WO2003004064A2 - Products for treating ovarian cancers and for preventing relapses - Google Patents

Abstract

The invention relates to products for treating ovarian cancers and for preventing relapses. Said products contain (1) a non-viral nucleic acid vector comprising at least one imine polyethylene (IPE) or a derivative thereof, 2) a nucleic acid sequence comprising the gene bcl-xS or a functional fragment thereof and (3) at least one anti-tumoral agent which is active against ovarian cancer, as a combined preparation for sequential use in the treatment of ovarian cancer, the sequence of nucleic acid associated with said non-viral nucleic acid vector being administered after the anti-tumoral compound.

Description

 PRODUCTS SUITABLE FOR TREATING AND OVARY CANCER

PREVENT RELAPSES.

The present invention relates to products capable of treating ovarian cancer and preventing relapse. Ovarian cancers represent the leading cause of death from gynecological cancer and are the fourth leading cause of death from cancer in women and their incidence is only increasing (of the order of 4% per year). The severity of these cancers is linked in particular to their late discovery and their ability to develop strong drug resistance during chemotherapy cycles, which means that the death rate from this cancer has decreased little during the past fifteen years.

In France, the incidence rate of ovarian cancer is estimated at 1.1 / 100,000 and increases with age.

Little is known about the etiology of ovarian cancer; the majority of these cancers are due to a malignant transformation of the ovarian epithelium (epithelial cancers). They have particular growth properties: they can appear, both in the form of solid tumors invading the ovary, multicellular spheroids suspended in an ascites liquid in the peritoneal cavity or in the form of nodules disseminated on the peritoneum . Dissemination is also carried out by lymphatic or blood route, causing the appearance of lymph node, pulmonary or hepatic metastases.

Their usual therapy combines surgery and chemotherapy based essentially on platinum salts, such as cisplatin (cis-dichlorodiamino-platinum (II) or CDDP) or carboplatin (diaminecyclobutane-dicarboxylo-platinum or CBDCA). cisplatin, ^whose equivalent dose has been established (compared CBDCA / CDDP 4).

The various multidrug therapies using one of the platinum derivatives have constituted major progress in the treatment of these tumors. Both cisplatin and carboplatin are administered in doses ranging, depending on the product and depending on the case, from 100 to 650 mg / m ² per month, in the form of infusions in 1 to 24 hours (Poulain et al .. Int J. Cancer, 1998, 78, 454-463, M. Marone et al., Clin. Cancer Res., 1998, 4, 517-524, JR Liu et al, Gynecol. Oncol, 1998, 70, 398-403 , JK Taylor et al., Oncogene, 1999, 18, 4495-4504, and JK Taylor et al., Nature Biotechnol, 1999, 17, 1097-1 100).

In multidrug therapies, the main drugs currently used are, in addition to platinum derivatives, alkylating agents, such as mephalan, chlorambucil. cyclophosphamide or thiotepa. anthracyclines, such as adriamycin or taxanes. The following combinations of multidrug therapy are preferably recommended: CDDP 100 mg / m ² -cyclophosphamide 600 mg / m ² _, paclitaxel-carboplatin, cisplatin-carboplatin or cisplatin 100 mg / m ² -cyclo-phosphamide 300 mg / m ^" -carboplatin 300 mg / m. ^the excision as complete as possible, to the efficacy of adjuvant treatments. It is conventionally followed by 4 to 6 cycles of chemotherapy, spaced 28 days, mainly comprising, in combination or not, the above products and in particular platinum salts, cyclophosphamide and adriamycin, and radiotherapy can also be used, particularly in areas that are heavily affected.

Ovarian carcinomas are generally sensitive to first-line chemotherapy, but paradoxically patient survival remains short, due to the acquisition of chemoresistance during treatment, in the vast majority of cases. The resistance mechanisms are notably described in Poulain et al., 1998 (cited above); they relate more precisely:

- the alteration of the intracellular incorporation of CDDP, by reduction of the intracellular incorporation of platinum or by elevation of the intracellular levels of sulfur compounds (glutathione, thioredoxin), - modifications of the DNA repair systems,

- mitochondrial alterations or

- alterations in the expression of genes involved in the control of the cycle and / or regulation of apoptosis.

In particular, the role of homologous genes of the bcl-2 family in cell cycle control and ^apoptosis is put forward to explain some of the mechanisms of chemoresistance: . Bcl-2 overexpression is correlated with the chemoresistance of various types of tumors, including ovarian cancer.

. the protein Bcl-x, has two isoforms resulting from an alternative splicing: Bcl-xL, which has an anti-apoptotic function, similar to that of Bcl-2 and Bcl-xS (US Patent 5,834,309), which has a pro function -apoptotique.

. the acquisition of resistance to cisplatin, associated with the capacity of the treated cells to progress in the cell cycle beyond the Gl phase: the defect observed in the regulation of the cell cycle could intervene at the level of the inhibitor of cdk p2i ^{cl, / WAH} . Indeed, cisplatin exerts its cytotoxic effect by inducing cell death by apoptosis, resulting from a lethal damage of the AON ** 'moreover, this drug interrupts cell progression and thus leads to an accumulation of cells in phases specific to the cycle, usually before apoptosis. A dysfunction in the metabolism of nuclear p53 and of the cdk inhibitor p2l ^{clpl / WAF1 is observed} in cells resistant to cisplatin. A decrease in sensitivity to chemotherapy is linked to the alteration of the levels of Bcl-2, Bax or Bcl-x, which are involved in the mechanism of apoptosis (M. Marone et al, Clin. Cancer Res., 1998 , 4, 517-524).

