TREATMENT OF CANCER BY SYSTEMIC ADMINISTRATION OF DBAIT MOLECULES

Abstract

The present invention relates to the use of a DBait molecules by systemic routes without any combination with an endosomolytic agent.

Claims

1. A method of treating ovarian cancer comprising the administration, by a parenteral systemic route selected from intraperitoneal and intravenous administration to a subject having an ovarian cancer, a nucleic acid molecule of the following formula: ##STR00014## wherein N is a deoxynucleotide, n is an integer from 15 to 195, the underlined N refers to a nucleotide having or not a modified phosphodiester backbone, L is a linker, C is the molecule facilitating endocytosis selected from a lipophilic molecule or a ligand which targets cell receptor enabling receptor mediated endocytosis, L is a linker, m is an integer being 0 or 1 and p is 1; wherein the nucleic acid is to be used without combined administration of any quinoline endosomolytic agent.

2. The method according to claim 1, wherein the nucleic acid of formula (I) has one or several of the following features: N is a deoxynucleotide selected from the group consisting of A (adenine), C (cytosine), T (thymine) and G (guanine) and selected so as to avoid occurrence of a CpG dinucleotide and to have less than 80% sequence identity to any gene in a human genome; and/or the linked L is selected from the group consisting of hexaethyleneglycol, tetradeoxythymidylate (T4) and 1,19-bis(phospho)-8-hydraza-2-hydroxy-4-oxa-9-oxo-nonadecane; and/or m is 1 and L is a carboxamido polyethylene glycol; and/or C is selected from the group consisting of a cholesterol, single or double chain fatty acids and a ligand which targets cell receptor.

3. The method according to claim 1, wherein the nucleic acid molecule has one of the following formulae: ##STR00015## wherein the underlined nucleotide refers to a nucleotide having or not a phosphorothioate or methylphosphonate backbone, the linked L is selected from the group consisting of hexaethyleneglycol, tetradeoxythymidylate (T4) and 1,19-bis(phospho)-8-hydraza-2-hydroxy-4-oxa-9-oxo-nonadecane; m is 1 and L is a carboxamido oligoethylene glycol, C is selected from the group consisting of dioleoyl, octadecyl, folic acid, tocopherol and cholesterol.

4. The method according to claim 1, wherein the nucleic acid is ##STR00016## and wherein the underlined nucleotide refers to a nucleotide having a phosphorothioate backbone, the linked L is 1,19-bis(phospho)-8-hydraza-2-hydroxy-4-oxa-9-oxo-nonadecane; m is 1 and L is a carboxamido tetraethylene glycol, C is cholesterol.

5. The method according to claim 1, wherein the nucleic acid is to be administered by intravenous route.

6. The method according to claim 5, wherein the nucleic acid is to be administered by injection, intravenous drip, bolus or pump.

7. The method according to claim 1, wherein the nucleic acid is administered in combination with antitumor treatment.

8. The method according to claim 1, wherein the nucleic acid is administered in combination with radiotherapy and/or chemotherapy.

9. The method according to claim 1, wherein the nucleic acid is administered in combination with a DNA damaging anti-tumor agent.

10. The method according to claim 9, wherein the DNA damaging anti-tumor agent is selected from the group consisting of an inhibitor of topoisomerases I or II, a DNA crosslinker, a DNA alkylating agent, an anti-metabolic agent and inhibitors of the mitotic spindles.

11. The method according to claim 1, wherein the nucleic acid is to be used in combination with a platinum drug selected from the group consisting of oxaliplatin, carboplatin and cisplatin.

12. The method according to claim 2, wherein L is carboxamido triethylene or tetraethylene glycol.

13. The method according to claim 2, wherein C is selected from the group consisting of octadecyl, oleic acid, dioleoyl acid, stearic acid, tocopherol, cholesterol, folic acid, galactose, mannose, oligosaccharide of galactose and/or mannose, RGD, bombesin, integrin and transferrin.

