Inhibition of OCT2, MATE1 and MATE2-K as a possible mechanism of drug interaction between pazopanib and cisplatin.

We hypothesized that pazopanib is an inhibitor of cisplatin renal transporters OCT2, MATE1 and MATE2-K based on previous studies demonstrating an interaction between tyrosine kinase inhibitors and these transporters. Because several combinations of targeted therapies and cytotoxics are currently in development for cancer treatment, such an interaction is worth investigating. Experiments on HEK293 cells stably transfected to express OCT2, MATE1, MATE2-K or an empty vector (EV) were conducted. The inhibitory effect of pazopanib on these transporters was measured using the uptake of fluorescent substrate ASP+ and cisplatin in the different cell lines. The effect of pazopanib on cisplatin-induced cytotoxicity was also evaluated. A decrease of ASP+ uptake was observed in OCT2-HEK, MATE1-HEK and MATE2K-HEK cell lines after addition of pazopanib at increasing concentrations. Pazopanib inhibited cisplatin specific uptake in OCT2-HEK, MATE1-HEK and MATE2K-HEK lines. Cytotoxicity experiments showed that co-incubation of cisplatin with pazopanib multiplied up to 2.7, 2.4 and 1.6 times the EC50 values of cisplatin in OCT2-HEK, MATE1-HEK and MATE2K-HEK cell lines respectively, reaching about the same values as in EV-HEK cells. To conclude, pazopanib inhibits OCT2, MATE1 and MATE2-K, which are involved in cisplatin secretion into urine. The combination of these two drugs may lead to an interaction and increase the cisplatin-induced systemic toxicity. Given the wide variability of plasma pazopanib concentrations observed in vivo, the interaction may occur in a clinical setting, particularly in overexposed patients. The existence of a drug-drug interaction should be investigated when pazopanib is associated with a substrate of these transporters.

Genotyping of a family with a novel deleterious DPYD mutation supports the pretherapeutic screening of DPD deficiency with dihydrouracil/uracil ratio.

Despite the growing evidence that dihydropyrimidine dehydrogenase deficiency (DPD, encoded by the DPYD gene) confers a higher risk of developing severe toxicity, most patients are not screened for DPD deficiency before fluoropyrimidine treatment. We report here the genetic and phenotypic analyses of DPD in a family related to a patient who died after a first cycle of 5-fluorouracil and in 15 additional retrospective patients having a partial DPD deficiency (as measured by plasma dihydrouracil/uracil ratio). The patient with lethal toxicity was found to be a compound heterozygote for two DPYD mutations: a novel 8-bp duplication (c.168_175dupGAATAATT, p.Phe59Ter) and c.1679T>G (Ile560Ser). The patient's dihydrouracil/uracil ratio indicates complete DPD deficiency. The novel mutation was found in two members of the patient's family. Deleterious DPYD mutations were identified in 9 out of the 15 patients. The relationship between genotype and dihydrouracil/uracil values in the 22 patients of the present study was significant (P = 0.01).

Trastuzumab induced in vivo tissue remodelling associated in vitro with inhibition of the active forms of AKT and PTEN and RhoB induction in an ovarian carcinoma model.

BACKGROUND: The incidence of ovarian cancer has been increasing worldwide and it is currently the leading cause of death from gynaecological malignancy. Unlike breast cancer, the prognostic role of the human epidermal growth factor receptor-2 (HER-2) in ovarian carcinoma remains controversial. METHODS: The aim of this preclinical study was to further characterise the biological, molecular and cellular effects of trastuzumab (Herceptin) using NIH-OVCAR-3 and derived cell lines both in vitro and in vivo. RESULTS: In vitro assessments have shown that trastuzumab treatment inhibited total and phosphorylated HER-2. This was associated with inhibition of the phosphorylated form of phosphatase and tensin homologue (PTEN), mitogen-activated protein kinase and AKT, and the total level of p27(kip). Inhibition of PTEN is associated with phosphorylated MEK1/2 upregulation, suggesting a specific inhibition of the protein phosphatase function of PTEN. Moreover, trastuzumab induced the upregulation of RhoB. These molecular modifications promote inhibition of cell migration and potentially restoration of tumour cell contact inhibition. RhoB induction in NIH-OVCAR-3 control cell lines mimics the molecular and cellular trastuzumab long-time exposition effects. RhoB inhibition in NIH-OVCAR-3 long-time exposed to trastuzumab cell line reverses the cellular and molecular effects observed in this model. In vivo examinations have shown that these changes are also associated with the restoration of structural, morphological and normal functions of the peritoneum of an ovarian carcinoma mouse model. CONCLUSION: These results provide an indication of the mechanisms underlying the anti-tumour activity of trastuzumab that strongly implicate RhoB in an ovarian carcinoma model that does not show HER-2 amplification or overexpression. These findings highlight that trastuzumab effects involve a possible cross-talk between RhoB and PTEN in the early stages of tumour re-growth in a model of micrometastatic ovarian cancer.

