BACKGROUND
the tyrosine kinase inhibitor imatinib , belonging to the 2-phenylaminopyrimidine class, selectively inhibits bcr/abl  <cit> , pdgfr , c-kit  <cit> , and c-fms  <cit>  kinase activity.
as imatinib is commonly used without chemotherapeutic agents, few reports have evaluated the therapeutic effect of concomitant administration of imatinib and chemotherapy either in mice xenografted tumors or cancer patients.
using a human small cell lung cancer  xenograft in nude mice, we previously reported enhanced tumor growth inhibition following chemotherapy  in combination with imatinib and showed that this effect was not dependent on c-kit expression level  <cit> .
the increase of conventional antineoplastic agent-induced tumor growth inhibition was also exclusively observed when imatinib and chemotherapy were administered concomitantly.
various mechanisms can be proposed to explain this effect, namely  an increase of drug uptake by imatinib-induced reduction of tumor interstitial fluid pressure and an increase of transcapillary transport  <cit> ,  inhibition of tumor angiogenesis combined with the antitumor effect of chemotherapy  <cit> , and  certain intracellular events induced by the two concomitant therapeutic modalities that remain to be determined.
however, our in vivo experiments showed a higher toxicity of combined imatinib and chemotherapy than for imatinib or chemotherapy alone, exclusively when both treatments were administered concomitantly.
various mechanisms for the toxicity of combined therapy can be proposed, particularly pharmacokinetic interactions between imatinib and chemotherapeutic agents that could also explain the increase of chemotherapy-induced tumor growth inhibition, as numerous data have shown that imatinib induces cytochrome p <dig> inhibition and that this inhibition decreases elimination of chemotherapeutic agents such as etoposide, ifosfamide, and topotecan  <cit> .
in order to explore these pharmacokinetic interactions, we therefore investigated the in vivo impact of imatinib on the pharmacokinetics of chemotherapy and its efficacy on two different human cancers xenografted into immunodeficient mice: a sclc  <cit>  and an ebv-associated b-cell lymphoproliferation  <cit> .
we showed that imatinib significantly increases the auc  of etoposide in mice via a decrease of its fecal excretion.
finally, in one of the two xenografted models used, we confirmed the imatinib-induced increase of tumor growth inhibition after chemotherapy with agents such as etoposide  and gemcitabine.
these results therefore support the initiation of further prospective phase i and ii clinical trials combining etoposide-based chemotherapy and imatinib in cancer patients, such as malignant lymphoma patients, with careful toxicologic monitoring.
methods
in vivo experiments in immunodeficient mice bearing human tumors
female nude or scid mice, weighing  <dig> g to  <dig> g, 6– <dig> weeks old, were bred in the animal facilities , maintained under specific pathogen-free conditions with artificial lighting  and fed with a regular diet and water ad libitum.
the care, housing, and handling of the mice were performed in accordance with the recommendations of the french ethics committee and under the supervision of authorized investigators.
for curative therapeutic trials, the tumor-bearing mice were randomly divided into equivalent groups of  <dig> to  <dig> animals and mice were treated at different times after transplantation.
two different human tumors were used: the sclc <dig> small cell lung cancer xenografted in nude mice  <cit> , and the ly- <dig> ebv-associated human b-cell lymphoma xenografted in scid mice  <cit> .
sti <dig>  was diluted in  <dig> μl of h2o and administered at a total dosage of  <dig> mg/kg per day in one intraperitoneal injection on different days, as indicated.
etoposide  and ifosfamide  were diluted in  <dig> μl of  <dig> % sodium chloride and administered at a dose of  <dig> mg/kg and  <dig> mg/kg, respectively, in one daily intraperitoneal injection on days  <dig> to  <dig> of treatment.
fluconazole  was diluted in  <dig> μl of water and administered at a total dosage of  <dig> mg/kg per day in one intraperitoneal injection on days  <dig> to  <dig> of treatment.
gemcitabine  was diluted in  <dig> μl of  <dig> % sodium chloride and administered in one weekly intraperitoneal injection at a dosage of  <dig> mg/kg per day.
the control group received  <dig> % sodium chloride injections according to the same schedule as experimentally treated mice.
all mice were weighed once weekly.
tumor growth was monitored by measuring two perpendicular diameters with calipers.
tumor volume  and relative tumor volume  were calculated as follows:
v = a <dig> × b/ <dig>
where a is the width  and b the length  of the tumor in millimeters.
rtv = vx/vi,
where vx is the mean tumor volume in cubic millimeters at any given time and vi is the mean initial tumor volume in cubic millimeters at the start of treatment  <cit> .
mice were ethically sacrificed when the tumor volume reached  <dig>  mm <dig> in the control group.