Description

DESCRIPTION OF THE FIGURES

[0128] FIG. 1: DT01 significantly increases sensitivity to CT in a CRC (HT29) liver metastatic model Intrahepatic tumor bearing NMRI.sup.NU/NU mice were treated as described in the Material and Methods, and sacrificed 22 days post treatment. Livers were sampled for macroscopic and microscopic examination. (A) Sequence of DT01 and CT therapy. CT was administered 2 or 4 hours post DT01 treatment. (B) Mean liver tumor volume (mm.sup.3) in each treatment group. (C) Representative macroscopic and HES sections of liver tumors in each group. (D) Tumor necrotic component assessed through HES staining. Necrosis is expressed as a proportion (%) of the total tumor surface of the tissue section analyzed. (E-F) Immunohistochemistry. Proliferation of the viable tumor component and the average micro-vessel density were determined by Ki67 (D) and CD31 (E) staining, respectively. Results are expressed as an averageSEM.

[0129] FIG. 2: Association of DT01 with CT significantly decreases the peritoneal tumor volume Intrahepatic tumor bearing NMRI.sup.NU/NU mice were treated as described in FIG. 4, and sacrificed 22 days post treatment. The presence of peritoneal metastasis was visually monitored during the duration of treatment and measured at the time of sacrifice. (A) Mean peritoneal tumor volume (mm.sup.3) in each treatment group. (B) Representative macroscopic images of peritoneal tumors in each group. Results are expressed as an averageSEM.

[0130] FIG. 3: Comparison of systemic and local administration in two TNBC xenografted models. NMRI.sup.NU/NU mice grafted in the fat pad with TNBC cell lines were treated by SC or IP injections of Dbait during 5 consecutive days for one cycle (left panel) or three cycles (right panel) separated each by two weeks without treatment. Doses of each Dbait injection is indicated in the legend. The Data represent the mean relative tumor volume (Vt/Vi) at the different time after beginning of treatment.

[0131] FIG. 4: DT01 association to carboplatin significantly decreases tumor growth in TNBC model. Median tumor volume per treatment group (error bars in DT01 treated group and DT01+ carboplatin group indicate the standard error of the mean, SEM).

[0132] FIG. 5: DT01 association to carboplatin significantly increases survival in TNBC model. Kaplan-Meier representation of animal survival in MDA-MB-231 model.

[0133] FIG. 6: Scheme of treatment in FIGS. 4 and 5.

EXAMPLES

Example 1

DT01 as a Novel Therapeutic Strategy for Chemo-Sensitization of Colorectal Liver Metastasis

[0134] Metastatic liver disease from colorectal cancer (CRC) is a significant clinical problem. This is mainly attributed to non-resectable metastases that frequently display low sensitivities to available chemotherapies and develop drug resistance partly via hyperactivation of some DNA repair functions. Combined therapies have shown some disease control however, there is still a need for more efficient chemotherapies to achieve eradication of CRC liver metastasis.

[0135] The inventors investigated the tolerance and efficacy of Dbait in association with conventional chemotherapy. In vitro, Dbait treatment increases sensitivity of HT29 and HCT116 CRC cell lines. In vivo, the pharmacokinetics, biodistribution and the efficacy of the cholesterol conjugated clinical form of Dbait, DT01, were assessed. The chemosensitizing abilities of DT01 were evaluated in association with oxaliplatin and 5-fluorouracil in intrahepatic HT29 xenografted mice used as a model for CRC liver metastasis. The high uptake of DT01 indicates that the liver is a specific target. The inventors demonstrated significant anti-tumor efficacy in a liver metastasis model with DT01 treatment in combination with oxaliplatin and 5-fluorouracil (mean: 501 vs 872 mm.sup.2, p=0.02) compared to chemotherapy alone. The decrease in tumor volume is further associated with significant histological changes in necrosis, proliferation, angiogenesis and apoptosis. Repeated cycles of DT01 do not increase chemotherapy toxicity. Combining DT01 with conventional chemotherapy may prove to be a safe and effective therapeutic strategy in the treatment of metastatic liver cancer.

[0136] The aims of this study were to firstly demonstrate the efficacy of DT01 in vitro, secondly to assess the pharmacokinetics and the distribution of DT01 in the liver, and thirdly to demonstrate the concomitant impact of systemic DT01 administration in combination with conventional chemotherapy (oxaliplatin with 5-fluorouracil) in a CRC metastatic liver tumor model.