The Bcl-2 gene (for B cells lymphomas) was discovered thanks to its constitutive expression in certain human follicular lymphomas due to a translocation t (14,18) (q32, q32) placing it close to the strong promoter of the gene immunoglobulins. The protein Bcl-2 protects the cell against apoptosis induced by a wide range of stimuli. It is expressed in the mitochondrial outer membrane, the endoplasmic reticulum, and the nuclear envelope. The proteins of the Bcl-2 family have a more or less extensive structural homology, sometimes even restricted to two particular domains BH1 and BH2, involved in the dimerization of these proteins, which constitutes an essential element for the regulation of their activity. : these proteins can have opposite roles, since some are involved in the induction of apoptosis (Bax, Bcl-xS, Bad, Bak, Bik), while others (Bcl-2, Bcl-xL, Mcll, Bfll, Al) are implicated in its repression. Their homo- or dimerization allows an equilibrium shift from a situation of cell survival and growth towards an induction of programmed cell death (JR Liu et al., Gynecol. Oncol., 1998, 70, 398-403; JK Taylor et al., Nature Biotechnoi, 1999, 17, 1097-1100).

Resistance to apoptosis, which plays an important role in tumors that are refractory to chemotherapy, is regulated by the ratio of anti-apoptotic proteins / pro-apoptotic proteins.

J.K. Taylor et al., Nature Biotechnoi., (Cited above), hypothesize that by manipulating the levels of these different proteins, cells can become sensitive to apoptosis in response to anti-tumor agents. They show in particular that by modifying the Bcl-xL / Bcl-xS ratio in the cell, by using an antisense oligonucleotide, one can obtain a sensitization of the cells to apoptosis.

Given that an overexpression of Bcl-xL is associated with a decrease in apoptosis in tumors, resistance to anticancer agents and an unfavorable clinical course, they hypothesize that a pharmacological intervention which decreases Bcl-xL levels and increasing Bcl-xS levels could be an interesting alternative chemotherapy.

In this context, the use of members of this family has been recommended for therapeutic purposes.

It has been proposed, in order to increase the expression of Bcl-xS, to inhibit the use of the 5 'splicing site in exon II of TARN bcl-xL, so as to redirect the splicing machinery , using antisense oligonucleotides (JK Taylor et al., Nature Biotechnoi .. 1999, cited above): A549 cells (human lung carcinoma cell lines), transfected with lipofectin (control), the oligonucleotide ISIS 22783 (US Patent 6,172,216) in the presence of lipofectin or a control oligonucleotide in the presence of lipofectin show that the treatment of the cells with the oligonucleotide ISIS 22783, which targets exon 1 of the transcript Bcl-xL and is capable of modifying the ratio Bcl-xS / Bcl-xL from 17% to 293% without reducing the amount of total Bcl-x RNA in A549 cells, results in a 2-fold increase in apoptosis, after treatment with cisplatin.

However, this document JK Taylor et al. does not allow to conclude:. to the effect of Bcl-xS; Indeed, it is notably specified in this article that, taking into account other tests, in which the same stimulation of apoptosis was obtained, only by reduction of Bcl-xL, it would seem that the decrease tion of the levels of Bcl-xL has a more significant effect on the induction of apoptosis that increasing the ^expression of Bcl-xS and

. the role of Bcl-xS in ovarian tumors; none of the cells used in this document come from ovarian tumors. ^Other treatments have also been proposed:

- J.S. Han et al. (Springer Semin. Immunopathol., 1998, 19, 279-288) recommend using a viral vector (adenovirus) expressing the bcl-xS gene, which diffuses only at limited distances in solid tumors and which is a functional inhibitor of Bcl-2 and Bcl-xL. The use of such a vector is particularly recommended, because of its low diffusion properties, in the treatment of cancers which induce dissemination in a cavity: bladder, ovarian cancer (peritoneal cavity).

- AP Simoes-Wust et al. (Int. J. cancer, 2000, 87, 582-590) describe an antisense treatment bcl-xL to induce apoptosis in breast cancers (carcinomas). The oligonucleotide used is 2'-O-methoxy-ethoxy nucleotide 4259 which targets nucleotides 687-706 of the bcl-xL mRNA, a sequence which is not found in the pro-apoptotic bcl-xS transcript. Such an oligonucleotide has several advantages: it is specific for bcl-xL, the modifications at the 5 ′ and 3 ^* ends by 2′-MOE residues allowing it to present a significantly increased affinity for its target and stability in serum. Treatment of MCF7 cells with this oligonucleotide 4259 at a concentration of 600 nM for 20 hours decreases the levels of bcl-xL mRNA and of corresponding protein by 80% and 50%, respectively.

This type of treatment is recommended only in the breast, since the ^expression of Bcl-2 and Bcl-xL (anti-apopto- ticks members of the family) is often increased in primary breast cancers.

With regard to the protocol used, it is specified that the oligonucleotide is administered in the form of a complex with lipofectin.

- PJ Beale et al. (Br. J. Cancer, 2000, 82, 2, 436-440) sought to determine whether the levels of proteins of the Bcl-2 family are correlated with the sensitivity or the resistance to treatment with platinum derivatives, in ovarian tumors (carcinomas). In particular, they consider that: (1) overexpression of the Bcl-2 protein in ovarian tumors confers sensitivity to cisplatin rather than resistance;

(2) no significant correlation is observed when comparing the sensitivity to cisplatin in vitro with Bax, Bcl-xL or Bak levels; (3) the ^introduction of Bax, in lines resistant to cisplatin or Bcl-xL into sensitive lines does not result in significant changes in chemosensitivity.