pharmacokinetics of etoposide  and/or ifosfamide in mice
in order to evaluate pharmacokinetic interactions between sti <dig>  and chemotherapeutic agents , various concentrations of the two cytotoxic agents were measured.
plasma, urine, and fecal concentrations of etoposide were determined by a validated high performance liquid chromatography  method with u.v.
detection  <cit> .
standard samples were prepared from a stock solution of  <dig> μg/ml etoposide which was added to drug-free pooled plasma or urine.
the analytes were extracted from plasma, urine and feces by liquid-liquid extraction into aqueous phase with dichloromethane.
all 24-hour fecal samples were weighed and homogenized with  <dig> ml of water.
plasma concentrations of ifosfamidewere determined by a validated gas chromatography assay  with nitrogen-phosphorus detection  <cit> .
the analytes were extracted from plasma and urine by liquid-liquid extraction into aqueous phase with ethyl acetate.
evaluation of c-kit mrna expression
total rna extraction and cdna synthesis were performed as previously described  <cit> .
briefly, total rna was extracted from crushed tumor samples  by rna plus® kit .
one μg of total rna was reverse transcribed in a final volume of  <dig> μl containing 1× reverse transcriptase buffer ,  <dig> units of rnase inhibitor ,  <dig> mm dtt,  <dig> units of superscript ii rnase h-reverse-transcriptase , and  <dig>  mm random hexamers .
the reaction mix was then incubated at 42°c for  <dig> min.
c-kit transcripts were quantified using real-time quantitative reverse transcription-pcr assays.
primers and probes were chosen with the assistance of primer express software .
the nucleotide sequence was then blasted against dbest and nr  to confirm the total gene specificity of the nucleotide sequences chosen as primers at probes.
each primer couple was positioned in different exons  of the gene in order to avoid amplification of contaminating genomic dna.
pcr reactions were performed using an abi prism  <dig> sequence detection system  and core reagent kit .
real-time detection was performed using oligonucleotide probes containing a fluorescent dye at its 5'-end and a quencher at its 3'-end .
fluorescent probes were synthesized by applied biosystems, and primers were synthesized by invitrogen .
the nucleotide and probe sequences are as follows: c-kit: upper primer 5'-aagcagatttcagagagcacca-3', lower primer 5'gctgccgacagaattgatcc-3' and probe 5'actccaacttagcaaactgcagccccaa-3'.
transcripts of tbp  <cit>  were also quantified as endogenous rna of reference genes to normalize c-kit expression.
the thermal cycling conditions comprised an initial denaturation step at 95°c for  <dig> min, then  <dig> cycles at 95°c for  <dig> sec and an annealing temperature depending upon the target.
for c-kit quantification, a calibration curve for mrna expression was generated using serial dilutions of l <dig> human colon carcinoma cell line mrna expressing high levels of c-kit mrna to assess pcr efficiency.
all measurements were performed under blind conditions and in duplicate.
two negative "non-template" controls were included in each amplification run.
results were expressed as n-fold differences in target gene expression relative to the reference gene  and the calibrator was called "ntarget".
it was determined as follows: n target = e, where the Δct values of the sample and calibrator were determined by subtracting the average ct values of the target gene from the average value of the reference gene  and e is the efficiency of pcr measured using the slope of the calibration curve.
for c-kit mrna quantification, the positive control, corresponding to a case of uveal melanoma exhibiting strong c-kit overexpression, was assessed with sclc samples.
statistical analysis
statistical analysis of variance  was performed to assess the difference between auc0– <dig> h  of etoposide in the various treatments.
a mann-whitney u-test was used to assess the in vivo effect of the various treatments on the growth of xenografted tumors in nude or scid mice.
u values were considered significant when the probability of a difference was less than or equal to  <dig> .
values were considered significant when the probability of a difference was less than or equal to  <dig> .
RESULTS
impact of sti <dig> on the in vivo xenografted tumor growth of etoposide  chemotherapy
sclc6-bearing nude mice received intraperitoneal injections of etoposide  on days 1– <dig>  with or without sti <dig> administered on days 1– <dig> by intraperitoneal injections at a total dose of  <dig> mg/kg daily, and with or without intraperitoneal injections of fluconazole  on days 1– <dig>  each treatment was initiated on day +  <dig> after tumor transplantation.
we observed a brief but non-significant inhibition of slcl <dig> tumor growth after etoposide administration compared to the control group , and this inhibition was not increased by concomitant injections of etoposide + sti <dig> or etoposide + sti <dig> + fluconazole .
as previously performed for the sclc <dig> tumor , we first evaluated the antitumor effect of sti <dig> alone in the scid mice xenografted ly- <dig> lymphomatous tumors.