[0137] Results

[0138] Dbait Treatment Increases Sensitivity of Colon Cancer Cell Lines to Chemotherapy

[0139] The inventors have previously shown that Dbait acts by activating DNA-PK kinase, which phosphorylate numerous targets including the histone variant H2AX. They first confirmed the activity of Dbait in two CRC cell lines (HCT116 and HT29) by monitoring the pan-nuclear phosphorylation of H 2AX.

[0140] To first investigate the effects of Dbait on cell survival to chemotherapy, the inventors determined the number of living cells, at different time points after treatment with Dbait or oxaliplatin (OXA) and 5-fluorouracil (5-FU) or a combination of Dbait with chemotherapy (CT) (FIG. 1B). As already observed in fibroblasts (Quanz, 2009, PloS one, 4, e6298), Dbait alone appears to have no effect on cell proliferation in both cell lines. Treatment with OXA and 5-FU resulted in a decrease in cell proliferation. However, the level of proliferation was significantly reduced by day 9 in cells transfected with Dbait prior to chemotherapy treatment in both HCT116 and HT29 cell lines compared to chemotherapy alone (p<0.001 and p<0.02, respectively). These differences become apparent particularly at later time points (>5 days after treatment) indicating that the increase of efficacy with Dbait may be a slow process.

[0141] To confirm the chemosensitization effect of Dbait in combination with OXA and 5-FU, clonogenic survival assays were performed on HCT116 and HT29. HCT116 cells showed approximately 30% (p<0.01) lethality after Dbait treatment alone revealing their dependency in repair activity for survival whereas no significant effect was noted in HT29. Since the sensitivity of HCT116 to Dbait was not detected during the first 8 days of proliferation, this result suggests that the cells growing with Dbait accumulate lethal lesions that impair their survival later on. Treatment with chemotherapy alone (OXA/5-FU) resulted in a significant decrease in the survival of HCT116 (p<0.001) whereas only a trend was observed with the HT29 cell line (p=0.08). However, combination of Dbait with chemotherapy resulted in a significant reduction in survival in both cell lines (p=0.05). HCT116 and HT29 differ by many parameters including their P53 status (HCT116 being proficient whereas HT29 is mutated). In this instance, despite some differences in their sensitivity to standalone Dbait treatment both cell lines were equally sensitive to the combination of CT with Dbait.

[0142] Pharmacokinetic and Biodistribution Analyses of Intraperitoneal Vs Intravenous Administration of DT01

[0143] To avoid transfectant adjuvant toxicity, all in vivo studies were performed with DT01, a Dbait-cholesterol conjugate facilitating the cellular uptake of these molecules without added toxicity. To determine the best route for systemic administration of DT01 mice were treated with either a single intraperitoneal (IP) or an intravenous (IV) dose of 5 mg of DT01. IP administration resulted in a C.sub.max of 578g/ml, a T.sub.max of 1 hour and an AUC.sub.0-6 of 799 whereas IV led to a C.sub.max of 1,917 g/ml, a T.sub.max of 0.08 hours and an AUC.sub.0-6 of 799. Pharmacokinetic analyses revealed that following IP injection, the plasmatic exposure of DT01 was longer than that of IV bolus injection with an AUC corresponding to approximately 70% of the AUC with IV administration.

[0144] The inventors used a fluorescent labelled cy5-DT01 molecule to monitor the biodistribution in excised whole-organs. Both cy5-DT01 and DT01 have similar properties in terms of pharmacokinetics and DNA-PK activation. The maximal DT01 fluorescence was observed in the liver, intestines, and the kidneys by both routes with the highest intensities observed in the liver and intestines following IP administration. The high fluorescence emitted by the kidneys and urine observed in mice suggest that DT01 is preferentially eliminated by the kidneys. Although there was no measurable DT01 in the blood 6 hours after injection, significant amounts of DT01 were still detectable in the liver indicating a specific retention in this organ.

[0145] As already demonstrated in vitro, DT01 activation of DNA-PK in tissue can be revealed by the phosphorylation of the histone H2AX. The inventors monitored DT01 activity by analyzing distribution of H2AX phosphorylation in livers bearing HT29 grafted tumors. Interestingly, a high level of -H2AX was specifically observed in the tumor and not in the surrounding healthy tissues indicating a preferential uptake or activity of the DT01 molecules in the tumor cells of the liver.