[Hyperthermic intraoperative intraperitoneal chemotherapy (HIPEC): evaluation, prevention and policies to avoid occupational exposure for operating room personnel]. / Chimiohyperthermie intrapéritonéale : évaluation, prévention et gestion des risques professionnels au bloc opératoire.

To develop a treatment strategy for peritoneal carcinomatosis using a combination of extended peritoneal resections, local destructive procedures and hyperthermic intraperitoneal chemotherapy creates great concern between healthcare workers involved in these procedures. New professional risks exist: risk of exposure to cytotoxic drugs, environmental risks (inhalation of smoke, aerosolization of chemotherapy agents). Information, education and training of healthcare workers is mandatory in order to ensure proper evaluation, prevention, and management of professional exposure risks in coordination with the occupational health office.

Preclinical study of an ex vivo gene therapy protocol for hepatocarcinoma.

Preclinical studies in several animal models as well as clinical trials have shown a reduction in tumor growth following immunotherapy with interleukin-12 (IL-12). This cytokine is appropriate to test in therapeutic clinical trials to treat hepatocarcinoma (HC), a pathology often associated with hepatitis B or C-induced cirrhosis. The local delivery into the liver would be achieved through ex vivo gene transfer using retroviral (rv) vectors in autologous fibroblast carriers. In support of this clinical trial, a rv vector has been constructed to express coordinately both chains p35 and p40 of human IL-12. Here, we have tested good manufacturing practices (GMP) clinical lots of viral vectors derived from the transfected packaging cell line, PG13rvIL-12. We have also devised methods to facilitate the isolation of fibroblasts from freshly harvested skin specimens, enhance their outgrowth in large-scale cultures and assay IL-12 production following transduction, without any selection and irradiation. Twenty-four human skin specimens were processed to obtain fibroblast suspensions that were typically maintained for up to 8 or 12 passages. The mean +/-s.d. overall time for obtaining the required number of transduced cells for the highest IL-12 need was 40 days. The procedure, in accordance with the French medical agency for gene therapy clinical trials, is now ready to begin a clinical trial.

Selective inhibition of HER2 inhibits AKT signal transduction and prolongs disease-free survival in a micrometastasis model of ovarian carcinoma.

Although first-line chemotherapy induces complete clinical remission in many cases of epithelial ovarian cancer, relapse usually occurs 18-28 months from diagnosis owing to micrometastases. The present study aimed to evaluate the effect of trastuzumab on disease-free and overall survival in a specially designed murine model of ovarian cancer (OVCAR-3), which mimicked the natural history of human micrometastatic disease. Trastuzumab can cure the mice if started soon after induction chemotherapy. It can modestly inhibit the proliferation through mitogen-activated protein kinase signal transduction and clearly inhibit AKT phosphorylation, which is involved in survival pathway. As OVCAR-3 cell lines show no HER2 amplification or overexpression, these results warrant further studies to assess the efficacy of trastuzumab in the early stage of relapse in cancer models other than those overexpressing HER2.

Cellular determinants of oxaliplatin sensitivity in colon cancer cell lines.

Oxaliplatin (L-OHP) is a new platinum analogue that has shown antitumour activity against colon cancer both in vitro and in vivo and is now used in the chemotherapeutic treatment of metastatic colon and rectal cancer. L-OHP like cisplatin (CDDP), is detoxified by glutathione (GSH)-related enzymes and forms platinum (Pt)-DNA adducts lesions that are repaired by the nucleotide excision repair system (NER). We investigated the cytotoxicity and the pharmacology of L-OHP and CDDP on a panel of six colon cell lines in vitro. We showed that GSH and glutathione S-transferase (GST) activity were not correlated to oxaliplatin cytotoxicity. Pt-DNA adducts formation and repair were correlated with CDDP, but not with L-OHP cytotoxicity. The determination of ERCC1 and XPA expression, two enzymes of the NER pathway, by reverse transcriptase-polymerase chain reaction (RT-PCR), demonstrated that ERCC1 expression was predictive of L-OHP sensitivity (r(2)=0.67, P=0.02) and XPA level after oxaliplatin exposure was also correlated to L-OHP IC(50) (r(2)=0.5; P=0.04). The knowledge of such correlations could help predict the sensitivity of patients with colon cancer to L-OHP.

Combination of oxaliplatin and irinotecan on human colon cancer cell lines: activity in vitro and in vivo.

