1-3 and 8-10 (incorporating a 5-day interval). The cytotoxic treatment (5-FU, 25 mg/kg intraperitoneally) was administered on days 1-5 in group 4 as well as in cytotoxic control group 3. The dual vehicle control group (group 1) was administered with the test agent vehicle (water) on days 1-3 and 8-10 and with the 5-FU vehicle (saline) on days 1-5. All the mice in both studies were weighed 3 times per week. Well-being was checked daily: any animal with more than 15% weight loss was considered unwell and killed if the weight loss was greater than 20%. If an animal became unwell, any treatment of that animal was suspended. If there was no recovery, the mouse was killed. Tumor sizes were measured for up to 28 days after the first dose of CR3294 or vehicle.

Statistics

For the evaluation of statistical differences between experimental groups, one-or twoway analysis of variance (ANOVA) followed by Holm-Sidak test for multiple comparisons was used. Data without homogeneity of variance were analyzed with Mann-Whitney Rank Sum Test or Kruskal-Wallis one-way ANOVA, and multiple post-hoc comparisons were performed using Dunn's method. In studies of proliferation and apoptosis, the common median for all the animals (at each cell position in the crypt) was calculated. Next, the individual values for each animal were classified as being below or above the median, and the Chi squared test was applied on the tabulated frequencies. All analyses were done with the SigmaStat version 3.5 software, and a significance level of 0.05 was used throughout.

Results

In vitro studies

First, we tested the hypothesis that CR3294 has the ability to inhibit key molecular events in the onset of mucositis. In these in vitro studies, we used human PMNL and PBMC because leukocytes are a rich source of both ROS and cytokines.

PMA rapidly increased ROS production by PMNL, as measured by chemiluminescence.

CR3294 inhibited this response in a concentration dependent manner, with an IC 50 value of 2.9±0.2 µM and a maximal inhibition of about 76% at 10 µM. The effects of treatment and time, and their interaction, were all significant (P < 0.01, two-way ANOVA). Post-hoc analysis showed significant differences across all groups from 6 minutes onward (Fig. 1).

To assess the impact of CR3294 on cytokine release, human PBMC were stimulated with 1 µg/mL LPS for 4 hours. Under these conditions, CR3294 concentrationdependently inhibited the release of TNF-α, IL-6, and IL-8. The IC 50 values ranged between 6 and 9 µM. Moreover, CR3294 potently inhibited the release IL-1β, with an IC 50 of about 100 nM. Similar results were obtained in two independent experiments, each done in duplicate, and significant inhibitions were generally seen at concentrations of 3 µM or higher.

In vivo studies

Effects of CR3294 on the mucosal epithelium

Next, we studied whether orally administered CR3294 could protect crypt cells in the intestinal mucosa against damage induced by chemotherapy or radiation. We tested different dosing schemes and found that CR3294 was especially effective when given as a preventive measure-i.e., 72, 48, and 24 hours before cytotoxic treatment. Under these conditions, none of the animals receiving 5-FU died before the scheduled sacrifice time (4 days after the last dose of 5-FU). All mice, except those in the untreated control group, lost weight to similar extents and with similar kinetics (not shown). CR3294 protected the mucosa against 5-FU-induced toxicity, and there was a clear doseresponse effect. The corrected number of surviving crypts per circumference in the group treated with 20 mg/kg CR3294 was nearly 3-fold that in vehicle-treated mice (18.1±1.9 vs. 6.5±0.9, Protection Index 2.8, P < 0.001; Table 1). Conversely, no conclusive effects of CR3294 were seen in a model of radiation-induced mucositis. The Protection Index ranged from 1 to 1.9 in three different experiments, and there was no clear dose-response relationship.

More experiments were run to determine whether CR3294 protected the epithelium by increasing cell proliferation or by decreasing the sensitivity of crypt cells-either all cells or a particular cell type in the crypt hierarchy-to 5-FU-induced apoptosis. As expected, sections of small intestine from control mice showed high levels of proliferation: about half of the cells in the mid-crypt region were labeled with BrdU.

