ITPD20100272A1 - GT APTAMERIC OLIGONUCLEOTIDS AND THEIR USE AS ANTI-HUMAN AGENTS - Google Patents

Description

Description of the industrial invention entitled:

GT aptameric oligonucleotides and their use as anticancer agents

Field of invention

The invention concerns new aptameric oligonucleotides of length 75 nucleotides (nt) and of GT sequence able to exert a specific and selective antiproliferative action in human tumor cells and to bind / inactivate the proteins of the eukaryotic elongation factor eEF1A family. The invention also concerns the therapeutic use of these oligonucleotides as new anticancer agents with antiproliferative activity in highly aggressive tumors and the diagnostic use as reagents for the specific detection of proteins of the eEF1A family.

State of the art

Among the members of the eukaryotic elongation factor family eEF1A, two genes are constitutively transcribed: EEF1A1 on chromosome 6q14.1 and EEF1A2 on chromosome 20q13.3. The eEF1A proteins belong to the superfamily of G proteins; are among the most abundant proteins in mammalian cells and participate in the protein translation process, bringing aminoacyl-tRNA (aa-tRNA) to the A site of ribosomes in the form of ternary complex eEF1A-GTP-aa-tRNA (Scaggiante B . et al., 2008). Both proteins eEF1A1 (Homo sapiens eukaryotic translation elongation factor 1 alpha 1: NM_001402.5 NP_001393.1) and eEF1A2 (Homo sapiens eukaryotic translation elongation factor 1 alpha 2: NM_001958.2 NP_001949.1) also have non-canonical functions: they are involved in many other essential cellular processes such as cytoskeletal remodeling and cell migration, modulation of kinase activity and phosphorylation, control of translation fidelity, modulation of ubiquitinylated protein degradation, cell response to stress and heat shock and activity conformation modulation of proteins (Scaggiante B. et al., 2008; Koiwai K et al., 2008; Zhong D et al., 2009; Jeganathan S et al., 2008; Leclercq TM et al., 2008; Panasyuk G et al., 2008).

In mammals, eEF1A1 is ubiquitously expressed, while the expression of eEF1A2 occurs in specialized tissues such as skeletal muscle, heart and central nervous system (Scaggiante B et al., 2008). Both eEF1A1 and eEF1A2 proteins are involved in the transformation and progression of cancer cells. In particular, the expression of the EEF1A2 gene in tissues other than skeletal muscle, heart and central nervous system is associated with the development of tumors and is related to their aggressiveness in ovarian, breast, pancreatic and hepatocellular cancers. (Scaggiante B. et al., 2008; Schlaeger C et al., 2008; Cao H et al., 2009; Sun Y et al., 2008; Lee et Surh, 2009). Furthermore, in hepatocarcinoma the overexpression of eEF1A1 / 2 correlates with an increase in the proliferation rate and eEF1A1 / 2 are more expressed in undifferentiated cells with a more aggressive phenotype (Grassi G et al., 2007). It has been shown that eEF1A2 determines the migration and invasiveness of breast cancer cells (Scaggiante B. et al, 2008). Furthermore, both eEF1A1 and eEF1A2 are interactors of the â € œgene human testis-specific Y-encodedâ € (TSPY) and it has been suggested that the TSPY-eEF1A1 / 2 complex promotes and supports the neoplastic transformation of the germ cells of the testis, but also probably that of prostate cells or other types of somatic tissues (Kido T and Lau YF, 2008). Overexpression of the EEF1A1 gene has been associated with increased proliferation and transformation of cells (Scaggiante B. et al., 2008; Scaggiante B. and Manzini G., 2010). The eEF1A1 protein regulates the half-life of osteopontin mRNA (OPN), a phosphoprotein whose overexpression is characteristic of advanced metastatic tumors, acting as a transactivating factor and promoting the invasiveness of hepatocarcinoma cells (Zhang J et al ., 2009). An increase in eEF1A1 is associated with resistance to cisplatin-resistant head and neck cancers. An isoform of eEF1A1 is present in human hematopoietic tumors, but not in normal cells. Deregulation of eEF1A1 in rodent cells exposed to chemical and physical carcinogens promotes neoplastic transformation. The decreased expression of eEF1A1 in leukemic cells of promyelocytes subjected to the action of the differentiating drug â € œAll-trans-retinoic acidâ € (ATRA) contributes to the survival of malignant cells. The overexpression of eEF1A1 contributes to the survival of breast cancer cells and is implicated in the invasiveness of these cells, while in mouse pro-B cells it confers resistance to apoptosis following stress (Scaggiante B et al., 2008 ; Scaggiante B. and Manzini G., 2010).