mice bearing tumors measuring approximately  <dig> mm <dig> were treated from day  <dig> until sacrifice of the animals by an intraperitoneal injection of sti <dig> at a dose of  <dig> mg/kg.
a slight but non-significant tumor growth inhibition was temporarily observed for a few days after initiation of treatment .
thereafter, scid mice bearing ly- <dig> tumors were treated by intraperitoneal injections of etoposide  on days 1– <dig>  with or without intraperitoneal injection of sti <dig> at a dose of  <dig> mg/kg on days 1– <dig>  and with or without intraperitoneal injections of fluconazole  on days 1– <dig>  each treatment was initiated at day +  <dig> after tumor transplantation.
we observed a spectacular enhancement of etoposide-induced tumor growth inhibition by concomitant administration of sti <dig> , and this inhibition was not enhanced by concomitant administration of fluconazole .
moreover, we observed three early deaths in the combined etoposide + sti <dig> group, but not in the etoposide + sti <dig> + fluconazole group.
finally, in order to confirm the value of combining sti <dig> with other chemotherapy modalities, mice bearing ly- <dig> tumors measuring approximately  <dig> mm <dig> received weekly intraperitoneal injections of gemcitabine , with or without intraperitoneal injections of sti <dig> at a total dose of  <dig> mg/kg daily from day  <dig> until sacrifice of the animals.
we observed an enhancement of gemcitabine-induced tumor growth inhibition by concomitant and continuous administration of sti <dig> .
impact of sti <dig> on the pharmacokinetics of combined etoposide  and ifosfamide chemotherapy regimen in mice
to study the impact of sti <dig> on the in vivo pharmacokinetics of chemotherapy, c <dig> black <dig> mice were treated on days  <dig> to  <dig> by concomitant intraperitoneal injections of etoposide  and ifosfamide , with or without sti <dig> , and serum was collected from an eye vein on days  <dig> and  <dig>   <dig>   <dig>   <dig> and  <dig> hours after injections.
we observed an increase of the cmax of both etoposide and ifosfamide on days  <dig> and  <dig> when mice were treated by chemotherapy combined with sti <dig> compared to chemotherapy alone .
to confirm this result and to focus on etoposide pharmacokinetics, cd <dig> mice were treated on days 1– <dig> by intraperitoneal injections of  <dig> mg/kg/d etoposide, with or without intraperitoneal  <dig> mg/kg/d of sti <dig>  samples were collected on days  <dig> and  <dig>   <dig> ,  <dig>   <dig> and  <dig> hours after injections.
we observed a significant increase of the mean auc0– <dig> h  of etoposide  when mice were treated with etoposide + sti <dig> .
in order to evaluate the role of cyp3a <dig> on the pharmacokinetic interaction between etoposide and sti <dig>  cd <dig> mice were treated on days 1– <dig> by intraperitoneal injections of etoposide , with or without sti <dig> , and with or without the cyp3a <dig> inhibitor, fluconazole .
the samples were collected on days  <dig> and  <dig>   <dig> ,  <dig>   <dig> and  <dig> hours after injections.
a summary of etoposide pharmacokinetic parameters  is presented in table  <dig>  we observed a highly significant increase of the mean auc0– <dig> h of etoposide  when mice were treated with etoposide + sti <dig> + fluconazole , as compared to either etoposide + sti <dig>  or etoposide + fluconazole  combinations .
in both experiments, after administration of vp <dig> with sti <dig>  vp <dig> concentrations peaked at  <dig>  ±  <dig>  ng/ml, followed by a bi-exponential decline .
vp <dig> concentrations reached  <dig>  ±  <dig>  ng/ml when vp <dig> was administered with sti <dig> and fluconazole.
in both cases, the pharmacokinetic curve was best described as bi-exponential as demonstrated by the concentration-time curve of vp <dig> administered with sti <dig> .
to explain the impact of sti <dig> on the pharmacokinetics of etoposide, we studied the liver and kidney clearance of etoposide by assaying the chemotherapeutic agent in urine and feces of cd <dig> mice treated on day  <dig> by one intraperitoneal injection of etoposide  with or without sti <dig> .
samples were collected for  <dig> hours after injection.
we observed a significant decrease of fecal excretion of etoposide when mice were treated with either etoposide + sti <dig>  or etoposide + sti <dig> + fluconazole  .
in contrast, no significant impact of sti <dig> was observed on the renal clearance of etoposide, with  or without fluconazole .
evaluation of c-kit mrna expressions
the level of c-kit mrna expression was quantified and compared to the level of mrna expression of a known c-kit protein overexpressing melanoma.
as previously reported  <cit> , the two sclc <dig> and ly- <dig> xenografts express very low levels of c-kit mrna .
discussion
in conclusion, we have shown that imatinib increased the auc of etoposide in mice and that this effect was mediated by a reduction of its fecal excretion.