Twenty-four hours after the last dose of CR3294 (20 mg/kg), there were no significant changes in the frequency or distribution of BrdU labeling (Fig. 2a). One or two doses (6 hours apart ) of 5-FU induced progressively higher rates of crypt cell apoptosis.

Pretreatment with 20 mg/kg of CR3294 did not affect the levels of apoptosis induced by 5-FU (Fig. 2b).

Effect of CR3294 on 5-FU-induced diarrhea

We then assessed in a proof-of-concept study whether CR3294, at the dose (20 mg/kg) that significantly decreased mucosal damage, also reduced the incidence and/or severity of diarrhea in mice exposed to 5-FU. Nearly all mice (13 out of 15) receiving 5-FU and vehicle developed diarrhea starting from day 4. The number of mice without diarrhea for the entire study was 3-fold higher in the group treated with CR3294 than in vehicletreated animals. Moreover, pretreatment with CR3294 reduced the cumulative symptom score by 50% (Fig. 3). Moderate-to-severe diarrhea (grade 2-3) was more frequent in the vehicle-treated group than in the group treated with CR3294 (13 vs. 4 observations; nearly 70% reduction). Similar results were found when we analyzed the 7-day study period during which all mice were alive (Fig. 4). Statistical analysis showed a significant difference (P < 0.05, Mann-Whitney Rank Sum Test) between the two groups in the median diarrhea score.

Animals died between days 8 and 13 after 5-FU challenge. Thus, CR3294 did not improve survival, although the onset of death was slightly delayed in the group receiving the active treatment (4 deaths during days 8 and 9 vs. 7 deaths during the same period in the vehicle-treated group).

Tumor proliferation models

To rule out adverse effects of CR3294 on tumor growth or 5-FU activity, we finally undertook two studies in xenograft models of human colorectal (HT29)/breast (MDA-MB-231) cancer. In the first study, the mean volume of HT29 and MDA-MB-231 tumors grew about 10-and 7-fold, respectively, over 1 month. Two weeks of treatment with CR3294 20 mg/kg once-daily had no effect on the rate of tumor growth (Fig. 5a and 5b) and did not affect animal weight. In the second study, 5-FU inhibited the growth of HT29 xenografts, and CR3294 did not hamper the response to the chemotherapy drug (Fig. 6).

Discussion

Severe alimentary mucositis has a major impact on patients undergoing chemotherapy or radiation therapy. There is clearly an unmet medical need for drugs that reduce the rate and severity of GI mucositis and diarrhea [START_REF] Keefe | Emerging drugs for chemotherapyinduced mucositis[END_REF]. Indeed, clinical practice guidelines call for sustained efforts to develop better therapeutic strategies, targeted to the mechanisms of mucosal injury [START_REF] Keefe | Updated clinical practice guidelines for the prevention and treatment of mucositis[END_REF]. The present experimental study demonstrates that CR3294 improves the signs and symptoms of GI mucositis by blocking key molecular events in the pathogenesis of this condition.