Overexpression of the EEF1A1 gene is related to resistance to methotrexate in many human solid and hematopoietic tumors (Selga E et al, 2009). During the alkalization of cancer cells, a common phenomenon in transformation, an increase in the levels of eEF1A1 / 2 mobilized by actin promotes and supports the invasiveness of neoplastic cells (Kim J et al, 2009).

Oligodeoxyribonucleotides GT phosphodiesteric or partially phosphorothioates with a length of between 20 and 60 nucleotides have been shown to be effective in specifically, selectively and dose-dependently inhibiting the growth of human tumor cells (WO 97/20924; Scaggiante B et al., 1998; Morassutti C et al., 1999a; Morassutti C et al., 1999b). The GT oligonucleotide sequences that function as aptamers are ligands of the eEF1A proteins with the ability also to recognize a specific isoform produced only by leukemic tumor cells (Dapas B. et al., 2003). These GT aptamers, in particular one of length 51 nucleotides, if carried by polycations, are active in nanomolar concentrations in inhibiting the proliferation of hematopoietic tumor cells (Scaggiante B. et al, 2005). It has been shown that GT aptamers from 23 to 27 nucleotides in length are active as anticancer agents if they do not give rise to particularly stable structures such as the quadruplexes of G (Scaggiante B et al, 2006). An aptameric GT sequences of length 27 nucleotides is able to increase the therapeutic index of conventional antineoplastic drugs in hematopoietic and solid tumors (Dapas B et al, 2002). These oligonucleotides have been shown to be active on many solid and hematopoietic human tumors. However, their activity has never been studied in cells with a high degree of malignancy such as hepatocarcinoma. In this type of tumor, proteins of the eEF1A family have been shown to be overexpressed and to contribute to tumor progression (Grassi G et al., 2007).

On the other hand, as is known, hepatocarcinoma is a type of tumor that finds little possibility of pharmacological interventions and therefore new formulations useful for controlling its expansion / progression are extremely important. Equally felt is the need to find molecules and formulations of molecules that are able to effectively control the growth of undifferentiated tumors and therefore their dissemination in the body and that have limited side effects or in any case have a favorable risk / benefit ratio compared to the severity of the disease and the possibility of being treated with methods currently in use.

Summary

In looking for a possible therapeutic modality to treat tumors with a high degree of aggressiveness, such as hepatocarcinoma, and still orphans of adequate therapy, the inventors have identified in the length of these aptameric GT oligonucleotides the substantial characteristic in determining the antiproliferative activity of the same. In particular, the inventors have found that the oligonucleotide having a length of 75 nucleotides has the ability to inhibit the proliferation of undifferentiated human hepatocarcinoma cells in a surprisingly specific and dose-dependent manner after a single administration of nanomolar doses.

Therefore, in a first aspect, the invention relates to a molecule consisting of an aptameric phosphorodiester oligonucleotide having SEQ ID NO.1 TG (TTTG) 18T (TGTTTGTTTGTTTGTTTGTTTGTTTGTTTGTTTGTTTGTTTGTTGTGTTGTTGTGTGTTGTGTGTTGTGTTGTGTTGTGTTGTGTTGT This sequence is hereinafter identified as GT75.

The invention also extends to derivatives of this aptameric oligonucleotide GT75 having SEQ ID NO.1 with different GT oligodeoxynucleotide sequences with orientation 5â € ™ -3â € ™ or 3â € ™ -5â € ™, which do not give rise to structures compatible with the quadruplexes of G and the oligoribonucleotides corresponding to the GT75 oligodeoxynucleotide sequence and derived oligodeoxynucleotide sequences. The derivatives of these oligodeoxynucleotide or oligoribonucleotide sequences with biochemical or chemical modifications suitable for stabilization, compartmentalization or localization in situ are still included in the invention. Such derivatives with biochemical or chemical modifications include alkylates, phosphorothioates, and / or conjugates with peptides, polycations or PEGs, as well as optical isomers (explanelmer).

In a second aspect, the object of the invention is the therapeutic use of the aptameric oligonucleotide having SEQ ID NO.1 TG (TTTG) 18T or its derivatives as an antitumor agent for the treatment of tumor pathologies and in particular for highly aggressive and / or undifferentiated tumors. Therefore, pharmaceutical compositions which comprise this SEQ ID NO.1 oligonucleotide or its derivatives for the treatment of highly aggressive and / or undifferentiated tumors are a further object of the invention.

In another aspect, the invention relates to the diagnostic use of the aptameric oligonucleotide having SEQ ID NO.1 TG (TTTG) 18T or its derivatives as reagents for diagnostic kits for the detection of proteins of the eEF1A family for the diagnosis / prognosis of highly aggressive tumor pathologies and for monitoring the effectiveness of the chosen chemotherapy treatment.

In a further aspect, therefore, the invention relates to the diagnostic kit comprising the aptameric oligonucleotide of SEQ ID NO.1 TG (TTTG) 18T or its derivatives for the diagnosis and / or prognosis of tumor pathologies and for the monitoring of € ™ effectiveness of the therapeutic treatment.