in contrast, imatinib did not appear to influence the urinary excretion of etoposide, probably due to sample collection conditions, and concomitant administration of the cyp3a <dig> inhibitor fluconazole with imatinib did not modify the pharmacokinetics of etoposide plus imatinib alone.
we also demonstrated a slight tumor growth inhibition induced by imatinib administered alone in one in vivo ebv-associated b-cell lymphomatous xenograft, and this effect was observed despite the absence of c-kit receptor mrna expression.
finally, when mice bearing human xenografted tumors were treated concomitantly by imatinib and chemotherapy, an increase of the chemotherapy-induced antitumor effect was observed in the lymphoma model but not in a small cell lung cancer model, and this antitumor effect was not influenced by concomitant administration of fluconazole.
our in vivo b-cell lymphoma studied by rt-pcr was negative for c-kit mrna expression.
this result was concordant with that reported in the literature, where c-kit expression, studied by rt-pcr or immunohistochemical methods, was mainly observed in multiple myeloma, cd30-positive anaplastic large cell lymphoma, and hodgkin's disease, as shown in table  <dig> <cit> .
moreover, very few data have been published on the therapeutic efficacy of imatinib on lymphoproliferative diseases.
imatinib demonstrated a minimal effect in anaplastic large cell lymphoma with npm-alk fusion protein  <cit>  and hodgkin lymphoma  <cit>  cell lines.
in contrast, despite an in vitro antiproliferative effect of sti <dig> on a multiple myeloma cell line  <cit> , no response was observed in a phase ii trial of imatinib in patients with refractory/relapsed myeloma  <cit> .
abbreviations: l., lymphoma; mm, multiple myeloma; dlbcl, diffuse large b-cell lymphoma; hd, hodgkin's disease.
the combination of imatinib and chemotherapy has not yet been reported in the literature.
however, despite a large number of in vitro studies, very few data are available on in vivo effect of combined imatinib and chemotherapeutic agents.
these reports evaluated combinations of imatinib and zoledronate  <cit> , troxacitabine  <cit> , epothilone b  <cit> , and paclitaxel  <cit> .
in our study, we showed that the imatinib-induced increase of etoposide auc enhances the tumor growth inhibition of a human xenografted lymphomatous tumor, but not that of an in vivo human small cell lung cancer tumor.
this observation argues in favor of an increased in vivo efficacy of etoposide combined with imatinib, showing that chemotherapy resistance could be reversed by the combination with imatinib in some cancer situations, such as our ebv-associated b-cell lymphomatous model and several other small cell lung cancer xenografts previously reported  <cit> .
a number of published clinical studies have used various chemotherapeutic regimens in various cancer indications.
all these data are summarized in table  <dig> <cit> .
because of the heterogeneity of these series, it is impossible to clearly define the impact of these combinations on patient outcome.
however, they provide a good overview of the toxicities.
some observed side effects can be mainly attributed to the use of antineoplastic drugs, namely hematologic toxicity, nausea and vomiting, infections, and diarrhea, while other side effects could be attributed to the use of imatinib, such as fatigue, peripheral edema, skin eruptions, neuropathy, and liver dysfunction.
only one phase i study of imatinib mesylate combined with doxorubicin and gemcitabine in patients with small cell lung carcinoma showed a high rate of dose-limiting toxicity that required early discontinuation of the trials  <cit> .
* nausea-vomiting.
* fatigue.
* abdominal pain.
* peripheral edema.
* fatigue.
* nausea.
* nausea-vomiting.
* reduction of lvef.
* diarrhea.
* thrombosis.
* infections.
* nausea.
abbreviations: hyper-cvad, cyclophosphamide + vincristine + doxorubicin + dexamethasone + methotrexate + cytarabine; lvef, left ventricular ejection fraction.
CONCLUSIONS
in summary, imatinib potentiates the effects of etoposide in cancer cells such as lymphoma cell lines.
these results therefore show that further prospective phase i and ii clinical trials with combinations of etoposide-based chemotherapy and imatinib are therefore warranted in some cancer patients, such as malignant lymphoma patients, with careful toxicologic monitoring.
authors' contributions
kr, fl, and sw contributed to the pharmacokinetics experiments.
ig, vb, re, and mg contributed to the in vivo experiments.
pdc contributed to the c-kit mrna expression determination.
mfp contributed to the correction of the manuscript.
dd and fl contributed to the direction of the experiments.
dd conceived of the study.
all authors read and approved the final manuscript.