[0146] DT01 Significantly Increases Sensitivity to OXA and 5-FU In Vivo

[0147] To explore the interest of associating DT01 with the frontline treatment for metastatic CRC, the inventors used a HT29 xenografted liver tumor model, since previous reports and the in vitro data demonstrate this line to be highly chemo-resistant mainly due to the V600E BRAF mutation. The animals were treated with OXA and 5-FU, a treatment close to the traditional FOLFOX protocol for patients, using two different schedules based on biodistribution data (FIG. 1A). The two schedules consisted of either two or four-hour intervals between the two treatments, since the maximum DT01 levels in the liver were observed at one and three hours after treatment.

[0148] As previously observed in vitro, the tumors were highly resistant to CT alone and DT01 had only a moderate effect when administered alone (FIGS. 1B, 1C). Interestingly, the association of DT01 to OXA and 5-FU significantly decreased the liver tumor size in both combination treated groups compared to CT alone when administered at two (mean volume: 525.80 vs 872.01 mm.sup.2, p=0.03) and four hours (mean volume: 501.05 vs 872.01 mm.sup.2, p=0.02) before CT (FIGS. 1B, 1C). This effect was not observed when DT01 was associated with a single chemotherapy agent, either OXA or 5-FU. Detailed blinded histological analyses including measures of the viable tumor area, necrosis and apoptosis were assessed in haematoxylin-eosin-saffron (HES) stained sections, by an experienced pathologist. Both groups with DT01 and CT combined treatment showed higher treatment efficacy than the groups receiving single treatment, with a marked increase in necrosis in the group treated with a four-hour interval between CT and DT01 (p<0.0001) than two hours (p<0.01), compared to CT alone (FIG. 1D). Furthermore, a high apoptotic index was apparent in both groups treated with DT01 and CT. Similar to other histological parameters, the extent of apoptosis was elevated in animals treated with a four-hour delay (p<0.0001). These histological findings were not apparent in the DT01 or chemotherapy alone treated groups.

[0149] For many solid tumors, proliferation and microvascularization are indispensable prerequisites for tumor development and metastasis. To further investigate these parameters, immunostaining for Ki67 and CD31, markers of cell proliferation and angiogenesis respectively, were performed in the viable tumor component (FIGS. 1E, 1F). Ki67 immunoreactivity indicated that tumors treated with either DT01 or chemotherapy alone were densely packed with a high degree of proliferation.

[0150] Treatment with a two-hour interval between DT01 and CT resulted in a moderate decrease in proliferating cells (p=0.02) (FIG. 1E). Strikingly, immunoreactivity of Ki67 was 10-fold reduced in the group treated with a four-hour interval between DT01 and CT (p<0.001) (FIG. 1E). In this group, immunoreactivity was detected only in the tumor rim due to the high degree of necrosis observed in the center core region of the tumor. In addition, diminished intratumoral vessel densities were detected in groups treated with a combination of DT01 and CT, compared to CT alone (FIG. 1F). However, the mean microvessel density was even more notably reduced in the group treated with a four-hour interval (p<0.001) compared to two hours (p=0.02). Despite similarities in the anti-tumor effect on tumor growth at both the two and four-hour treatment schedules, histologically the efficacy was significantly more pronounced at the four-hour time point, in terms of necrosis, apoptosis, proliferation and angiogenesis.

[0151] Unexpectedly, tumors treated with a delay of four hours between DT01 and CT and sampled 22 days post treatment presented with a proportion of lysed hepatocytes within the tumor and slight edema in the adjacent non-malignant liver, in the absence of further clinical signs of toxicity such as loss of weight. Histological analyses did not reveal morphological signs of toxicity in the other groups (FIG. 1). In addition, liver enzyme tests did not reveal significant differences between the control and the combination treated groups.

[0152] Interestingly, no further edema was observed when animals receiving the same treatment were sacrificed between 30-65 days. This suggests that the edema observed at day 22, is reversible over time. Despite the significant tumor efficacy observed 22 days post treatment, tumors monitored after this time point resumed progression. Histological analysis revealed that the proliferative component reached 50% at 30-45 days post treatment, only slightly below the level observed in non-treated tumors.