Brief description of the figures

Figure 1. Dose-response effect of GT75 on cell growth measured with the MTT test as absorbance of the U2SO line after 10 days of culture in comparison with the CT75 oligonucleotide.

Figure 2. Dose-response effect of GT75 on cell growth measured with the MTT assay as absorbance of the MO59J line after 10 days of culture in comparison with the CT75 oligonucleotide.

Figure 3. Dose-response effect of GT75 on cell growth measured with the MTT test as absorbance of the LNCaP, DU-145 and PC-3 lines after 10 days of culture in comparison with the CT75 oligonucleotide.

Figure 4. Effect of idarubicin at different doses after 10 days of JHH6 hepatocarcinoma cell culture measured with the MTT assay as absorbance.

Figure 5. Effect of bortezomib (velcade) at different doses after 10 days of culture of JHH6 hepatocarcinoma cells as measured by the MTT assay as absorbance.

Figure 6. Effect of the association between GT75 and idarubicin (single treatment scheme 1: transfection with oligonucleotides day 1, drug incubation day 2) at the indicated doses of idarubicin after 10 days of culture of JHH6 hepatocarcinoma cells treated with GT75 or CT75 or untreated NT and measured with the MTT test as absorbance.

Figure 7. Effect of the association between GT75 and velcade (single-dose regimen 1 as above) at indicated doses of velcade after 10 days of culture of GT75 or CT75-treated or non-NT treated JHH6 hepatocarcinoma cells and measured with the test of MTT as absorbance.

Figure 8. Effect of the association between GT75 and idarubicin (single-dose regimen 2: transfection with oligonucleotides day 1, drug incubation day 3) at the indicated doses of idarubicin after 10 days of culture of JHH6 hepatocarcinoma cells treated with GT75 or CT75 or untreated NT and measured with the MTT test as absorbance.

Figure 9. Effect of the association between GT75 and velcade (single-dose regimen 2 as above) at indicated doses of velcade after 10 days of culture of GT75 or CT75-treated or non-NT treated JHH6 hepatocarcinoma cells and measured with the test of MTT as absorbance.

Figure 10. Effect of the association between GT75 and idarubicin (single treatment scheme 3: transfection with oligonucleotides day 1, drug incubation day 4) at the indicated doses of idarubicin after 10 days of culture of JHH6 hepatocarcinoma cells treated with GT75 or CT75 or untreated NT and measured with the MTT test as absorbance.

Figure 11. Effect of the association between GT75 and velcade (single treatment scheme 3 as above) at indicated doses of velcade after 10 days of culture of GT75 or CT75 treated or non-NT treated JHH6 hepatocarcinoma cells and measured with the test of MTT as absorbance.

Figure 12. Effect of the association between GT75 and idarubicin (single-dose regimen 4: transfection with oligonucleotides day 1, drug incubation day 8) at the indicated doses of idarubicin after 10 days of culture of JHH6 hepatocarcinoma cells treated with GT75 or CT75 or untreated NT and measured with the MTT test as absorbance.

Figure 13. Effect of the association between GT75 and velcade (single treatment scheme 4 as above) at indicated doses of velcade after 10 days of culture of GT75 or CT75 treated or untreated NT JHH6 hepatocarcinoma cells and measured with the test of MTT as absorbance.

Detailed description of the invention

The invention concerns an aptameric oligonucleotide having SEQ ID NO.1 TG (TTTG) 18T (hereinafter referred to as GT75) and its derivatives having different GT sequences with 5â € ™ -3â € ™ or 3â € ™ -5â orientation € ™, which do not give rise to structures compatible with the quadruplexes of G. The invention also extends to derivatives of these oligodeoxynucleotides, including those having different GT sequences, consisting of the oligoribonucleotides corresponding to the phosphodiester oligodeoxynucleotide sequences. The derivatives of these oligonucleotide sequences are also included in the invention, whether they are oligodeoxy- or oligoribonucleotides, chemically and / or biochemically modified as known to an expert in the art to increase their stability and cellular penetration in situ (see review Grassi M et al 2010), for example phosphorothioates, alkylates, methylates, etc., and / or conjugates with peptides, polycations or PEGs and optical isomers.

In order to verify if the length of the oligonucleotide, together with the sequence, was the determining factor for the antiproliferative activity and for the specificity on highly aggressive and undifferentiated tumor cells, the GT75 oligonucleotide was compared with oligonucleotides GT of similar sequence but of shorter length, i.e. oligonucleotides of SEQ ID NO. 2 TG (TTTG) 6T (hereafter referred to as GT27) and SEQ ID NO. 3 TG (TTTG) 12T (hereinafter referred to as GT51) and with CT sequence oligonucleotides and same sequence and length as SEQ ID NO. 4 TC (TTTC) 18T (hereafter referred to as CT75) or shorter than SEQ ID NO. 5 TC (TTTC) 6T (hereafter referred to as CT27) and SEQ ID NO.6 TC (TTTC) 12T (hereafter referred to as CT51).