Other treatments to alleviate the phenomenon of chemoresistance, recommend potentiating the cytotoxicity of cisplatin and carboplatin in ovarian cancers, which makes it possible to recover sensitivity to treatment:

. by amphotericin B (AmB) (Poulain et al., Cancer Chemother. Pharmacol., 1997. 40, 385-390); however, the cisplatin-AmB association is too nephrotoxic for clinical use, while the carboplatin-AmB association does not exhibit such nephrotoxicity. Other modulators, such as methyl xanthines, are recommended, in combination with AmB, to attenuate the nephrotoxicity of the cisplatin-AmB association or

. by the cipl-wafl gene (Lincet et al., Cancer Letters, 2000, 161, 17-26).

However, the various treatments recommended in the prior art, for ovarian cancers, do not make it possible to avoid the chemo-resistance observed or to eliminate recurrences or relapses in the long term.

Consequently, the Applicant has set itself the goal of providing a treatment which is more suitable and which better meets the needs of the practice than the treatments proposed in the prior art, in particular in that it avoids long-term relapses or relapses term.

The subject of the present invention is products containing (1) a non-viral nucleic acid vector comprising at least one polyethylene imine (PEI) or a derivative of PEI, (2) a nucleic acid sequence comprising the bcl-xS gene or a functional fragment thereof and (3) at least one anti-tumor agent active in ovarian cancer, as a combined preparation for sequential use in the treatment of ovarian cancer, the acid sequence nucleic associated with said vector ^of non-viral nucleic acid is administered after the anti-tumor compound.

Such a sequential combination makes it possible, unexpectedly, to solve both the problem of chemoresistance and the problem of long-term relapses or relapses.

According to the invention:

the non-viral vector is preferably selected from the group consisting of PEIs of molecular weight less than 25 kDa, in particular linear PEIs of molecular weight less than 25 kDa, PEIs onto which a sugar, an antibody or a nucleotide are grafted , a lipid, a vitamin or a peptide, such as the integrin binding peptide (RGD); said PEIs are optionally combined with polyethylene glycol (PEG) or with another hydrophilic polymer or else with folic acid.

Linear PEIs which can be used as a non-viral vector are notably described in O. Boussif et al., (PNAS, 1995, 92, 7297-7301), L. Poulain et al., (Cancer Gene Ther., 2000, 7, 4, 644-652), American Patent 6,013,240 in the name of Rhone-Poulenc Rorer, JS Rémy et al, (Adv. Drug Delivery Rev., 1998, 30, 85-95), S. Ferrari et al., (Gene Ther, 1997, 4, 1100-1106), D. Goula et al., (Gene Ther., 1998, 5, 1291-1295), F. Labat-Moleur et al., (Gene Ther., 1996, 3, 1010-1017). Thio derivatives of PEI of about 25 kDa, conjugated to the integrin binding peptide (PEI-RGD) are described in P. Erbacher et al., (Gene Ther., 1999, 6, 138-145); galactosylated PEIs are described in MA Zanta et al., (Bioconjugate Chem., 1997, 8, 839-844); and the coupling of membrane ligands to PEI is described in R. Kircheis et al., (Gene Ther., 1997, 4, 409-418) - the anti-tumor agent active in ovarian cancer is preferably selected in the group consisting of platinum derivatives, taxanes, topoisomerase inhibitors 1 and 2 and the associations of these different products; said anti-tumor agents are optionally combined with other anti-cancer agents and / or with amphotericin B or caffeine derivatives. The plasmid Bcl-xS combined with the antitumor agent significantly increases the death of cancer cells so that relapses or relapses are significantly reduced.

Said products are advantageously combined within the framework of a sequential use in the treatment of ovarian cancers.

In fact, the order of administration of the products is essential: the vectored nucleic sequence must imperatively be administered after the antitumor compound.

The combination of a vectorized nucleic acid sequence (Bcl-xS associated with a PEI, as defined above) and at least one antitumor agent, administered in the order indicated, has the following advantages:

- potentiation of the activity of the antitumor agent, which reduces the dose of ^the antitumor agent

- elimination of the effect of chemoresistance to the antitumor agent and restoration of a level of sensitivity of the cells to the antitumor agent, entirely satisfactory and

- elimination of long-term recurrences or relapses.

More specifically, the sequential combination of antitumor agent and in particular cisplatin then bcl-xS gene vectorized by a non-viral vector based on PEI, preferably of molecular weight less than or equal to 25 kDa, makes it possible to destroy a sufficient number of tumor cells. by apoptosis, to avoid recurrence, that ^one can not obtain with the Bcl-xS gene alone or with the antitumor agent alone.

The PEI / DNA complexes will preferably be administered by the intraperitoneal route, although the intravenous route remains possible. The complexes are preferably prepared in a 5% glucose solution using an N / P ratio of 5 as detailed in Poulain et al, Cancer Gene Ther .; 2000, 7, 4, 644-652. Three injections 12 hours apart are preferably considered.

In addition to the foregoing provisions, the invention also comprises other provisions, which will emerge from the description which follows, which refers to examples of implementation of the method which is the subject of the present invention as well as to the accompanying drawings, which : - Figure 1A illustrates the modifications, induced by exposure to 5 μg / ml of CDDP, of the cell cycle of the sensitive ovarian tumor cell lines IGROV1 and OAW42.

FIG. 1B illustrates the recovery time from a normal state of proliferation of the sensitive ovarian tumor cell lines IGROV1 and

OAW42, after exposure to 5 μg / ml CDDP.