 the tyrosine kinase inhibitor imatinib, belonging to the 2-phenylaminopyrimidine class, selectively inhibits bcr/abl, pdgfr, c-kit, and c-fms kinase activity. as imatinib is commonly used without chemotherapeutic agents, few reports have evaluated the therapeutic effect of concomitant administration of imatinib and chemotherapy either in mice xenografted tumors or cancer patients. using a human small cell lung cancer xenograft in nude mice, we previously reported enhanced tumor growth inhibition following chemotherapy in combination with imatinib and showed that this effect was not dependent on c-kit expression level. the increase of conventional antineoplastic agent-induced tumor growth inhibition was also exclusively observed when imatinib and chemotherapy were administered concomitantly. various mechanisms can be proposed to explain this effect, namely an increase of drug uptake by imatinib-induced reduction of tumor interstitial fluid pressure and an increase of transcapillary transport, inhibition of tumor angiogenesis combined with the antitumor effect of chemotherapy, and certain intracellular events induced by the two concomitant therapeutic modalities that remain to be determined. however, our in vivo experiments showed a higher toxicity of combined imatinib and chemotherapy than for imatinib or chemotherapy alone, exclusively when both treatments were administered concomitantly. various mechanisms for the toxicity of combined therapy can be proposed, particularly pharmacokinetic interactions between imatinib and chemotherapeutic agents that could also explain the increase of chemotherapy-induced tumor growth inhibition, as numerous data have shown that imatinib induces cytochrome p inhibition and that this inhibition decreases elimination of chemotherapeutic agents such as etoposide, ifosfamide, and topotecan. in order to explore these pharmacokinetic interactions, we therefore investigated the in vivo impact of imatinib on the pharmacokinetics of chemotherapy and its efficacy on two different human cancers xenografted into immunodeficient mice: a sclc and an ebv-associated b-cell lymphoproliferation. we showed that imatinib significantly increases the auc of etoposide in mice via a decrease of its fecal excretion. finally, in one of the two xenografted models used, we confirmed the imatinib-induced increase of tumor growth inhibition after chemotherapy with agents such as etoposide and gemcitabine. these results therefore support the initiation of further prospective phase i and ii clinical trials combining etoposide-based chemotherapy and imatinib in cancer patients, such as malignant lymphoma patients, with careful toxicologic monitoring. methods in vivo experiments in immunodeficient mice bearing human tumors female nude or scid mice, weighing g to g, 6– weeks old, were bred in the animal facilities, maintained under specific pathogen-free conditions with artificial lighting and fed with a regular diet and water ad libitum. the care, housing, and handling of the mice were performed in accordance with the recommendations of the french ethics committee and under the supervision of authorized investigators. for curative therapeutic trials, the tumor-bearing mice were randomly divided into equivalent groups of to animals and mice were treated at different times after transplantation. two different human tumors were used: the sclc small cell lung cancer xenografted in nude mice, and the ly- ebv-associated human b-cell lymphoma xenografted in scid mice. sti was diluted in μl of h2o and administered at a total dosage of mg/kg per day in one intraperitoneal injection on different days, as indicated. etoposide and ifosfamide were diluted in μl of % sodium chloride and administered at a dose of mg/kg and mg/kg, respectively, in one daily intraperitoneal injection on days to of treatment. fluconazole was diluted in μl of water and administered at a total dosage of mg/kg per day in one intraperitoneal injection on days to of treatment. gemcitabine was diluted in μl of % sodium chloride and administered in one weekly intraperitoneal injection at a dosage of mg/kg per day. the control group received % sodium chloride injections according to the same schedule as experimentally treated mice. all mice were weighed once weekly. tumor growth was monitored by measuring two perpendicular diameters with calipers. tumor volume and relative tumor volume were calculated as follows: v = a × b/ where a is the width and b the length of the tumor in millimeters. rtv = vx/vi, where vx is the mean tumor volume in cubic millimeters at any given time and vi is the mean initial tumor volume in cubic millimeters at the start of treatment. mice were ethically sacrificed when the tumor volume reached mm in the control group. pharmacokinetics of etoposide and/or ifosfamide in mice in order to evaluate pharmacokinetic interactions between sti and chemotherapeutic agents, various concentrations of the two cytotoxic agents were measured. plasma, urine, and fecal concentrations of etoposide were determined by a validated high performance liquid chromatography method with u.v. detection. standard samples were prepared from a stock solution of μg/ml etoposide which was added to drug-free pooled plasma or urine. the analytes were extracted from plasma, urine and feces by liquid-liquid extraction into aqueous phase with dichloromethane. all 24-hour fecal samples were weighed and homogenized with ml of water. plasma concentrations of ifosfamidewere determined by a validated gas chromatography assay with nitrogen-phosphorus