The extended sequences of the GT and CT aptamer oligonucleotides are shown below:

GT75 (SEQ ID NO.1) TGTTTGTTTGTTGTTTGTTTGTTGTTTGTTTGTTTGTTTGTTTGTTTGTTTGTT TGTTTGTTTGTTGTTTGT;

GT27 (SEQ ID NO.2)

TGTTTGTTTGTTTGTTTGTTTGTTTGT;

GT51 (SEQ ID NO.3) TGTTTGTTTGTTTGTTTGTTTGTTTGTTTGTTTGTTTGTTTGTTTGTTGT;

CT75 (SEQ ID NO.4)

TCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTT CTTTCTTTCTTTCTTTCT;

CT27 (SEQ ID NO.5)

TCTTTCTTTCTTTCTTTCTTTCTTTCT;

CT51 (SEQ ID NO.6)

TCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCT.

The comparison showed that GT75 demonstrated the ability to inhibit the proliferation of undifferentiated human hepatocarcinoma cells in a surprisingly specific and dose-dependent manner after a single administration of nanomolar doses. Conversely, GT oligonucleotides of less than 75 nt length, and in particular GT27 and GT51, did not demonstrate any significant growth inhibition activity of undifferentiated hepatocellular carcinoma cells. GT75 has also been shown to be active on other lines of solid human tumors that do not find effective therapeutic treatment such as osteosarcoma and glioblastoma. In a model of adenocarcinoma cells of the prostate with different degrees of aggression, GT75 proved to be very active in the most undifferentiated ones, confirming the result of its surprising antiproliferative action on highly aggressive human tumors.

Based on the results obtained, the SEQ ID NO. 1 and its derivatives can therefore be used as antiproliferative agents for the treatment of undifferentiated tumors with a high degree of aggressiveness and in particular for the treatment of tumors chosen from hepatocarcinoma, osteosarcoma, glioblastoma, adenocarcinoma of the prostate, melanoma, colonadenocarcinoma, leukemia / lymphomas and lung cancer.

Furthermore, GT75 has also been shown to be able to significantly increase the therapeutic index of conventional antineoplastics with the result of inhibiting the growth of hepatocarcinoma at subtoxic doses and with different formulation schemes of the active molecules. This potentiation effect is particularly interesting, because it allows GT75 to be used in combined action with conventional antineoplastic drugs at doses that minimize the risk of side effects in multiple types of tumor pathologies for which the conventional antineoplastic drug is considered to be therapy of election.

The present invention therefore also relates to a therapeutic method comprising the administration to a patient suffering from highly aggressive and / undifferentiated tumor pathologies, of the aptameric oligonucloeotide of SEQ ID NO. 1 and its derivatives according to the invention, with single administration or in combination with other antineoplastic drugs thereof.

Therefore the present invention extends to pharmaceutical compositions in which the active ingredients according to the invention are combined with suitable excipients and / or diluents or with other active ingredients for systemic or local administration. The active ingredients according to the invention are also preferably conveyed by drug-delivery systems such as liposomes, lipid nanoparticles and / or biodelivery systems and / or molecular â € œtargetingâ € systems mediated by protein sequences (for example â € œRGDâ € domains) at their site of action, i.e. the tumor, whether primary or secondary.

Furthermore, given the ability of aptamers to recognize proteins of the eEF1A family, further use of GT75 appears in the diagnostic sector through the use of oligomers of this type labeled with fluorescent substances or dyes for histological tests or for Aptamer DNA-based assays. .

The diagnostic method can also be of a prognostic type for the evaluation of tumor progression. In fact, if the histological examination shows overexpression of the eEF1A proteins, this may be related to a good / bad prognosis together with the scores already accredited and in use for the typing of the tumor stages. For diagnostic purposes in particular, the aptameric oligonucleotide GT75, conjugated to a fluorochromic or colorimetric detection system, can be anchored to a support where proteins extracted from biopsy samples are put in contact with the aptamer GT 75 or its derivative in a highly stringent solution that guarantees the specificity of the reaction. If specific proteins are present, they bind to the aptamer and are measured by means of the appropriate binding detection signal. Alternatively, the aptamer conjugated with a fluorophore or a chromophore can be used as an antibody (aptabody) on paraffinized histological samples or on fresh histological samples for histochemical analysis for diagnostic and prognostic purposes. Furthermore, the aptamer suitably conjugated at both ends with a fluorophore and a quencer can be used to quantify very precisely the eEF1A proteins present in a tumor sample by means of techniques such as FRET.