- Figure 2 is a histogram showing the evolution of the response of the sensitive IGROV1 cell line over time, after exposure to 5 μg / ml of CDDP. - Figure 3 A illustrates the changes, induced by exposure to

5 μg / ml of CDDP, from the cell cycle of IGROV1-R10 and SKOV3 resistant cell lines.

- Figure 3B illustrates the recovery time from a normal state of proliferation of the IGROVl and OAW42, IGROV1-R10 and SKOV3 cell lines, after exposure to 5 μg / ml of CDDP.

- Figure 4 illustrates the evolution of the response of the resistant cell line SKOV3 over time, after exposure to 5 μg / ml of CDDP.

FIG. 5 illustrates the modifications, induced by exposure to 20 μg / ml of CDDP, of the cell cycle of the sensitive cell lines IGROVl and OAW42 (A and C) and of the resistant cell lines IGROV 1 -RI 0 and SKOV3 (B) .

- Figure 6A illustrates the basal expression of bcl-xL and bcl-xS in the IGROV1 (OVl) and OAW42 (OA) cell lines and the IGROV1-R10 (OVR) and SKOV3 (SK) cell lines.

FIG. 6B illustrates the presence of the proteins Bcl-X [_ and Bcl-xs in the cell lines IGROVl (OVl) and OAW42 (OA) and the cell lines

IGROVl -RIO (OVR) and SKOV3 (SK) evaluated by Western blot.

- Figure 7A illustrates the expression of bcl-xL and bcl-xS in the cytoplasm of ovarian tumor cells demonstrated by immunohistochemistry.

- Figure 7B illustrates the expression of the proteins Bcl-xL and Bcl-xS in samples of ovarian tumors, evaluated by Western blot. FIG. 8A illustrates the modifications of the expression of bcl-x in the cell lines IGROV1, OAW42 and IGROV1-R10 and SKOV3, 6 h after exposure to 5 and 20 μg / ml of CDDP.

FIG. 8B illustrates the modifications in the level of the proteins Bcl-xL and Bcl-xS in the cell lines IGROVl, OAW42 and IGROV1-R10 and SKOV3,

24 hours after exposure to 5, 20 and 100 μg / ml of CDDP.

FIGS. 9 and 10 illustrate the effectiveness of a combinatorial protocol combining chemotherapy (with different concentrations of CDDP) then non-viral transfer of the bcl-x gene to overcome the chemoresistance of ovarian cancers; these figures illustrate the number of viable (ordered) cells after transfection of SKOV3 cells with a plasmid Bcl-xS, 24 h after exposure to CDDP 5 μg / ml.

- Figure 11A illustrates the results obtained with a combinatorial protocol in which SKOV3 cells are transfected with bcl-xS and then exposed to 5 μg / ml of CDDP, for 2 h. - Figure 11B illustrates the effectiveness of a combinatorial protocol in which SKOV3 cells are exposed to 5 μg / ml of CDDP for 2 h, then transfected with bcl-xS.

FIG. 12 illustrates the results obtained with a protocol in which the gene transfer precedes the exposure to cisplatin by 24 h (a protective effect can be observed after exposure to 5 μg / ml of CDDP alone). In this case, the transfection of Bcl-xS does not increase the cytotoxicity of CDDP and even protects the cells against this agent. These results clearly show the importance of the order of administration of the products.

FIG. 13A illustrates the analysis, by the ribonuclease protection test, of the expression of the mRNAs of genes of the bcl-2 family in the ovarian tumor cells IGROV1, IGROV1-R10, OAW42 and SKOV3, 6 hours after exposure to 5 and 20 μg / ml of CDDP.

- Figure 13B illustrates the analysis, by the ribonuclease protection test, of the expression of mRNAs of genes of the bcl-2 family in SKOV3 ovarian tumor cells transfected with the bcl-xS gene.

- Figure 14 illustrates the effect on nuclear morphology of SKOV3 cells of treatment with: cisplatin alone, the bcl-xS gene or an empty plasmid (pcDNA) alone, or the combination of exposure to cisplatin with transfer of the bcl-xS gene, transfection following exposure to the anti-tumor agent.

- Figure 15 illustrates the dissemination, morphology (B) and histology (C) of ovarian tumors obtained at different times (21, 28 or 53 days) after the injection of SKOV3 cells in the swiss-nude mouse, by comparison with tumors observed in a patient with ovarian cancer (A).

- Figure 16 illustrates the analysis of gene transfer in vivo in ovarian tumors, after intraperitoneal injection of PEI / pCMV-luciferase complexes - Figure 16 A illustrates the PCR analysis of the location of the luciferase gene, 24 h after intraperitoneal injection of 100 μg of plasmid pCMV-Luc complexed with linear PEI of 22 kDa (L-PEI), prepared in 5% glucose. R: kidney; F: liver; PT: peritoneum; D: diaphragm; P: lung; C: heart; RA: spleen; PC: pancreas; TO: ovarian tube; M: muscle. FIG. 16B illustrates the analysis by PCR of the location of the luciferase gene, 24 h after three successive injections (in 36 h) of 100 μg of plasmid pCMV-Luc complexed with linear PEI of 22 kDa (L-PEI), prepared in 5% glucose. R: kidney; F: liver; PT: peritoneum; D: diaphragm; P: lung; C: heart; RA: spleen; PC: pancreas; TO: ovarian tube; M: muscle; Tum: ovarian tumor. FIG. 16C illustrates the analysis by luminometry of the luciferase activity, 24 h after three successive injections (in 36 h) of 100 μg of plasmid pCMV-Luc complexed with linear PEI of 22 kDa (L-PEI). prepared in 5% glucose. The results are expressed in relative light units (RLU) per mg of protein. R: kidney; F: liver; PT: peritoneum; D: diaphragm; P: lung; C: heart; RA: spleen; PC: pancreas; TO: ovarian tube; M: muscle; Tum: ovarian tumor.