[0153] To confirm that combination treatment did not induce additional toxicity to the liver, the inventors analyzed the tolerability of DT01 in association with OXA or 5-FU for extended treatment cycles. They determined the toxicity of escalating doses of DT01 (total doses of 30, 50 or 80 mg) following systemic administration for two cycles (5xDT01 administrations per treatment cycle) associated to OXA or 5-FU in a cohort of 50 mice. No loss of weight was observed in animals during or post treatment. Similarly, other clinical signs of toxicity such as diarrhea or behavioral changes were not noted in these mice. At autopsy 6 weeks post the second cycle of treatment, all abdominal organs, the thoracic cavity and contents appeared normal. No major variations in liver weights or histology were observed between the vehicle and combination treated groups.

[0154] These results suggest that the reversible edema detected after combined treatment in animals bearing hepatic tumor is likely an acute reaction to the tumor response to efficient combination treatment.

[0155] Peritoneal Metastasis Treatment

[0156] CRC often metastasizes to the liver and the peritoneum. Interestingly 90% of the mice intrahepatically xenografted with CRC tumors developed peritoneal metastasis. This property allowed the inventors to monitor the effect of DT01 not only on liver tumors but also on peritoneal metastasis. Animals receiving a combination of DT01 and chemotherapy displayed significantly decreased peritoneal tumor volumes when compared to chemotherapy alone at both the two (mean volume: 300.31 vs 867.20 mm.sup.2, respectively, p<0.01) and four hour time intervals (mean volume: 259.51 vs 867.20 mm.sup.2, respectively, p<0.01) (FIG. 2A, 2B). Although a slight decrease in tumor volume was observed in the group treated with DT01 alone, this did not reach statistical significance.

[0157] Discussion

[0158] Approximately 50% of patients with CRC will present either with liver and/or peritoneal metastases or develop them throughout the course of their disease. A majority of patients with CRC hepatic metastases present with non-resectable disease and systemic chemotherapy represents the main if not the only form of therapy. However, the therapeutic window of chemotherapy is limited due to tumor resistance and high toxicity to non-targeted tissue. In such clinical situations, an aggressive chemotherapy regimen alone may not only fail to improve survival, but may also adversely affect the quality of life. Consequently the mortality of these patients remains high. Therefore development of new agents' specifically targeting DNA repair to circumvent chemoresistance and sparing healthy tissues is imperative in the treatment of these cancers. DT01 is an attractive drug candidate based on its central role in DNA repair.

[0159] In the present study, the inventors showed for the first time that systemic DT01 treatment sensitizes CRC cells to conventional chemotherapies by in vitro and in vivo assays. In a CRC metastatic model, they demonstrated significant anti-tumor efficacy in the liver and the peritoneum (regarded as a terminal condition) with DT01 treatment in combination with OXA and 5-FU. It is of interest to note, that the significant anti-tumor effect was limited to DT01 association with both OXA and 5-FU and not with single agent chemotherapy. This demonstrates that in agreement with the clinical conventional setting, combination with DT01 must be associated to double chemotherapy rather than single-agent chemotherapy in the treatment of CRC metastases. This study further highlights that tumors receiving double chemotherapy combined with DT01 restart proliferation and re-growth at later time points (post 22 days). Therefore repeated cycles of treatment would be necessary to achieve long term disease control similar to current conventional chemotherapy protocols. This would be possible as no added toxicity was observed with DT01 alone or in combination with OXA or 5-FU.

[0160] DT01 preferentially accumulate in the liver and intestines after systemic injection. Although the entire liver appeared to be uniformly fluorescent after Cy5-DT01 injection, the activation of DNA-PK revealed by the phosphorylation of H2AX was observed exclusively in tumor cells and not in the healthy tissue surrounding the tumor. This observation indicates that either DT01 does not enter non-tumor cells and/or that DT01 is not active in healthy liver tissue. DT01 was specifically designed by cholesterol conjugation firstly, in order to increase the bioavailability and secondly, to play on the difference in the substrate uptake between cancer and normal cells. Low density lipoproteins (LDL) are a major component of the cholesterol pathway. High requirement for LDL by malignant cells and thus the consequent overexpression of LDL receptors has been shown in many types of cancer cells making tumor cells specific targets of DT01. Additionally, an extensive analysis of normal and cancerous human tissues by immunohistochemistry revealed that either DNA-PKcs or Ku80 were consistently absent in the liver and the mammary epithelium, a specific post-transcriptional regulation that was not found in the other tissues and most of the tumors. Taken together, these data highlight that DT01 is likely to be an efficient drug for the treatment of liver cancers.