Typically the diagnostic method can include at least the phases of:

to. provide a biological biopsy sample;

b. contact the biological sample with the SEQ ID NO.1 oligonucleotide or its derivative, optionally labeled;

c. detect the reaction between the SEQ ID NO oligonucleotide. 1 or a derivative thereof and the proteins of the eEF1A family present in the biological sample;

d. measure the signal of detection of the occurred reaction.

The diagnostic kit for the determination of the expression of eEF1A proteins using the aptameric oligonucleotide GT75 or one of its derivatives may comprise at least: a container containing the aptameric oligonucleotide GT75 of SEQ ID NO.1 or one of its derivative, optionally labeled (preferably with fluorophores and / or chromophores), a container, a sample of the eEF1A proteins to be used as competitive ligands, optionally labeled, and a package leaflet with instructions.

Here are some examples of implementation of the invention and evaluation of the benefits deriving from the aptameric oligonucleotide GT75 of the invention, provided by way of non-limiting example of the invention itself.

EXAMPLES

Example 1: synthesis of oligonucleotides (GT) ne (CT) n (n = 75, 51, 27)

The deoxyribonucleotide sequences of the GT75 and CT75 phosphorodiester oligonucleotides were synthesized with the known chemical method of phosphoramidites (Mag M and Engels, 1988) and purchased from Eurofins MWG / operon. The oligonucleotides before use on cells were deprotected, desalted and purified from all toxic products by HPSF and their purity checked with MALDI-TOF MS.

The oligonucleotides thus prepared were tested on different human cell lines: undifferentiated human hepatocarcinoma, androgen-dependent adenocarcinoma of the prostate, androgen-independent adenocarcinoma of the prostate, osteosarcoma and glioblastoma according to the experimental methods described below.

Cell Line Cultivation Methods and Oligonucleotide Administration All human cell lines were cultured in complete medium containing 10% FBS and supplemented with 10 U / ml penicillin, 10 µg / ml streptomycin and 2 mM L-glutamine. The media, supplements and FBS were purchased from Invitrogen.

The cell lines used and the respective media are indicated below:

- JHH6 undifferentiated hepatocarcinoma, RPMI medium;

- LNCaP androgen-dependent adenocarcinoma of the prostate and DU-145 and PC-3 androgen-independent adenocarcinoma of the prostate (tumor cell aggression: PC-3> DU-145> LNCaP), RPMI medium;

- U2OS osteosarcoma, DMEM medium;

- MO59J glioblastoma, DMEM medium.

Unless otherwise indicated, the cells were seeded at the following densities in 200 µl of the appropriate complete medium in 96 microtiter wells:

- cells of the JHH6, DU-145, PC-3, U2OS lines at a density of 10 <3>; - the cells of the MO59J line at a density of 2.5x10 <3>;

- the cells of the LNCaP line at a density of 4 x10 <3>.

After 12 h of incubation at 37 ° C, 5% CO2 to allow adhesion, the cells were transfected with the synthesized oligonucleotides as described in example 1. The oligonucleotides were administered at the concentrations indicated in the examples below, using the method of transfection with cationic liposomes and in particular with Lipofectamine as described by Yu JY et al., 2002. Lipofectamin 2000 (Invitrogen) was hydrated in 100 µl of Optimem (Invitrogen), then mixed in a ratio of 1: 1 (w / w) with the oligonucleotides and the mixture was incubated for 20 min at room temperature. The liposome / oligonucleotide mixture was then diluted with optimem to obtain the desired concentration of oligonucleotides / 100 µl of medium. 100 µl of medium containing the oligonucleotides and the transfectant were added to the cells seeded in 96 well microtiter to replace their complete medium. After 3 hours of incubation at 37 ° C, the medium with the oligonucleotides and transfectant was replaced with 200 µl of fresh complete medium. The cell medium was replaced with fresh medium every 72 h of culture for the first two intervals and subsequently after 48 h, with the following scheme:

- day 0 sowing;

- 1st day transfection and medium change after 3 h;

- 4th day land change;

- 7th day land change;

- 9th day land change.

Cell growth was monitored with the MTT test (Scudiero DA et al., 1988) in the indicated times (3rd day, 6th day and 10th day from the administration of the oligonucleotide). The cells were added 20 µl of MTT (4 mg / ml) to each well. The cells were incubated for 4 hours at 37 ° C and 5% CO2, then the medium was removed and the cells solubilized in 200 µl of DMSO. The color development of the tetrazolium salts was read at 540-690 nm in a microtiter plate reader. Example 2: effect on antiproliferative activity in JHH6 undifferentiated human hepatocarcinoma cells - comparison between (GT) n and (CT) n (n = 75, 51, 27)

The comparison between GT75 and shorter GT sequences was performed on undifferentiated human hepatocarcinoma cells JHH6 according to the methods of cell culture cultivation and administration of oligomers described above, measuring the effect in different conditions of concentration of the oligomers, cell density and observation times from the administration of the oligomers.