It should be understood, however, that these examples are given by way ^of illustration of the object of the invention, which they do not in any way constitute a limitation. Example 1: Materials and Methods Cell Lines

The lines IGROVl (Bénard et al., Cancer Res., 1985, 45, 4970-4979), OAW42 (Wilson et al .. J. Natl. Cancer Inst., 1984, 72, 513-521) and SKOV3 (Fogh et al., J. Nall. Cancer Inst., 1977, 59, 221-226) are lines established from human adenocarcinoma of the ovary. The IGROVl -RIO line, obtained by successive treatments of the IGROVl line with increasing doses of cisplatin, has a resistance to cisplatin 10 times that of the parental line (Poulain et al., Int. J. Cancer. 1998, 78 , 454-463). as demonstrated by the results of the XTT test.

The IGROV1 and SKOV3 lines are cultivated in RPMI 1640 medium (GIBCO-BRL) containing 10% fetal calf serum (GIBCO-BRL), 2 mM L-glutamine (GIBCO-BRL), 33 mM sodium bicarbonate and 20 mM Hepes. The OAW42 line is cultivated in DMEM medium (GIBCO-BRL) containing 10% fetal calf serum, 2 mM L-glutamine (GIBCO-BRL), 1 mM sodium pyruvate and 20 IU / 1 of bovine insulin (GIBCO-BRL).

The cells are cultured at 37 ° C. in a humid atmosphere, in the presence of 5% CO? and divided twice a week by trypsination. IGROV1 cells, of polygonal appearance (characteristic of epithelial cells), grow in a monolayer and are capable, once the confluence is reached, of forming domes released in the medium and of re-adhering to a new support. These cells are tumorigenic in nude mice. The growth curve of the IGROVl line has a latency phase followed by an exponential phase before reaching a plateau after 6 days of culture. At the start of the exponential phase, the population of cells in suspension represents 1/5 of the total population, while at the end of this phase it represents 1/10 of the total population. The doubling time of the cells is 27 hours.

OAW42 cells (Wilson et al., 1984, supra) have a similar appearance to that of IGROV1 cells, although larger, but do not have the same growth characteristics. Indeed, their doubling time is of the order of 40 hours, and the confluence causes a stop of growth and a progressive degeneration of the culture in the absence of transplanting.

The SKOV3 line is derived from a human ovarian adenocarcinoma (Fogh et al., 1977, cited above). These cells, which only grow in monolayers and hardly support confluence, have a fibroblastic appearance. Their growth curve shows that they have a doubling time of 26 h. Treatment with cisplatin

The cisplatin [cis-diamino-dichloro-platinum II (CDDP)] supplied by Roger-Bellon (Cisplatyl ™) is diluted to 1 mg / ml in sterile distilled water and stored at - 20 ° C. The cells in the exponential growth phase are treated for 2 hours at 37 ° C. with different concentrations of CDDP in medium without serum. After treatment, the cells are rinsed and incubated in culture medium.

XTT test

The cells are seeded on a 96-well plate (10 ⁴ cells / well) then 2 days later, during the exponential growth phase, the cells are treated with increasing doses of CDDP. The cytotoxicity of cisplatin is measured by the XTT-PMS staining test, which measures cell viability, 6 days after exposure to CDDP, using the protocol described by Scudiero et al., Cancer Res., 1988, 48, 4827-4833. RT-PCR

1 μg of total RNA, extracted using the RNAeasy kit (QIAGEN) are back transcribed into cDNA, in the presence of 200 IU of reverse transcriptase (Superscript II, GIBCO-BRL), following the manufacturer's instructions. The cDNA obtained is amplified using the primers 5'-CGCAGAAGGGGTCCTGGTGA-3 '(SEQ ID NO: 1) and 5'-CAGCTCCTTCTTCTGCTCCGGGGT-3' (SEQ ID NO: 2), as described in Maronne et al. supra. The PCR reaction is carried out in the presence of 2 mM MgCl ₂ , 250 mM dNTP, 10 pmol of each of the primers and 5 IU of Taq polymerase (EUROBIO) under the following conditions: denaturation at 94 ° C for 40 sec, hybridization at 58 ° C for 60 sec and extension to 72 ° C for 40 sec (30 cycles), using an Amplitron ^R thermal cycler (BIOBLOCK). After electrophoresis of the products obtained in 2% agarose gel, two distinct fragments, corresponding respectively to Bcl-xS and Bcl-xL are observed. The fragment corresponding to the long form is much more abundant than that corresponding to the short form. Immunoblotting After 6 hours of treatment with CDDP, frozen tumor cells or grinds are lysed for 30 min on ice, in a buffer containing 150 mM NaCl, 50 mM Tris-HCl, pH 8.0, 1% Triton X-100, 4 mM PMSF, 2 mM aprotinin and 5 mM EDTA. The lysates are clarified by centrifugation at 10,000 g for 15 min at 4 ° C and the protein concentration is measured by the Bradford test (BIO-RAD). A fraction of cell extract (100 μg of protein) is separated by gel electrophoresis of 10% polyacrylamide-SDS, transferred to a nitrocellulose membrane by electrotransfer, 30 minutes at 500 mA in Tris-glycine-methanol (Hybond, AMERSHAM ). The membrane is incubated overnight at 4 ° C. in a T-TBS buffer (132 mM NaCl, 20 mM Tris-HCl pH 7.6 and 0.1% Tween 20) containing 5% of skimmed milk powder. The membrane is incubated, 1 h at room temperature, with the primary antibody (anti-Bcl-x, L / S 651186 E, PHARMINGEN) diluted 1/1000 in the T-TBS buffer containing 5% skim milk powder . After 3 washes with the T-TBS buffer, the membrane is incubated, 1 h at room temperature, with the secondary antibody conjugated to peroxidase (antibody NA 934, AMERSHAM) diluted 1/5000 in the same buffer as the antibody primary. After 4 washes with the T-TBS buffer and a washing with water, the immunoreaction is revealed by chemiluminescence (ECL kit, AMERSHAM). Colored low molecular weight markers (BIORAD) serve as a reference. immunohistochemistry