[0161] In conclusion, there is an urgent need for new treatment options targeting secondary hepatic malignancies, a rapidly progressive disease with a poor prognosis and an alarming rate of mortality. The present study demonstrated that combining systemic administration of DT01 with conventional chemotherapy can be a safe and effective therapeutic strategy in the treatment of CRC metastasis of the liver and the peritoneum.

[0162] Materials and Methods

[0163] Cell Culture, Constructs, Dbait Molecules, Immunofluorescence and Western Blotting

[0164] CRC cell lines; HT29 (mutated p53, ATCC: HTB-38) and HCT116 (wild-type p53, ATCC: CCL-247) were purchased directly from ATCC. These cells were authenticated by ATCC by generating human short tandem repeat profiles by simultaneously amplifying multiple STR loci and amelogenin (for gender determination) using the Promega PowerPlex Systems. These cells were cultured in the laboratory for less than 6 months from the date of purchase in DMEM medium supplemented with 10% fetal bovine serum, 1% sodium pyruvate, 100 mg/ml streptomycin and 100 mg/ml penicillin (Invitrogen, Carlsbad, Calif.), when the current study was performed. HT29 cell line stably expressing luciferase was established in-house using a pGL4.5 luciferase reporter vector (luc2/CMV/Hygro) (Promega). HT29 luciferase cells were supplemented with 200g/m1 hygromycin B. All cell lines were additionally subjected to mycoplasma testing in-house and were free of mycoplasma contamination (Biovalley, France).

[0165] Cells were transfected with 2.5 gs of Dbait (5-GCTGTGCCCACAACCCAGCAAACAAGCCTAGA-(H) TCTAGGCTTGTTTGCTGGGTTGTGGGCACAGC-3 SEQ ID No 9) (Eurogentec, Belgium) where H is a hexaethyleneglycol linker and underlined nucleotides are phosphorothioates. The cells were sham transfected with an 8bp oligonucleotide control (8H) complexed with 11 kDa polyethyleneimine (PEI) as previously described (Quantz et al, 2009, PloS one, 4, e6298).

[0166] H2AX immunofluorescence was performed as described previously using a monoclonal anti-phospho-Histone H2A.X (Ser139) Antibody, clone JBW301 (1:500 dilution; 05-636, Millipore, USA) (9).

[0167] In Vitro Proliferation Assay

[0168] Cells were seeded at a density of 310.sup.4 cells/60 mm dishes and transfected with Dbait. Following treatment, cells were washed and left untreated or treated with a combination of 5 M of oxaliplatin (OXA, Sigma) and 2.5 M of 5-fluouracil (5-FU, Sigma) and live cell counts were performed on days 1, 3, 5, 6, 7 and 9.

[0169] Clonogenic Assay

[0170] Cells were transfected with Dbait and left untreated or treated with 5 M of OXA and 2.5 M of 5-FU for 1 hr. The cells were diluted, allowed to grow for 14 days and the clones were stained with crystal violet and counted.

[0171] In Vivo Experiments

[0172] The current study was carried out in strict accordance with the European Union guidelines for animal care. All animal experimentation was approved by the ethics committees of the Institut Curie and the French ministry. Surgical procedures were performed under anesthesia with local analgesia to minimize suffering.

[0173] Animals

[0174] Six week old female NMRI.sup.Nu/Nu mice (Janvier, France) weighing 20-22 g were housed in specific pathogen free environment on a 12 h light and 12 h dark schedule with food and water ad libitum. No more than 6 animals were housed per cage and they were acclimated for at least one week prior to initiating in vivo studies.

[0175] Intrahepatic HT29L Grafting

[0176] HT29 Luciferase (HT29L) cells were implanted by direct injection of cell suspensions (110.sup.6/10 L of PBS) onto the upper surface of the left lobe. Tumor growth was monitored through bioluminescence analysis (IVIS, Caliper sciences).

[0177] DT01 Molecule

[0178] For in vivo studies, DT01 (Dbait with a cholesterol tetraethylene glycol incorporated at the 5-end) was used (Agilent technologies, Boulder, Colo.).