detection. the analytes were extracted from plasma and urine by liquid-liquid extraction into aqueous phase with ethyl acetate. evaluation of c-kit mrna expression total rna extraction and cdna synthesis were performed as previously described. briefly, total rna was extracted from crushed tumor samples by rna plus® kit. one μg of total rna was reverse transcribed in a final volume of μl containing 1× reverse transcriptase buffer, units of rnase inhibitor, mm dtt, units of superscript ii rnase h-reverse-transcriptase, and mm random hexamers. the reaction mix was then incubated at 42°c for min. c-kit transcripts were quantified using real-time quantitative reverse transcription-pcr assays. primers and probes were chosen with the assistance of primer express software. the nucleotide sequence was then blasted against dbest and nr to confirm the total gene specificity of the nucleotide sequences chosen as primers at probes. each primer couple was positioned in different exons of the gene in order to avoid amplification of contaminating genomic dna. pcr reactions were performed using an abi prism sequence detection system and core reagent kit. real-time detection was performed using oligonucleotide probes containing a fluorescent dye at its 5'-end and a quencher at its 3'-end. fluorescent probes were synthesized by applied biosystems, and primers were synthesized by invitrogen. the nucleotide and probe sequences are as follows: c-kit: upper primer 5'-aagcagatttcagagagcacca-3', lower primer 5'gctgccgacagaattgatcc-3' and probe 5'actccaacttagcaaactgcagccccaa-3'. transcripts of tbp were also quantified as endogenous rna of reference genes to normalize c-kit expression. the thermal cycling conditions comprised an initial denaturation step at 95°c for min, then cycles at 95°c for sec and an annealing temperature depending upon the target. for c-kit quantification, a calibration curve for mrna expression was generated using serial dilutions of l human colon carcinoma cell line mrna expressing high levels of c-kit mrna to assess pcr efficiency. all measurements were performed under blind conditions and in duplicate. two negative "non-template" controls were included in each amplification run. results were expressed as n-fold differences in target gene expression relative to the reference gene and the calibrator was called "ntarget". it was determined as follows: n target = e, where the Δct values of the sample and calibrator were determined by subtracting the average ct values of the target gene from the average value of the reference gene and e is the efficiency of pcr measured using the slope of the calibration curve. for c-kit mrna quantification, the positive control, corresponding to a case of uveal melanoma exhibiting strong c-kit overexpression, was assessed with sclc samples. statistical analysis statistical analysis of variance was performed to assess the difference between auc0– h of etoposide in the various treatments. a mann-whitney u-test was used to assess the in vivo effect of the various treatments on the growth of xenografted tumors in nude or scid mice. u values were considered significant when the probability of a difference was less than or equal to. values were considered significant when the probability of a difference was less than or equal to.  impact of sti on the in vivo xenografted tumor growth of etoposide chemotherapy sclc6-bearing nude mice received intraperitoneal injections of etoposide on days 1– with or without sti administered on days 1– by intraperitoneal injections at a total dose of mg/kg daily, and with or without intraperitoneal injections of fluconazole on days 1– each treatment was initiated on day + after tumor transplantation. we observed a brief but non-significant inhibition of slcl tumor growth after etoposide administration compared to the control group, and this inhibition was not increased by concomitant injections of etoposide + sti or etoposide + sti + fluconazole. as previously performed for the sclc tumor, we first evaluated the antitumor effect of sti alone in the scid mice xenografted ly- lymphomatous tumors. mice bearing tumors measuring approximately mm were treated from day until sacrifice of the animals by an intraperitoneal injection of sti at a dose of mg/kg. a slight but non-significant tumor growth inhibition was temporarily observed for a few days after initiation of treatment. thereafter, scid mice bearing ly- tumors were treated by intraperitoneal injections of etoposide on days 1– with or without intraperitoneal injection of sti at a dose of mg/kg on days 1– and with or without intraperitoneal injections of fluconazole on days 1– each treatment was initiated at day + after tumor transplantation. we observed a spectacular enhancement of etoposide-induced tumor growth inhibition by concomitant administration of sti, and this inhibition was not enhanced by concomitant administration of fluconazole. moreover, we observed three early deaths in the combined etoposide + sti group, but not in the etoposide + sti + fluconazole group. finally, in order to confirm the value of combining sti with other chemotherapy modalities, mice bearing ly- tumors measuring approximately mm received weekly intraperitoneal injections of gemcitabine, with or without intraperitoneal injections of sti at a total dose of mg/kg daily from day until sacrifice of the animals. we observed an enhancement of gemcitabine-induced tumor growth inhibition by concomitant and continuous administration of sti. impact of sti on the pharmacokinetics of combined etoposide