Once the 4 μm sections have been made in the paraffin blocks, they are spread on polysilanized slides so as to promote the adhesion of the tissue to the slide during the various steps which follow. The slides can be stored between two cardboard papers away from air and light until use. The slides are then dewaxed by successive passages of 5 minutes in 3 toluene baths, then in alcohol baths at 100 ° (x2), 95 °, 70 ° and 50 °.

They are then incubated in a bath of 1% TBS-H ₂ O2 for 10 min, before undergoing 3 passages of 4 '15 "in the 750W microwave in 10 mM citrate buffer pH 6.

After blocking the endogenous peroxidase activity by incubation in a 1% solution of hydrogen peroxide in PBS, the sections are transferred to PBS buffer. After a 20 min pre-incubation in TBS buffer containing 20% goat serum, the slides are incubated, 1 h at room temperature, with the primary anti-Bcl-x antibody (S 18 antibody, SANTA-CRUZ) diluted 1/500 in PBS buffer containing 0.5% bovine serum albumin (BSA). After extensive washes, the immune complexes are amplified using the HRP duet kit (DAKO), following the manufacturer's instructions. Finally, the immunoperoxidase activity is revealed by incubation, 5 min at room temperature, in a DAB solution (10 mg / ml, SIGMA) in a 50 mM Tris-HCl buffer, pH 7.6, 0.03%. H ₂ O ₂ . The slides are quickly counter-stained with hematoxylin. Plasmid transfection protocol

The transfection is carried out as described in Poulain et al., Cancer Gene Ther., 2000, 7, 4, 644-652.

Ribonuclease protection test (RPA or Ribonuclease protection assay)

The ribonuclease protection test (RPA) was carried out with hAPOl probes (RiboQuant ™ Multi Probe RNAse kit, Pharmingen, Becton-Dickinson), according to the supplier's instructions.

Analysis of the nuclear morphology The cells of the SKOV3 line resistant to CDDP, previously seeded in 24-well plates, are exposed for two hours to 5 μg / ml (C5) or 20 μg / ml (C20) of cisplatin. 24 hours later, these cells undergo transfection with LPEI / bcl-Xs plasmid complexes prepared in 5% glucose (N / P ratio = 5.2 g of plasmid per well). The nuclear morphology is observed 5 days later by staining of the nuclei with DAPI.

Analysis of gene transfer in vivo, in ovarian tumors, by PEI / naked DNA complexes

10 million SKOV3 cells were injected intraperitoneally into 6-week-old Swiss-nude mice. 28 days later, the plasmids pCMV-Luc and pCMV-β-gal were injected intraperitoneally in the form of L- complexes PEI / DNA (N / P ratio = 5) prepared in 5% glucose (100 or 250 μg of plasmid as a single injection or three injections of 100 μg of plasmid in 36 hours).

The distribution of the plasmid pCMV-Luc in the various organs was evaluated by PCR. The DNA was extracted using the DNeasy tissue kit (QIAGEN). lμg of DNA was then subjected to a PCR using two specific primers [Luci L, 5'-GCGCCATTCTATCCGCTGGA-3 '(SEQ ID NO: 3); Luci R, 5'- CTATCGAAGGACTCTGGCAC-3 '(SEQ ID NO: 4)].

^The luciferase expression was evaluated by measuring its activity by luminometry technique. The previously frozen bodies were crushed by using ^an Ultra Turrax in lysis buffer (Luciferase assay System, Promega, Cergy Pontoise, France). The lysates were then subjected to three freeze / thaw cycles and clarification. The protein concentration was measured using the Bradford test and the luciferase activity was quantified using the Promega kit and a lumino meter (Mediator PhL, Vienna, Austria), as described in Poulain et al. (Supra).

The expression of β-galactosidase was analyzed 24 hours after the injection of plasmid, using the β-gal staining kit (Invitrogen), following the manufacturer's instructions. Example 2: Efficacy of chemotherapy on ovarian tumor cell lines (IGROVl, OAW42).

The ovarian tumor cells IGROV1 and OAW42 described in Example 1 are exposed for 2 h to different concentrations of CDDP. Changes in their cell cycle and the recovery time from a normal state of proliferation are observed. The elongation of the S phase and the accumulation of cells in the G2-M phases are noted (FIG. 1A). The main differences concern the induction of apoptosis and the long-term survival of the treated cells. While highly sensitive cells (OAW42) are accumulated in phase S and massively enter apoptosis as early as 48 h after exposure to 5 μg / ml of CDDP, less sensitive cells (IGROVl) do not enter into apoptosis before 72 h at 96 h after ^exposure to 5 g / ml of CDDP. However, in both cases, almost all of the cells die after 4 days; a very small number of cells are then maintained in a quiescent state for several weeks, before recovering a normal growth model and the regeneration of a population capable of proliferating. The time before recovering from a proliferation state is 2 to 4 weeks for IGROV1 cells and is 8 weeks for OAW42 cells (Figure 1B). After 4 weeks, the effect of exposure to 5 μg / ml of CDDP is no longer visible on the histograms of IGROV1 cells (FIG. 2).