The in vitro and in vivo combination of oxaliplatin and irinotecan was investigated in a panel of four human colon cancer cell lines and their counterpart xenografts. In vitro and in vivo experiments demonstrated a synergistic or additive interaction in three cell lines (HCT-116, HCT-8 and HT-29) and an antagonism in SW-620 cells. Since there were clearly opposite interactions depending on the cell line, we further investigated cellular determinants possibly involved in the interaction between the two drugs in HCT-8 and SW-620 cells. Irinotecan slowed down the early platinum-DNA adducts repair (1 h after oxaliplatin exposure) in the presence of irinotecan only in HCT-8 cells (p=0.03, n=3). Moreover, a decrease of the expression of two proteins of the nucleotide excision repair (NER) system, ERCC1 and XPA, was observed. None of these effects was seen in SW-620 cells. Irinotecan induced apoptosis with an increase of poly(ADP-ribose) polymerase (PARP) cleavage in SW-620 cells (60 versus 7% basal level). Pretreatment of these cells with oxaliplatin abolished the increase in PARP cleavage induced by irinotecan (29%). In HCT-8 cells, a very little PARP cleavage was observed whatever the drug treatment. The persistence of platinum-DNA adducts in the presence of irinotecan could be due to a direct impact of irinotecan on NER gene expression or to an indirect effect on topoisomerase I activity. Complementary studies are required to determine if the cellular parameters identified in this study could be translated at the clinical level to predict clinical response after combined treatment with oxaliplatin and irinotecan in humans.

Schedule-dependent activity of topotecan in OVCAR-3 ovarian carcinoma xenograft: pharmacokinetic and pharmacodynamic evaluation.

Topotecan is a topoisomerase (Topo) I inhibitor used in ovarian carcinoma chemotherapy. Topo I inhibitors are thought to be more cytotoxic using protracted schedules of administration. We tested this hypothesis on a preclinical model: human ovarian carcinoma OVCAR-3 implanted i.p. Nude mice were treated i.p. with a total dose of topotecan of 12.5 mg/kg delivered in 1, 5, 10, 20, 40, or 80 daily injections. The toxicity was maximal when the total dose was delivered within 5 and 10 days of treatment. However, the efficacy was the greatest (all of the mice cured) in the 20-day schedule using 0.625 mg/kg/day, hence, making this latter schedule the most efficient without any major toxicity. A pharmacokinetic study was conducted to identify parameters related to the efficacy and toxicity of topotecan in our model. The use of a population pharmacokinetic approach allowed us to define a therapeutic window: maintaining plasma concentrations above 0.2 microM for >10 h was necessary for an optimal antitumor effect and avoiding plasma concentrations >0.7 microM allowed a manageable toxicity. Finally, Topo I activity was monitored in ascites from animals treated with different topotecan administration schedules. The optimal schedule defined above allowed for sustained inhibition of Topo I activity associated with a greater antitumor activity. These in vivo data constitute a rationale for clinical studies testing this type of administration.

Characterization of CPT-11 converting carboxylesterase activity in colon tumor and normal tissues: comparison with p-nitro-phenylacetate converting carboxylesterase activity.

Irinotecan (CPT-11) is a topoisomerase I inhibitor commonly used in the treatment of colorectal tumors. It is a prodrug, converted to an active metabolite, SN-38, by carboxylesterases (CEs). CEs are ubiquitary enzymes that react with numerous substrates. A specific CPT-11 converting enzyme was isolated from rat serum, with different kinetic properties than other CEs. We determined kinetic properties of specific CPT-11 CE activity (CPT-CE) in human normal liver and colon tumors. Km were very similar (3.4 microM in liver and 3.8 microM in colon tumors), but Vmax was higher in liver (2.7 pmol/min/mg protein) than in colon tumor (1.7 pmol/min/mg protein). CPT-CE and total CE (using p-nitro-phenylacetate as substrate) were weakly correlated in colon tumors. The large interpatient variability observed in liver CPT-CE activity could play a potential role in the pharmacokinetic variability observed with irinotecan.

CPT-11 converting carboxylesterase and topoisomerase activities in tumour and normal colon and liver tissues.

CPT-11 is a prodrug activated by carboxylesterases to the active metabolite SN-38 which is a potent inhibitor of topoisomerase I. CPT-11 is of clinical interest in the treatment of colorectal cancer. We evaluated the activities of CPT-11 converting carboxylesterase (CPT-CE) and topoisomerase I (topo I) in 53 colorectal tumours, in eight liver metastases and in normal tissue adjacent to the tumours. Both CPT-CE and topo I activities were widely variable in the malignant and the normal tissue of patients with colorectal carcinomas. CPT-CE was only two to threefold lower in primary tumours compared to normal liver, suggesting that a local conversion to SN-38 might occur in tumour cells. CPT-CE was similar in liver and in normal colon tissues. Levels of topo I in tumour ranged from 580 to 84 900 U mg protein(-1) and was above 40 000 U mg protein(-1) in 11 of 53 patients. Similarly, a very high ratio (> 5) between tumour and normal tissues were observed in 12 of 53 patients. An inverse correlation was observed between the topo I activity and the clinical stage of disease. Clinical studies are in progress in our institution to explore a possible relationship between CPT-CE and topo I activities in tumour cells and the response to CPT-11-based chemotherapy in patients with colorectal cancer.