[0179] Pharmacokinetics of DT01

[0180] HT29L grafted mice were treated with a single intraperitoneal (IP, n=4) or intravenous (IV, n=3) injection of 5 mg of DT01. Blood samples were harvested prior to treatment and 1, 5, 10, 30 mins, 1 hr, 2 hrs, 4 hrs and 6 hrs post treatment. Plasma was recovered through centrifugation and assayed by ELISA.

[0181] Fluorescence Measurement of Organs

[0182] As the ELISA technique failed to produce reliable quantification in tissues, we used fluorescent imaging, a reliable technique for assessing molecule distribution (15). NMRI.sup.NU/NU mice were injected with 1 mg of the DT01 fluorescent molecule (DT01-Cy5) through IP (n=3) or IV (n=3) administration.

[0183] The fluorescent DT01 (DT01-Cy5) incorporates a cyanine 5 at the thymidine located immediately after the linker. Six hours after injection, fluorescence imaging was performed using a Typhoon scanner (GE Helathcare).

[0184] DT01 and Chemotherapy Treatment

[0185] HT29L grafted animals (n=49) were allocated into treatment groups and administered one cycle of treatment. DT01 was systemically administered through IP injection at a dose of 5 mgs/day for 5 consecutive days starting on day 0 (DO). OXA (6mg/kg, lx per cycle, Day 1) and 5-FU (25 mg/kg, 3x per cycle, Days 1-3) were administered 2 or 4 hours after DT01 treatment. These mice were sacrificed 22 days post treatment.

[0186] An additional group treated with DT01 and OXA/5-FU at the 4 hour interval (n=10) were kept after treatment until the termination guidelines were met to assess the duration of treatment efficacy.

[0187] Liver Function Assessment

[0188] Blood samples were obtained through submandibular bleeding in lithium heparin tubes (Sarstedt) on days 0, 4 and 18 post treatment. Plasma alanine transaminase (ALT), aspartate aminotransferase

[0189] (ASAT), alkaline phosphatase (ALP), glutamyl transpeptidase (GGT), amylase (AMYL) and total bilirubin (TBIL) were measured using an MS-Scan II (Melet Schloesing Laboratories, France).

[0190] Toxicity Assays

[0191] NMRI.sup.NU/NU mice (n=50) were treated with two cycles of DT01 at escalating doses of 3 mg/day (30 mg total), 5 mg/day (50 mg total) or 8 mg/day (80 mg total) through IP injection in combination with OXA or 5-FU. OXA and 5-FU were administered through systemic IP injection at doses of 16 mg/kg or 325mg/kg, 4 hours after DT01 treatment respectively. Animals were observed regularly for any adverse effects.

[0192] Histology

[0193] Hematoxylin, eosin, and saffron (HES) stained tumor sections were assessed by an experienced pathologist (Dr. Huerre, Institut Curie) in a blinded fashion. Viable and necrotic components (indicated by increased cell size, indistinct cell border, eosinophilic cytoplasm, loss or condensation of the nucleus, or associated inflammation) were expressed as a proportion (%) of the total tumor surface. Apoptosis was estimated (weak-<5%, moderate 5-10%, significant 10-20% and very significant 20-50%) from representative non-necrotic fields at high power.

[0194] Digitization and image capture was performed using a whole-slide scanning system (Philips digital pathology solutions).

[0195] Ki67 and CD31 Immunohistochemistry

[0196] Immunohistochemistry was performed using rabbit anti-Ki67 (ab28364, 1/500; Abcam, UK) and rabbit anti-CD31 (ab15580, 1/500; Abcam, UK) antibodies. This was followed by a secondary biotinylated goat anti-rabbit IgG antibody (BA-1000; Vector, USA) and revealed using a rabbit specific HRP/DAB (ABC) detection kit. Images were captured using a fluorescence microscope (Eclipse 90i, Nikon). The average Ki67 index was scored by establishing a ratio between Ki67 +ve and -ve cells, in five randomly selected microscopic fields per section. Average micro-vessel density was determined by CD31 staining. CD31 positive vessels were counted in five randomly selected microscopic fields per section.