and ifosfamide chemotherapy regimen in mice to study the impact of sti on the in vivo pharmacokinetics of chemotherapy, c black mice were treated on days to by concomitant intraperitoneal injections of etoposide and ifosfamide, with or without sti, and serum was collected from an eye vein on days and and hours after injections. we observed an increase of the cmax of both etoposide and ifosfamide on days and when mice were treated by chemotherapy combined with sti compared to chemotherapy alone. to confirm this result and to focus on etoposide pharmacokinetics, cd mice were treated on days 1– by intraperitoneal injections of mg/kg/d etoposide, with or without intraperitoneal mg/kg/d of sti samples were collected on days and, and hours after injections. we observed a significant increase of the mean auc0– h of etoposide when mice were treated with etoposide + sti. in order to evaluate the role of cyp3a on the pharmacokinetic interaction between etoposide and sti cd mice were treated on days 1– by intraperitoneal injections of etoposide, with or without sti, and with or without the cyp3a inhibitor, fluconazole. the samples were collected on days and, and hours after injections. a summary of etoposide pharmacokinetic parameters is presented in table we observed a highly significant increase of the mean auc0– h of etoposide when mice were treated with etoposide + sti + fluconazole, as compared to either etoposide + sti or etoposide + fluconazole combinations. in both experiments, after administration of vp with sti vp concentrations peaked at ± ng/ml, followed by a bi-exponential decline. vp concentrations reached ± ng/ml when vp was administered with sti and fluconazole. in both cases, the pharmacokinetic curve was best described as bi-exponential as demonstrated by the concentration-time curve of vp administered with sti. to explain the impact of sti on the pharmacokinetics of etoposide, we studied the liver and kidney clearance of etoposide by assaying the chemotherapeutic agent in urine and feces of cd mice treated on day by one intraperitoneal injection of etoposide with or without sti. samples were collected for hours after injection. we observed a significant decrease of fecal excretion of etoposide when mice were treated with either etoposide + sti or etoposide + sti + fluconazole. in contrast, no significant impact of sti was observed on the renal clearance of etoposide, with or without fluconazole. evaluation of c-kit mrna expressions the level of c-kit mrna expression was quantified and compared to the level of mrna expression of a known c-kit protein overexpressing melanoma. as previously reported, the two sclc and ly- xenografts express very low levels of c-kit mrna. discussion in conclusion, we have shown that imatinib increased the auc of etoposide in mice and that this effect was mediated by a reduction of its fecal excretion. in contrast, imatinib did not appear to influence the urinary excretion of etoposide, probably due to sample collection conditions, and concomitant administration of the cyp3a inhibitor fluconazole with imatinib did not modify the pharmacokinetics of etoposide plus imatinib alone. we also demonstrated a slight tumor growth inhibition induced by imatinib administered alone in one in vivo ebv-associated b-cell lymphomatous xenograft, and this effect was observed despite the absence of c-kit receptor mrna expression. finally, when mice bearing human xenografted tumors were treated concomitantly by imatinib and chemotherapy, an increase of the chemotherapy-induced antitumor effect was observed in the lymphoma model but not in a small cell lung cancer model, and this antitumor effect was not influenced by concomitant administration of fluconazole. our in vivo b-cell lymphoma studied by rt-pcr was negative for c-kit mrna expression. this result was concordant with that reported in the literature, where c-kit expression, studied by rt-pcr or immunohistochemical methods, was mainly observed in multiple myeloma, cd30-positive anaplastic large cell lymphoma, and hodgkin's disease, as shown in table. moreover, very few data have been published on the therapeutic efficacy of imatinib on lymphoproliferative diseases. imatinib demonstrated a minimal effect in anaplastic large cell lymphoma with npm-alk fusion protein and hodgkin lymphoma cell lines. in contrast, despite an in vitro antiproliferative effect of sti on a multiple myeloma cell line, no response was observed in a phase ii trial of imatinib in patients with refractory/relapsed myeloma. abbreviations: l., lymphoma; mm, multiple myeloma; dlbcl, diffuse large b-cell lymphoma; hd, hodgkin's disease. the combination of imatinib and chemotherapy has not yet been reported in the literature. however, despite a large number of in vitro studies, very few data are available on in vivo effect of combined imatinib and chemotherapeutic agents. these reports evaluated combinations of imatinib and zoledronate, troxacitabine, epothilone b, and paclitaxel. in our study, we showed that the imatinib-induced increase of etoposide auc enhances the tumor growth inhibition of a human xenografted lymphomatous tumor, but not that of an in vivo human small cell lung cancer tumor. this observation argues in favor of an increased in vivo efficacy of etoposide combined with imatinib, showing that chemotherapy resistance could be reversed by the combination with imatinib in some cancer situations, such as our ebv-associated b-cell lymphomatous model and several other small cell lung cancer xenografts previously reported. a number of