CR3294 is an anti-inflammatory agent that inhibits the expression of iNOS and cyclooxygenase 2 (COX-2) [START_REF] Tafani | Induction of autophagic cell death by a novel molecule is increased by hypoxia[END_REF]. Recent findings suggest a role for iNOS and COX-2 in the onset of mucositis [START_REF] Sonis | The pathobiology of mucositis[END_REF][START_REF] Leitao | Role of nitric oxide on pathogenesis of 5fluorouracil induced experimental oral mucositis in hamster[END_REF][START_REF] Sonis | The relationship between mucosal cyclooxygenase-2 (COX-2) expression and experimental radiation-induced mucositis[END_REF]. However, it is widely accepted that ROS initiate the cascade of events-including increased levels of inflammatory cytokines-that leads to mucosal damage [START_REF] Sonis | The pathobiology of mucositis[END_REF]. In this study, CR3294 inhibited the release of IL-1β, IL-6, IL-8, and TNF-α from stimulated leukocytes at concentrations similar to those required to inhibit ROS production by these cells. The link between oxidative stress and mucositis is confirmed by other studies in which ROS scavengers were tested as protective agents against radiation-induced damage [START_REF] Murphy | Efficacy of superoxide dismutase mimetic M40403 in attenuating radiationinduced oral mucositis in hamsters[END_REF][START_REF] Facorro | Oxidative study of patients with total body irradiation: effects of amifostine treatment[END_REF]. Palifermin, the only drug approved for the treatment of oral mucositis, is a human keratinocyte growth factor (KGF) produced by recombinant DNA technology. In addition to stimulating epithelial cell proliferation, KGF activates the transcription factor NF-E2-related factor 2 (Nrf2), which in turn increases the expression of ROS-scavenging enzymes [START_REF] Braun | Nrf2 transcription factor, a novel target of keratinocyte growth factor action which regulates gene expression and inflammation in the healing skin wound[END_REF]. Because the use of antiinflammatory agents is now regarded as a promising approach to reduce the severity of mucositis [START_REF] Lalla | Anti-inflammatory agents in the management of alimentary mucositis[END_REF], there is a strong rationale for developing new drugs with combined ROS-scavenging and anti-inflammatory properties. This is particularly relevant to GI mucositis, for which no therapy is currently approved although a variety of agents have been assessed in preclinical and clinical studies. Recently, alternative medicine approach gained attention. Probiotics have been shown to reduce intestinal inflammation and diarrhea in experimental models of GI mucositis in rats [START_REF] Bowen | VSL#3 probiotic treatment reduces chemotherapy-induced diarrhea and weight loss[END_REF][START_REF] Smith | Lactobacillus fermentum BR11 and fruct-oligosaccharide partially reduce jejunal inflammation in a model if intestinal mucositis inrats[END_REF][START_REF] Whitford | Effects of Streptococcus thermophilus TH-4 on intestinal mucositis induced by the chemotherapeutic agent, 5-Fluorouracil (5-FU)[END_REF].

Herbal extracts, due to their anti-inflammatory and anti-oxidative properties and in keeping with CR3294 activity, have been shown to decrease mucosal damage in preclinical and clinical studies [START_REF] Cheah | Grape seed extract protects IEC-6 cells from chemotherapy-induced cytotoxicity and improves parameters of small intestinal mucositis in rats with experimentally-induced mucositis[END_REF][START_REF] You | effect of extracts from indigowood root (Isatis indigotica Fort.) on immune responses in radiation-induced mucositis[END_REF].

The main goal of this study was to examine whether CR3294 has the potential to prevent GI mucositis and diarrhea. In a first set of in vivo experiments we found that CR3294 significantly protected crypt cells against damage induced by 5-FU.

Conversely, no definite effects were seen in an experimental model of radiation-induced mucositis. Different factors could account for the lack of conclusive results in this experimental setting. Firstly, the adopted protocol may be non-optimal. Further studies with different radiation schemes have been planned to test the above hypothesis.

Additionally, because X-rays and 5-FU target different cells in the intestinal crypts [START_REF] Ijiri | Further studies on the response of intestinal crypt cells of different hierarchical status to eighteen different cytotoxic agents[END_REF], it is conceivable that CR3294 protects a specific cell type in the crypt hierarchy. Unlike KGF, CR3294 did not stimulate epithelial cell proliferation, but similarly to KGF, the effectiveness of CR3294 was greatest when it was administered in a preventive mode [START_REF] Farrell | Keratinocyte growth factor protects mice from chemotherapy and radiation-induced gastrointestinal injury and mortality[END_REF]. This finding suggests that other mechanisms, besides ROS scavenging, contribute to the cytoprotective activity of this agent. One such mechanism may involve decreased sensitivity to 5-FU-induced apoptosis [START_REF] Hwang | Ferredoxin reductase affects p53-dependent, 5-fluorouracil-induced apoptosis in colorectal cancer cells[END_REF], but our experiments did not show significant differences in this regard. Alternatively, given that CR3294 can induce the formation of autophagosomes [START_REF] Tafani | Induction of autophagic cell death by a novel molecule is increased by hypoxia[END_REF], it is possible that CR3294 increased crypt survival by activating the autophagic pathway. Autophagy may indeed serve as a rescue mechanism whereby cells extend their lifespan under critical conditions [START_REF] Yorimitsu | Autophagy: molecular machinery for selfeating[END_REF]. Although analyzing autophagyrelated processes was beyond the scope of our present work, studies are under way to test the above hypothesis.