In a first assay the measured cell growth was evaluated with the MTT test at a single administration of 250nM oligomers on 2.5x10 <3> cells in 6 well microtiters after 3 and 6 days from culture. GT aptameric sequences less than 75-mer in length (27-mer and 51-mer) are compared to GT75 as means ± sd of absorbance and as% of cell growth with respect to control sequences CT75, CT51 and CT27 or to untreated samples (NTC). Cell growth was evaluated with the MTT test as previously indicated and therefore the measurements performed are absorbance measurements.

The results obtained are shown in Table 1 below.

Table 1. Comparative effect of a single 250nM dose of oligonucleotides at 3 and 6 days after transfection on 2.5x10 <3> JHH6 cells

day GT27 CT27 GT51 CT51 GT75 CT75 NTC

3 ° 0.518 0.540 0.202 0.212 0.035 0.212 0.895

s.d. ± 0.05 ± 0.02 ± 0.06 ± 0.07 ± 0.03 ± 0.09 ± 0.08% CT 95 - 95 - 16 -% NTC 58 60 22 24 4 24

6 ° 0.787 0.839 1.203 1.029 0.688 1.122 1.122

s.d. ± 0.12 ± 0.09 ± 0.09 ± 0.05 ± 0.06 ± 0.09 ± 0.09% CT 94 - 117 - 61 -% NTC 47 51 73 62 41 68

As can be seen from the table, only GT75 is capable of exerting a significant inhibition of the proliferation of JHH6 tumor cells. Furthermore, it should be noted that the growth inhibition derives from a specific biological effect, as can be deduced from the comparison with the control sequence CT75. It should also be noted that in the CT75 control the lower cell proliferation derives from the non-specific toxicity due to the transfection treatment.

In a second test, cell viability was measured at a dose of 125 nM on cells with a density of 10 <3> in 96 well microtiter and with observation at 3, 6 and 10 days after transfection.

Table 2 shows the comparison data, measured as absorbance, obtained between GT75 and shorter GT sequences and the relative control CT sequences. Note how only GT75 demonstrates a significant antiproliferative effect 10 days after the start of a single administration compared to the CT75 control.

Table 2. Comparative effect at single dose of 125nM of oligonucleotides at 3, 6 and 10 days after transfection on 10 <3> cells of JHH6 day GT27 CT27 GT51 CT51 GT75 CT75 NTC

3 ° 0.559 0.424 0.285 0.267 0.150 0.236 0.915

s.d. ± 0.048 ± 0.014 ± 0.028 ± 0.037 ± 0.026 ± 0.024 ± 0.067% CT 132 - 107 - 63 -% NTC 61 46 31 29 16 26

6 ° 1.41 1.61 0.74 0.77 0.033 0.42 2.03

s.d. ± 0.124 ± 0.189 ± 0.118 ± 0.316 ± 0.025 ± 0.062 ± 0.183% CT 87 - 96 - 7.8

% NTC 69 79 36 38 1.6 21

10 ° 2.473 2.242 2.216 2.045 0.441 1.643 2.055

s.d. ± 0.199 ± 0.127 ± 0.125 ± 0.067 ± 0.144 ± 0.189 ± 0.022% CT 110 - 108 - 27 -% NTC 120 109 108 99 21 80

Example 3: effect on antiproliferative activity in undifferentiated human hepatocarcinoma cells JHH6 at the density of 10 <3> cells at different doses of oligonucleotides (GT) ne (CT) n (n = 75, 51, 27)

The comparison assay was carried out as in example 2, using in this case the cell density of 10 <3> and scalar doses of 125, 250 and 500nM oligonucleotides. Tables 3-5 show the comparative dose-response effects between GT75 and GT of shorter length on JHH6 seeded at the density of 10 <3> cells in 96 well microtiter and monitored at different times from the single administration compared to the control oligomers. CT and untreated samples (NTC). The determinations are also in this case of absorbance.