When the sensitive cells are exposed to 20 μg / ml of CDDP, a strong blockage in the G0-G1 phase is observed and the cells enter en masse after 24 h to 48 h (FIG. 5 A).

In this situation, no recurrence occurs, the ovarian tumor population is completely eliminated.

Example 3: Chemoresistance of ovarian tumor cell lines (SKOV3, IGROV1-R10) The ovarian tumor cells SKOV3 and IGROV1 -RIO described in the example are exposed for 2 h to different concentrations of CDDP. Changes in their cell cycle and the recovery time from a normal state of proliferation are observed.

There is a slowing down of the progression of the cell cycle, the accumulation of cells in the G2-M phase and the induction of apoptosis (FIG. 3A). Each event seems to occur in a very accelerated way compared to sensitive cells. Thus, despite massive cell death, the time before the reappearance of cells able to proliferate is almost eliminated, and the reappearance occurs between approximately 5 and 7 days for SKOV3 cells, and between 8 and 12 days for IGROVl -RIO cells ( Figure 3B).

After this time, the histograms no longer show any trace of exposure to 5 μg / ml of CDDP (FIG. 4).

When resistant ovarian tumor cells are exposed to 20 μg / ml CDDP, they are able to progress in the cell cycle (Figure 5B). IGROVl -RIO cells accumulate in the G2-M phase, which is not the case for SKOV3 cells.

Most cells then undergo apoptosis after 48 h, but a small population of them remains capable of re-initiating a new cell cycle and re-colonizing a culture flask in approximately 4 to 5 weeks. Example 4: Basal expression of bcl-x in ovarian tumor lines.

By RT-PCR, the bcl-x mRNA was demonstrated in all the cell lines studied, but only in its long form (L form) (figure 6A). A Western blot analysis shows indeed that 2-3 bands ^of approximately 30 kDa, corresponding to the Bcl-xL protein, are present in all cell lines studied, whereas Bcl-xS protein remains undetectable (Figure 6B).

The level of the Bcl-xL protein is quite similar in the IGROV1, IGROVl -RIO and SKOV3 cell lines, but is visibly lower in the OAW42 cell line.

The expression of Bcl-x was also evaluated by histochemistry on a panel of 60 specimens of ovarian tumors. 90% of them express Bcl-x in the cytoplasm of tumor cells (Figure 7A). The Western blot carried out on these samples only revealed the long form of the protein, Bcl-xL (figure

7B).

Example 5: Changes in the expression of bcl-x after exposure to cisplatin of the various sensitive and resistant ovarian tumor cell lines. The cells are exposed for 2 h to different concentrations of CDDP, then the modifications of expression of the bcl-x mRNA are studied by RT-PCR (FIG. 8A) or by protection with ribonuclease (FIG. 13A).

In response to exposure to 20 μg / ml of CDDP, the level of mRNA is reduced as early as 6 h after exposure to CDDP in the cell lines OAW42 and IGROVl, while it is maintained at a high level in the cell lines SKOV3 and IGROVl -RIO (Figure 8 A and 13 A).

The same results were observed at the protein level 24 h after exposure (FIG. 8B).

In response to exposure to 5 μg / ml CDDP, the level of bcl-x mRNA and the level of protein remain stable in cell lines

IGROVl, IGROV1 -R10 and SKOV3, 6 h and 24 h, respectively, after the exposure.

In the case of the OAW42 cell line. the mRNA level remains stable 6 h after exposure but the protein level is greatly reduced 24 h after exposure.

These results show that the bel-X _L mRNA is strongly expressed in the basal state in all of the lines studied, and that exposure to cisplatin causes a strong decrease in this expression in the IGROV1 and

OAW42, both sensitive to CDDP. This decrease is correlated with the absence of recurrence in vitro. On the other hand, in the IGROV-R10 and SKOV3 lines, capable of evading treatment and of recurring rapidly in vitro, the expression of bel-XL is not modified in response to the CDDP.

Example 6: Level of expression of Bcl-x and sensitivity to CDDP. The level of expression of the Bcl-x protein is similar in IGROV1 cells. IGROV1-R10 and SKOV3, but significantly lower in OAW42 cells. the latter are also the most sensitive to CDDP (see example 1, FIG. 5A).

Also, the modifications of expression of Bcl-x have been studied, in response to CDDP in the various ovarian tumor cell lines.

The CDDP is capable of inducing a repression of the expression of Bcl-x in the sensitive cell lines (IGROVl, OAW42) but not in the resistant cell lines (IGROVl -RIO, SKOV3). However, this repression can be observed only in conditions where massive and early apoptosis is observed: IGROVl exposed to 20 μg / ml of CDDP and OAW42 exposed to 5 or 20 μg / ml of CDDP and in which no recurrence is observed.

The correlation between variations in bcl-x RNA and the level of protein after exposure to CDDP suggests that the level of protein is mainly controlled at the level of RNA. EXAMPLE 7 In Vitro Efficacy of the Combination of an Antitumor Agent (CDDP) and of the Non-Viral Transfer of a Gene Involved in the Induction of Apoptosis (bcl-x) to Overcome the Chemoresistance of Ovarian Cancers .

A. Exposure to the antitumor agent after transfection (Figure 11 A). In this sense, the combinatorial protocol is not effective.