Here we were interested in assessing whether mucosal protection could translate into a lower incidence of diarrhea, because CID is associated with dose reductions, dose delays, and excess hospitalizations and deaths. In our proof-of-concept study, pretreatment with CR3294 halved the cumulative diarrhea score in mice exposed to 5-FU. CR3294 was particularly effective against moderate-to-severe CID: animals treated with the 20 mg/kg dose were 70% less likely to experience grade 2-3 diarrhea than vehicle-treated mice. Severe diarrhea is common in patients receiving fluoropyrimidinebased chemotherapy [START_REF] Leichman | Phase II study of fluorouracil and its modulation in advanced colorectal cancer: a Southwest Oncology Group study[END_REF][START_REF] Wisinski | Chemotherapy-induced mucositis: focusing on diarrhea[END_REF][START_REF] Saltz | Understanding and managing chemotherapy-induced diarrhea[END_REF]. In the absence of effective drugs to avoid this adverse event, treatment has superseded prevention in the clinical setting, and high-dose loperamide is used for managing mild-to-moderate diarrhea. Unfortunately, loperamide is far less effective when it comes to severe episodes, those that may be life threatening [START_REF] Rothenberg | Mortality associated with irinotecan plus bolus fluorouracil/leucovorin: summary findings of an independent panel[END_REF][START_REF] Saltz | Understanding and managing chemotherapy-induced diarrhea[END_REF][START_REF] Benson | Recommended guidelines for the treatment of cancer treatment-induced diarrhea[END_REF].

CR3294 did not improve animal survival, even though it slightly delayed the onset of death after exposure to 5-FU. However, our proof-of-concept study was designed to detect an effect of the compound on diarrhea rate and severity rather than on death from GI syndrome. Damage to the GI tract, if sufficiently serious, would cause death within 6 to 7 days, whereas none of the mice in our study died before day 8 after 5-FU injection. So, mortality was presumably associated with damage to the hematopoietic system [START_REF] Potten | Protection of the small intestinal clonogenic stem cells from radiation-induced damage by pretreatment with interleukin 11 also increases murine survival time[END_REF], and clearly CR3294 did not limit chemical-induced toxicity to the bone marrow.

Likewise, CR3294 neither altered the antitumor activity of 5-FU nor affected the growth rate of tumor xenografts in both female and male mice. Candidate drugs for the prevention or treatment of mucositis must not protect tumors. For instance, other authors have demonstrated that palifermin does not stimulate the growth of tumors, even those expressing the KGF receptor [START_REF] Smith | Lactobacillus fermentum BR11 and fruct-oligosaccharide partially reduce jejunal inflammation in a model if intestinal mucositis inrats[END_REF][START_REF] Gibson | Palifermin reduces diarrhea and increases survival following irinotecan treatment in tumor-bearing DA rats[END_REF]. Regardless that CR3294 is unlikely to promote cell proliferation via KGF-dependent mechanisms, we ruled out unexpected yet possible untoward effects. A recent study has even shown that CR3294 reduces tumor growth in vivo [START_REF] Tafani | Induction of autophagic cell death by a novel molecule is increased by hypoxia[END_REF]. The variable results between that study and the present one might be due either to the different models used or to the different duration of treatment (8 vs.

weeks).