[0197] Statistical Analysis

[0198] In vitro experiments were performed with a minimum of two independent experiments. Two-sided unpaired t-tests were used for comparison of cell mortality and survival. Kruskal-Wallis tests were used to compare tumor volumes, and histological data. Error bars indicate standard error of the mean (SEM), except when specifically indicated. All statistical analyses were performed using StatEL software (adScience, France) and a P value of 0.05 was considered statistically significant.

Example 2

DT01 in Models of Triple Negative Breast Cancer (TNBC) and Its Potentiating Effect in a Co-Treatment with Carboplatin

[0199] DT01 Effect Alone on a TNBC Model

[0200] The objective of the present study was to demonstrate a systemic effect of DT01 alone in a model of breast cancer, in particular of triple negative breast cancer. The animal model is mice after 45 days of engraftment. Mice were subcutaneously grafted in mammary fat pad with MDA-MB-231 tumor cells.

[0201] In previous experiments, the inventors demonstrated that DT01 could control effectively tumor growth in all tested triple negative breast cancer models (mice engrafted with BC227, BC173, MDA-MB-468 and MDA-MB-231 cell lines) by local administration.

[0202] As shown in FIG. 3, the inventors compared intraperitoneal administration to local administration (intratumoral and peri-tumoral subcutaneous) in two TNBC xenografted models. They surprisingly observed that 3-5 fold more DT01 are required for similar efficacy than local administration.

[0203] The route of administration was intraperitoneal administration which mimics in mouse intravenous perfusion administration in human. The dose level of DT01 was 5 mg/animal/day. The DT01 intraperitoneal administration was performed during 3 sessions of 5 consecutive days with one week without treatment between each cycle. 13 mice were included, 7 of which receiving DT01. The control group received vehicle alone 0.9% NaCl.

[0204] No toxicity was observed. Intraperitoneal DT01 administration is well tolerated.

[0205] DT01 treatment showed a significant better tumor growth control and animal survival than the control group.

[0206] The MDA-MB-231 triple negative breast cancer model was chosen because it was the most resistant to DT01 treatment in previous experiments using intratumoral and peri-tumor subcutaneous administrations.

[0207] This experiment confirms that standalone administration of DT01 delays tumor growth in breast cancer tumor.

[0208] Effect of the combination of DT01 with carboplatin on a TNBC model

[0209] As shown in FIG. 6, each treatment cycle comprised of 5 consecutive days of administration with DT01 at a dose level of 5 mg/animal/day. Treatment was administered in 3 cycles with a 2 week gap between cycles either alone or in association with carboplatin. Only one dose of carboplatin was injected per cycle of DT01 treatment (2.sup.nd day of each cycle). The dose of carboplatin was 50 mg/kg per cycle. Treatments were performed over 7 weeks (3 cycles of treatment).

[0210] During the experiment no toxicity is observed. No sign of toxicity such as loss of weight in DT01 treated group. No increase in weight loss or toxicity in DT01+ carboplatin treated group was observed compared to the carboplatin group.

[0211] No abnormal death occurred during the 177 days of the experiment, except one in the carboplatin alone treated group.

[0212] Intraperitoneal DT01 administration is well tolerated.

[0213] Antitumor activity was evaluated by measuring tumor volume during and after treatment. DT01 was administered intraperitoneally during 3 sessions of 5 days treatment with two weeks of rest between each session. Carboplatin was administered once a week on the second day of each DT01 treatment cycle.

[0214] DT01+ carboplatin combination treatment showed a better tumor growth control compared to DT01 standalone treatment (FIG. 4). The curve was discontinued on the day of the first death in each group. In addition, the DT01+ carboplatin combination also increases the survival.

[0215] In this study, the treatment of DT01 combining with carboplatin is efficient and leads to a better tumor growth delay than single treatments.

[0216] Materials and Methods

[0217] DT01 Molecule

[0218] DT01, the cholesterol tetraethylene glycol incorporated form of Dbait was synthesized by automated solid-phase oligonucleotide synthesis (Agilent technologies, USA).

[0219] Cells & Animals

[0220] The MDA-MB231 cell line is derived from a human breast adenocarcinoma and can be ordered at the ATCC. The MDA-MB231 cells were grafted in the mammary fat pad with 10.10.sup.6 cells re-suspended in 0.1 ml of DMEM with no additive. The athymic nude mouse is immunodeficient, thus enabling the xenotransplantation and growth of human tumors.