Table 3. Comparative effect at 125nM dose of oligonucleotides at 3, 6 and 10 days post transfection on 10 <3> cells of JHH6 day GT27 CT27 GT51 CT51 GT75 CT75 NTC

3 ° 0.48 0.43 0.166 0.159 0.006 0.084 0.86

s.d. ± 0.04 ± 0.015 ± 0.08 ± 0.02 ± 0.01 ± 0.03 ± 0.07% CT 111 - 104 - 0.07 -% NTC 56 50 19 18 0.7 9.8

6 ° 1.36 1.28 0.674 0.596 0.018 0.292 2.02

s.d. ± 0.12 ± 0.14 ± 0.12 ± 0.08 ± 0.01 ± 0.06 ± 0.11% CT 106 - 113 - 6 -% NTC 67 63 33 29 0.9 14

10 ° 2.48 2.45 2.29 2.36 0.162 1.987 2.52

s.d. ± 0.03 ± 0.21 ± 0.11 ± 0.07 ± 0.03 ± 0.05 ± 0.10% CT 101 - 97 - 8.1 -% NTC 98 97 91 94 6.4 79

Table 4. Comparative effect at 250nM dose of oligonucleotides at 3, 6 and 10 days post transfection on 10 <3> cells of JHH6 day GT27 CT27 GT51 CT51 GT75 CT75 NTC

3 ° 0.153 0.145 0.021 0.019 -0.006 0.01 0.86

s.d. ± 0.03 ± 0.03 ± 0.01 ± 0.01 ± 0.01 ± 0.005 ± 0.07% CT 105 - 110 - -0.5 -% NTC 18 17 2.4 2.2 -0, 7 1.5

6 ° 0.696 0.659 0.1 0.14 -0.001 0.06 2.02

s.d. ± 0.06 ± 0.07 ± 0.003 ± 0.009 ± 0.01 ± 0.03 ± 0.11% CT 106 - 71 - -0.2 -% NTC 34 33 5 7 -0.05 3

10 ° 2.35 2.17 0.743 0.789 0.05 0.581 2.52 s.d. ± 0.13 ± 0.14 ± 0.15 ± 0.29 ± 0.06 ± 0.28 ± 0.10% CT 108 - 94 - 8.6 -% NTC 93 86 29 31 2 23

Table 5. Comparative effect at 500nM dose of oligonucleotides at 3, 6 and 10 days post transfection on 10 <3> cells of JHH6

GT27 CT27 GT51 CT51 GT75 CT75 NTC

3 ° 0.06 0.08 0.01 0.01 -0.01 0.016 0.86

s.d. ± 0.004 ± 0.03 ± 0.01 ± 0.003 ± 0.005 ± 0.02 ± 0.07% CT 75 - 100 - -62 -% NTC 7 9 1 1 1 2

6 ° 0.35 0.33 0.09 0.11 -0.01 0.01 2.02

s.d. ± 0.004 ± 0.05 ± 0.08 ± 0.03 ± 0.007 ± 0.007 ± 0.11% CT 106 - 82 - -1 -% NTC 17 16 4 5 -0.5 0.5

10 ° 1.51 1.36 0.34 0.32 0.01 0.298 2.52 s.d. ± 0.26 ± 0.34 ± 0.19 ± 0.17 ± 0.004 ± 0.13 ± 0.10% CT 111 - 106 - 3 -% NTC 60 54 13 13 0.4 12

It can be noted that only GT75 is able to exert a specific dose-dependent inhibition effect on the growth of JHH6.

Example 4: study of the antiproliferative effect of GT75 and CT75 in other tumor cell lines

The antiproliferative activity of GT75 was monitored after 10 days of cell culture. Furthermore, for each tumor line the transfection efficiency was evaluated and only the lines with incorporation of at least 90% of the oligomers were considered.

As illustrated in Figure 1, GT75 exerts a significant and specific dose-dependent antiproliferative effect on an osteosarcoma line, compared to the CT75 control.

Figure 2 illustrates the dose-response effect of GT75 on a glioblastoma line. Note the specific antiproliferative effect starting from the 125 nM dose.

Figure 3 illustrates the antiproliferative effect of a panel of prostate cancer lines with different degrees of aggression (PC-3> DU-145> LNCaP). Note that GT75 exerts a significant and specific antiproliferative effect only in the most aggressive prostate adenocarcinoma line.

These results indicate that GT75 is particularly active in undifferentiated and higher aggression index human tumor lines that are hormone independent (PC-3).

Example 5: effect of GT75 and CT75 in association with conventional antineoplastics in hepatocarcinoma cells

The antiproliferative effect of the combination of GT75 and two conventional antineoplastic drugs (idarubicin and bortezomib) was studied in the undifferentiated JHH6 hepatocarcinoma lineage. Idarubicin is an anthracycline whose mechanism of action is expressed, like the daunomycin from which it derives, by intercalating with DNA and inhibiting its transcription. Bortezomib (velcade) is instead a dipeptidyl derivative of boronic acid which acts by inhibiting the activity of the 26S proteasome which degrades ubiquitinylated proteins. The consequent effect is the inhibition of a whole cascade of molecular events that lead to cell death. GT75 and the antineoplastics of clinical use have been administered according to different therapeutic schemes in subtoxic doses that alone do not significantly alter cell growth to verify if the combined treatment is able to give antiproliferative activity on cell growth.