Cellular interactions of 5-fluorouracil and the camptothecin analogue CPT-11 (irinotecan) in a human colorectal carcinoma cell line.

CPT-11 (irinotecan) is a DNA topoisomerase I inhibitor active against metastatic colorectal carcinoma. We investigated, in a human colon carcinoma cell line, HT-29, the effects of CPT-11 and 5-fluorouracil (5FU) combinations. A strong synergism between CPT-11 and 5FU was observed after sequential exposure and only additivity or antagonism after simultaneous exposure. When cells were first exposed to 5FU, the product of cellular CPT-11 concentrations versus time (CxT) was 6895 +/- 1020 pmol x hr/10(6) cells, while it was 3875 +/- 121 pmol x hr/10(6) cells with CPT-11 alone (p < 0.01). The same phenomenon was observed with SN-38: 148.2 +/- 49.5 versus 83.4 +/- 23.6 pmol x hr/10(6) cells (p < 0.05). Consequently, the formation of protein-DNA complexes was 1.4 times greater with 5FU pretreatment than with CPT-11 alone (p = 0.03). Moreover, the incorporation of 5FU derivatives into DNA was multiplied by a factor of 1.5 24 hr after CPT-11 exposure. When cells were first incubated with CPT-11, the decrease in thymidylate synthase (TS) activity was identical to that obtained after 5FU exposure (1.09 to 0.023 pmol/min/mg protein), but this decrease persisted for 24 hr (0.014 pmol/min/mg protein) (p = 0.035). At the same time, a 1.8-fold increase in the incorporation of 5FU derivatives into DNA and a 2-fold increase in DNA-protein complex formation were evidenced. With the two sequential associations, we observed a persistent S-phase arrest, as compared with CPT-11 alone. These results suggest that CPT-11 and 5FU combinations are of clinical interest, and mechanisms of interaction between the two drugs seem to be multifactorial.

Sequence-dependent activity of the irinotecan-5FU combination in human colon-cancer model HT-29 in vitro and in vivo.

Irinotecan, a DNA-topoisomerase-I inhibitor, is active against metastatic colon carcinoma. We investigated the effects of irinotecan and 5FU combinations in human colon-carcinoma cell line HT-29, both in vitro and in vivo. Cytotoxicity of 24-hr exposure was evaluated by SRB technique and the nature of interactions were determined by median-effect analysis. Strong synergism between irinotecan and 5FU was observed after sequential exposure, and only additivity after simultaneous exposure. At 50% level of kill, the mean sums of fractional effects were 0.13 +/- 0.05 and 0.18 +/- 0.02 respectively for the 2 sequential schedules, indicating that the combined amount of the 2 drugs necessary to kill 50% cells was only 0.18 and 0.13 times respectively, as much as would be required if they demonstrated purely additive behavior. In nude-mice xenografts, schedule-dependent toxicity was observed: the schedule in which irinotecan was administered i.v. 6 hours before 5FU was the most toxic. Higher anti-tumoral activity was noted when 20 mg/kg/day of each drug was administered sequentially (a delay of 6 hr between the 2 drugs) to mice over 5 days, in comparison with simultaneous administration. In vivo data confirmed those obtained in vitro in the same human colon-cancer model. These results suggest that irinotecan and 5FU combinations are of clinical interest and that the schedule of administration is a critical parameter for chemotherapeutic efficacy.

A chemiluminescent microplate assay to detect DNA damage induced by genotoxic treatments.

A damaged DNA detection assay (3D assay) using plasmid DNA adsorbed on sensitized microplates as the substrate for an in vitro repair reaction is presented. DNA lesions are repaired by the excision repair pathway which implies an incision-excision reaction followed by DNA repair synthesis. In the 3D assay, we took advantage of (i) plasmid DNA adsorption on polylysine-coated microplates that allowed various DNA-damaging treatments; (ii) a protein extract that reproduced the repair reaction in vitro; (iii) incorporation of digoxigenylated deoxynucleotide monophosphate during the DNA polymerization step which was quantified by a chemiluminescent reaction. Under experimental conditions for quantitative DNA adsorption, a dose-response relationship between the extent of DNA modification and the repair synthesis activity was found. Optimization of the biochemical parameters with UVC light-induced DNA lesions allowed the detection of about one photoproduct per plasmid circle. This new assay that permits a quick and easy assessment of DNA damage is applicable to the screening of genotoxic compounds and to the testing of DNA-damaging treatments.