published clinical studies have used various chemotherapeutic regimens in various cancer indications. all these data are summarized in table. because of the heterogeneity of these series, it is impossible to clearly define the impact of these combinations on patient outcome. however, they provide a good overview of the toxicities. some observed side effects can be mainly attributed to the use of antineoplastic drugs, namely hematologic toxicity, nausea and vomiting, infections, and diarrhea, while other side effects could be attributed to the use of imatinib, such as fatigue, peripheral edema, skin eruptions, neuropathy, and liver dysfunction. only one phase i study of imatinib mesylate combined with doxorubicin and gemcitabine in patients with small cell lung carcinoma showed a high rate of dose-limiting toxicity that required early discontinuation of the trials. * nausea-vomiting. * fatigue. * abdominal pain. * peripheral edema. * fatigue. * nausea. * nausea-vomiting. * reduction of lvef. * diarrhea. * thrombosis. * infections. * nausea. abbreviations: hyper-cvad, cyclophosphamide + vincristine + doxorubicin + dexamethasone + methotrexate + cytarabine; lvef, left ventricular ejection fraction.  in summary, imatinib potentiates the effects of etoposide in cancer cells such as lymphoma cell lines. these results therefore show that further prospective phase i and ii clinical trials with combinations of etoposide-based chemotherapy and imatinib are therefore warranted in some cancer patients, such as malignant lymphoma patients, with careful toxicologic monitoring. authors' contributions kr, fl, and sw contributed to the pharmacokinetics experiments. ig, vb, re, and mg contributed to the in vivo experiments. pdc contributed to the c-kit mrna expression determination. mfp contributed to the correction of the manuscript. dd and fl contributed to the direction of the experiments. dd conceived of the study. all authors read and approved the final manuscript. 
altogether, these results therefore justify further prospective phase i and ii clinical trials with combinations of etoposide-based chemotherapy and imatinib in patients with certain cancers, such as malignant lymphoma, with careful toxicologic monitoring. slight tumor growth inhibition was induced by imatinib administered alone in one in vivo ebv-associated b-cell lymphomatous xenograft. in contrast, imatinib did not appear to influence the urinary excretion of etoposide, and concomitant administration of the cyp3a inhibitor, fluconazole, with imatinib did not modify the pharmacokinetics of etoposide plus imatinib alone. in contrast, an increase of the chemotherapy-induced antitumor effect was observed in the lymphoma model but not in a small cell lung cancer model when mice bearing human xenografted tumors were treated concomitantly by imatinib and chemotherapy. this antitumor effect was not influenced by concomitant administration of fluconazole. using a human small cell lung cancer xenografted in nude mice, we have previously reported enhanced tumor growth inhibition following chemotherapy in combination with imatinib. two different human tumors were used: sclc small cell lung cancer xenografted in nude mice, and ly- ebv-associated human b-cell lymphoma xenografted in scid mice. we therefore investigated the in vivo impact of imatinib on the pharmacokinetics and efficacy of chemotherapy. plasma, urine, and fecal concentrations of etoposide were determined by a validated high performance liquid chromatography method. plasma concentrations of ifosfamidewere determined by a validated gas chromatography assay with nitrogen-phosphorus detection. the auc0- h of etoposide was increased when mice were treated with etoposide + imatinib due to decreased fecal excretion.
 using a human small cell lung cancer xenografted in nude mice, we have previously reported enhanced tumor growth inhibition following chemotherapy in combination with imatinib. we therefore investigated the in vivo impact of imatinib on the pharmacokinetics and efficacy of chemotherapy. methods two different human tumors were used: sclc small cell lung cancer xenografted in nude mice, and ly- ebv-associated human b-cell lymphoma xenografted in scid mice. plasma, urine, and fecal concentrations of etoposide were determined by a validated high performance liquid chromatography method. plasma concentrations of ifosfamidewere determined by a validated gas chromatography assay with nitrogen-phosphorus detection.  slight tumor growth inhibition was induced by imatinib administered alone in one in vivo ebv-associated b-cell lymphomatous xenograft. in contrast, an increase of the chemotherapy-induced antitumor effect was observed in the lymphoma model but not in a small cell lung cancer model when mice bearing human xenografted tumors were treated concomitantly by imatinib and chemotherapy. this antitumor effect was not influenced by concomitant administration of fluconazole. the auc0- h of etoposide was increased when mice were treated with etoposide + imatinib due to decreased fecal excretion. in contrast, imatinib did not appear to influence the urinary excretion of etoposide, and concomitant administration of the cyp3a inhibitor, fluconazole, with imatinib did not modify the pharmacokinetics of etoposide plus imatinib alone.  altogether, these results therefore justify further prospective phase i and ii clinical trials with combinations of etoposide-based chemotherapy and imatinib in patients with certain cancers, such as malignant lymphoma, with careful toxicologic monitoring. 