The effect of different doses of the two conventional drugs was studied on the growth of 10 <3> JHH6 grown in 96 well microtiter according to the following therapeutic schemes:

- single administration regimen 1: transfection with oligonucleotides on day 1, incubation with drug on day 2;

- single treatment scheme 2: transfection with oligonucleotides on day 1, incubation with drug on day 3;

- single-dose treatment scheme 3: transfection with oligonucleotides on day 1, incubation with drug on day 4;

- single-dose regimen 4: transfection with oligonucleotides on day 1, incubation with drug on day 8.

As can be seen from figures 4 and 5, idarubicin does not significantly alter the growth of cells measured with the MTT test up to 5 ng / ml, while the velcade up to at least 15 nM.

As the standard subtoxic dose of GT75 was chosen that of 125 nM, which, under these experimental conditions, does not give more than 20% inhibition of cell growth. The subtoxic doses of idarubicin and velcade and that of GT75 are therefore used to study the effect of the combined treatment, as these alone are unable to produce a significant antiproliferative effect.

Due to the specificity of the action of the GT75, the experiments were also conducted in parallel with the CT75 control sequence. Cell growth was monitored with the MTT test after 10 days of culture.

Figure 6 shows the effects of the combination between GT75 and different subtoxic doses of idarubicin up to 5 ng / ml, while figure 7 shows that between GT75 and subtoxic velcade doses up to 15 nM with treatment scheme 1.

Figure 8 illustrates the effects of combining GT75 and different subtoxic doses of idarubicin up to 5 ng / ml, while figure 9 shows that between GT75 and subtoxic velcade doses up to 15 nM with treatment scheme 2.

Figure 10 illustrates the effects of combining GT75 and different subtoxic doses of idarubicin up to 5 ng / ml, while figure 11 shows that between GT75 and subtoxic velcade doses up to 15 nM with treatment scheme 3.

Figure 12 illustrates the effects of combining GT75 and different subtoxic doses of idarubicin up to 5 ng / ml, while figure 13 shows that between GT75 and subtoxic velcade doses up to 15 nM with treatment scheme 4.

From the results obtained, it can be concluded that GT75 is able to significantly and specifically increase the therapeutic index of the two drugs in all the different formulations indicated.

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Claims (15)

Claims 1. A molecule consisting of an aptameric oligonucleotide having SEQ ID NO.1 and its derivatives. 2. A molecule according to claim 1, wherein the derivatives of the aptameric oligonucleotide having the SEQ ID NO.1 are oligodeoxynucleotides with different GT sequences with orientation 5â € ™ -3â € ™ or 3â € ™ -5â € ™ which do not give rise to structures compatible with the quadruplexes of G. A molecule according to one of claims 1 and 2, wherein the derivatives of the oligodeoxynucleotides are the corresponding oligoribonucleotides. A molecule according to one of claims 1 to 3, wherein the derivatives are chemically and / or biochemically modified derivatives including alkylated derivatives, phosphorothioates and / or conjugated with peptides, polycations or PEGs and optical isomers (explanelmer). A molecule according to one of claims 1 to 4 for use as a medicament. A molecule according to one of claims 1 to 4 for use in the treatment of tumor pathologies. 7. A molecule according to claim 6, wherein the tumor pathologies are undifferentiated tumors. A molecule according to claim 6, wherein the tumor pathologies are hepatocarcinoma, osteosarcoma, glioblastoma, adenocarcinoma of the prostate, melanoma, colon adenocarcinoma, leukemias, lymphomas and lung carcinoma. 9. Pharmaceutical compositions comprising as the active principle a molecule according to one of claims 1 to 4 in combination with suitable excipients, and / or diluents and / or delivery systems for the same. 10. Pharmaceutical compositions according to claim 9, wherein said compositions are suitable for systemic and / or local administration. A molecule according to one of claims 1 to 4 for use as a reagent for the diagnosis and / or prognosis of tumor pathologies. A molecule according to one of claims 1 to 4 as a biological marker for monitoring the efficacy of the therapeutic treatment in tumor pathologies. 13. Diagnostic method for the diagnosis of tumor pathologies through the use of a molecule according to one of the claims from 1 to 4 for the detection of the expression of the eEF1A proteins. The method according to claim 13, comprising the steps of: to. provide a biological biopsy sample; b. contacting the biological sample with a molecule according to one of claims 1 to 4, optionally marked; c. detect the reaction between the molecule and the eEF1A family proteins present in the biological sample; d. measure the signal of detection of the occurred reaction. 15. Diagnostic kit comprising at least one container containing a molecule consistent according to one of claims 1 to 4, optionally labeled, a container containing a sample of proteins eEF1A, optionally labeled, and an illustrative leaflet for diagnostic and / or prognostic use in tumor pathologies and / or in monitoring the efficacy of therapeutic treatment in tumor pathologies.