In summary, our experimental study demonstrates that CR3294 acts on critical mechanisms of mucosal injury to reduce the signs of GI mucositis as well as the incidence and severity of 5-FU-induced diarrhea. These positive results warrant further investigation in humans because no drugs are currently approved for the prevention of CID. Phase I clinical trials are now under way to evaluate the safety profile of CR3294. were pretreated with CR3294 or its vehicle 72, 48, and 24 hours before receiving 2 doses (6 hours apart) of the cytotoxic agent. Scores were recorded as 0, 1, 2, or 3 where 0 is normal stools, 1 is loose stools, 2 is moderate diarrhea, and 3 is severe diarrhea CR3294 or its vehicle (water) was administered orally on days 1-3 and 8-10, whereas 5-FU (25 mg/kg) or its vehicle (saline) was administered intraperitoneally on days 1-5

Figure legends

  CR3294, N-[4-[[(pentylamino)thioxomethyl]amino]phenyl]-ethanimidamide, monohydrochloride, was synthesized by the Medicinal Chemistry Department of Rottapharm S.p.A. (Monza, Italy). Phorbol-12-myristate-13-acetate (PMA), luminol, HISTOPAQUE-1077, and lipopolysaccharide (LPS) were from Sigma (Milan, Italy);

  Purified cells were resuspended in RPMI-1640 culture medium containing 1% fetal bovine serum and adjusted to 2x10 6 cells/mL. Aliquots of cell suspension (200 µL) were added to duplicate wells of a flat-bottom 96-well plate containing CR3294 or vehicle (distilled water) and incubated for 1 hour at 37ºC in a humidified atmosphere of 5% CO 2 . Cytokine synthesis was induced by a 4-hour stimulation with 1 µg/mL LPS (from E. Coli, serotype 0127B7). To rule out cytotoxic effects, parallel plates were tested for viability by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. At the end of the incubation period, supernatants were collected and assayed for IL-1β, TNF-α, IL-6, and IL-8 by Bio-Plex technology (BioRad, Milan, Italy) according to the recommended procedures. Data were analyzed using the Bio-Plex Manager™ software from Bio-Rad (version 4.0), and results are given as IC 50 values (µM).

Fig. 1 Fig. 2 Fig. 3

 123 Fig. 1 Effects of increasing concentrations of CR3294 on ROS production by PMNL

Fig. 4 Fig. 5

 45 Fig. 4 Incidence of diarrhea by severity score. Animals (n=15 per group) were

Fig. 6

 6 Fig. 6 Tumor response to 5-FU in the presence or absence of CR3294. Male BALB/c

Table 1

 1 Effect of pretreatment with CR3294 on 5-FU-induced intestinal crypt death

	Tables
	Pretreatment a	Surviving crypts per	Protection Index, fold
		circumference b , mean ± SE	change compared to
			vehicle-treated controls
	Vehicle	6.5±0.9	-
	CR3294 2.5 mg/kg	9.9±1.0	1.5
	CR3294 5 mg/kg	11.7±2.5	1.8
	CR3294 10 mg/kg	11.8±1.2	1.8
	CR3294 20 mg/kg	18.1±1.9*	2.8
	Each treatment group comprised 8 mice
	a 72, 48, and 24 h before 2x450 mg/kg 5-FU
	*P < 0.001, Holm-Sidak method

b Corrected number, expressed as the following ratio: (mean crypt width in untreated controls/mean crypt width in treatment group) X mean number of surviving crypts in treatment group

Page 31 of 36 Cancer Chemotherapy and Pharmacology

Acknowledgments

The authors thank Mr. Mario Montagna for technical support and Mrs. Laura Radaelli for secretary assistance.

This study was funded by the Rottapharm Group. However, the Rottapharm Group as a corporate entity had no role in the conduct of the study; in the collection, analysis and interpretation of data; in the writing of the manuscript; and in the decision to submit the manuscript for publication. This work was part of a project co-financed in conjunction with the Italian Ministry of Health (Attività di ricerca finalizzata D.Lgs. 502/92).

Conflict of interest

All authors except Catherine Booth are employees of Rottapharm S.p.A. Catherine Booth is an employee of EpiStem Ltd., a research organization under contract with the Rottapharm Group.