B. Transfection after exposure to the anti-tumor agent.

SKOV3 ovarian tumor cells (Figures 9, 10, 11B and 14), resistant to CDDP, are exposed for 2 hours to different concentrations of CDDP. About 24 h after this exposure, the cells are transfected with the non-viral vector PEI (22kDa) carrying the bcl-xS gene. The viability and nuclear morphology of the cells exposed to this combinatorial protocol are then observed over time by comparison with cells which have not undergone any treatment, cells exposed to CDDP or transfected cells. The results are shown in Figures 9, 10 and 14 below.

The cells exposed to 5 μg / ml of CDDP or transfected have a reduced viability compared to the control 72 h after the start of the treatment but gradually recover a normal state of proliferation between 72 h and 168 h.

Cells undergoing the combinatorial protocol exhibit a greatly reduced viability 72 h after ^exposure to 5 g / ml of CDDP compared to other cells exposed to CDDP alone or transfected only; this reduced viability persists until 168 h. Cells exposed to 20 μg / ml of CDDP or transfected with bcl-x after exposure to 20 μg / ml of CDDP have a greatly reduced viability 72 h after treatment and this effect persists until 168 h after treatment.

However, the combinatorial protocol makes it possible to completely annihilate the population and, unlike the only exposure to 20 μg / ml of CDDP, no recurrence is possible, the cells being in a state incapable of evolving towards proliferation.

These results are confirmed by the observation of the nuclear morphology, 6 days after the treatment with CDDP (FIG. 14) which shows that the combination of empty plasmid / CDDP has an effect little different from that of CDDP alone (increase in the size of the nuclei without abundant cell death), while the combination 6c / -xS '/ CDDP leads to many nuclear condensations and fragments characteristic of cell death by apoptosis. Five days after the gene transfer, the cell population exposed to 20 μg / ml of CDDP no longer shows any sign of viability. Example 8: Characterization of the expression of mRNAs of members of the bcl-2 family in SKOV3 cells transfected with the bcl-xS gene.

SKOV3 cells were seeded in a 24-well plate

(40,000 cells per well) and the transfection was carried out 24 hours later with L-PEI / DNA complexes prepared in 5% glucose with an N / P ratio = 5.2 μg of plasmid / well. The total RNAs were extracted 24 hours after the start of the transfection on RNeasy columns (QIAGEN), according to the recommendations of the supplier then the expression of the mRNAs of the members of the bcl-2 family was analyzed by the ribonuclease protection test as described in Example 1.

The results presented in FIG. 13B show that the transfer of the bcl-xs gene results in the production of its mRNA and that it does not modify the expression of the other members of the bcl-2 family.

EXAMPLE 9 Efficiency of Gene Transfer in Vivo in Ovarian Tumors by the PEI / Naked DNA Complexes

A model of peritoneal carcinosis of human ovarian origin was established in the immunocompromised mouse, by injection of cells of the SKOV3 line as described in Example 1. The histological analysis of the tumors obtained in the mouse shows that they are very similar to those observed in patients with ovarian cancer (Figure 15).

The efficacy of PEIs for non-viral gene transfer in the tumor nodules obtained was evaluated using reporter genes coding for luciferase (pCMV-Luc) and for β-galactosidase (pCMV-β-gal), in mice with a 28-day tumor, as described in Example 1.

No toxicity was observed and the results of the gene transfer are as follows:

- Figure 16 A shows that the plasmid is distributed throughout the peritoneum and that it is capable of leaving the peritoneal cavity to reach organs such as the lungs or skeletal muscles.

- Figure 16B shows that luciferase is expressed in different organs, and more importantly in tumor nodules.

The location of the cells expressing the transgene was specified by analysis of the expression of ^β -galactosidase, 24 hours after the injection of the plasmid.

(PCMV-β-gal). The results obtained show that β-galactosidase is preferentially expressed in tumor cells, thus confirming the results obtained by luminometry.

As is apparent from the above, the invention is in no way limited to those of its modes of implementation, embodiment and application which have just been described more explicitly; on the contrary, it embraces all variants which may come to mind of the technician in the field, without departing from the scope, or the scope, of the present invention.

Claims

REVENDICATONS 1 °) Products containing (1) a non-viral nucleic acid vector comprising at least one polyethylene imine (PEI) or a derivative of PEI, (2) a nucleic acid sequence comprising the bcl-xS gene or a functional fragment thereof and (3) at least one anti-tumor agent active in ovarian cancer, as a combined preparation for sequential use in the treatment of ovarian cancer, the nucleic acid sequence associated with said vector of non-viral nucleic acid being administered after the anti-tumor compound. 2) products according to claim 1, characterized in that the non-viral vector is chosen from PEIs of molecular weight less than 25 kDa. 3 °) Products according to claim 1 or claim 2, characterized in that the non-viral vector is selected from the group consisting of linear PEIs of molecular weight less than 25 kDa and PEIs on which are grafted a sugar, an antibody , a nucleotide, a lipid, a vitamin or a peptide. 4 °) Products according to any one of claims 1 to 3, characterized in that said PEI are associated with polyethylene glycol (PEG) or another hydrophilic polymer or else with folic acid. 5 °) Products according to any one of claims 1 to 4, characterized in that the anti-tumor agent active on ovarian cancer is preferably selected from the group consisting of platinum derivatives, taxanes, topoisomerase 1 and 2 inhibitors and combinations of these different products. 6 °) Products according to any one of claims 1 to 5, characterized in that said anti-tumor agents are associated with other anticancer agents and / or with amphotericin B or caffeine derivatives.