WO1997020924A1

WO1997020924A1 - A class of oligonucleotides, therapeutically useful as antitumoural agents

Info

Publication number: WO1997020924A1
Application number: PCT/EP1996/005388
Authority: WO
Inventors: Bruna Scaggiante; Franco Quadrifoglio
Original assignee: Saicom S.R.L
Priority date: 1995-12-04
Filing date: 1996-12-04
Publication date: 1997-06-12
Also published as: ITMI952539A0; ITMI952539A1; AU1175497A; IT1277025B1

Abstract

A new class of phosphodiesteric oligonucleotides which exert a selective cytotoxic activity on tumoural cells, having the nucleotide sequence of formula (I): N-Tx-(GaTa')a''-(GbTb')b''-(GcTc')c''-(GdTd')d''-(GeTe')e''-(GfTf')f''-(GgTg')g''-N' with orientation 5'-3' or 3'-5', where N and N', equal or different from each other, are T or G; x ranges from 0 to 8; a, b, c, d, e, f and g, equal or different from each other, range from 0 to 10; a', b', c', d', e', f' and g', equal or different from each other, range from 0 to 30; and a'', b'', c'', d'', e'', f'' and g'', equal or different from each other, range from 0 to 16. Furthermore, pharmaceutical compositions containing at least one of said phosphodiesteric oligonucleotides and their therapeutic use as antitumoural agents.

Description

A CLASS OF OLIGONUCLEOTIDES, THERAPEUTICALLY USEFUL AS ANTITUMOURAL AGENTS

FIELD OF THE INVENTION

The present invention relates to a new class of phosphodiesteπc oiigonucleotides, which exert a selective cytotoxic activity on tumoural cells STATE OF THE ART

One of the main targets of the research on cancer is the identification of drugs able to act in a selective way on tumoural cells, without exerting harmful effects on the healthy ones Aπtitumoural drugs presently used in clinical trials do not discriminate neoplastic cells from the healthy ones, since their target is generally DNA replication or the interference with metabolites The difference of toxicity which is observed in tumoural cells and which permits the clinical use of said antitumoural drugs is due to the fact that the transformed cells replicate and metabolize more rapidly than the healthy ones The consequences of the lack of specific tumoural targets in the mechanism of action of traditional chemotherapeutic agents are unavoidable side effects at the systemic level In the last few years, oiigonucleotides have been studied as antitumoural agents As a matter of fact, with respect to traditional drugs, nucleic acids, which can be effectively taken up by cells via either a receptor-mediated endocytosis mechanism and/or pinocytosis exhibit higher possibilities of selectively acting on specific targets, such as the products of oncogenes or of drug-resistance genes, since their action is based on the specific sequence of bases with which the genetic information is codified However, the use of nucleotide sequences in human therapy is strongly limited by the short half-life period of natural oiigonucleotides in the serum and in the cells which is due to the presence of RNases This limitation has been overcome by using oiigonucleotides where the internucleoside phosphatidic chain is modified, for example obtaining phosphotπesters, phosphonates and phosphorothioates, which are more resistant to the attack of nucleases, moreover, in order to favour the penetration of oiigonucleotides into cells, on whose mechanism several hypotheses have been put forward, oiigonucleotides have been advantageously linked to poly-L-lysme chains or to cholesterol residues i

Recent experiments on rats have shown that oiigonucleotides, when intravenously, intrapeπtoneally or differently administered, can achieve pharmacologically active concentrations in the target organs and are very well tolerated at the systemic level (Vlassov V et al , FEBS LETTERS, 327 271 -

^ 274 1991 )

The above oligonucleotide sequences can act according to several mechanisms of action and cellular targets (Helene C and Toulme J J , Biochimica et Biophysica Acta, 1049 99-125, 1990) The approach which is currently more studied in the treatment of tumours consists

I O in the use of antisense oiigonucleotides, in this case, the stop of the translation of specific mRNAs is performed by mimicking the natural process of half-life regulation of the mRNA present in cells More precisely, an oligonucleotide which is complementary to a specific sequence of mRNA forms DNA-RNA partial hybrids which lead to the stop of the translation of the message and/or to their

15 degradation

However, the application of this strategy gives rise to remarkable difficulties and disadvantages which are essentially due to the low extra- and intra-cellular half- life period of oiigonucleotides with respect to the rapid turnover of mRNAs Another strategy, basing its mode of action at an earlier step with respect to the

20 antisense inhibition consists in the formation of the intermolecular DNA triple helix This kind of approach is less studied than the previous one but it offers the potentiality of performing a block directly at the transcription level Even in this case some applicability limitations are noticeable, mainly due to the need of identifying suitable regions of the gene where homopuπnic/homopyπdinic

25 sequences are present

More specifically, according to the above mechanism of action, the Applicant has found that the oligonucleotide having the sequence 5'- TGTGTTTTTGTTTTGTTGGTTTTGTTT-3', is able to inhibit the mRNA transcription of the mdr1 gene, in the MDR tumoural cell line which codifies a

30 transmembrane glycoprotein responsible for drug-resistance (Scaggiante B et al , FEBS Letters, 352 380-384, 1994) Finally, oiigonucleotides can be used also as a target of specific proteins It is known that interactions between single-stranded DNA and/or RNA and proteins are at the basis of essential regulation mechanisms of the replication, transcription repair and recombination of DNA or of the ripening and translation

> of mRNAs These proteins are present in all living organisms from prokaπotes to eukaπotes, and they are known as "single-strand DNA-binding proteins" (SSBs) Often the SSBs do not recognise specific sequences, but they recognize specific motifs (Holligsworth M A et al , Nucleic Acids Research, 22 1138-1146, 1994) A Aharoπi et al (Nucleic Acids Research, 1993 Vol 21 , N° 22, p 5521 -5528)

I O illustrate the ability of different DNA segments, among which the d(GT)ι o sequence to bind a new protein called PGB which has been identified in human fibroblasts, however no biological significance of the affinity of the above sequence for this protein is suggested So far, in the state of the art, no oligonucleotide sequences have been proposed

! 5 which can bind proteins with a specific and selective cytotoxic effect on neoplastic cells

SUMMARY OF THE INVENTION

The Applicant has now found a new class of phosphodiesteπc oiigonucleotides having a sequence of formula (I)

20 N-T_x-(G_aT_a.)_a '-(G_bT_b.)b'- (G_CT_C )c"-(G_dT_d ^>)d"-(G_eTe^<)e"-(GfT_f )_f-(GgTg-)g^»-N

(I) with orientation 5'-3 or 3'-5', where N and N', equal or different from each other, are T or G x ranges from 0 to 8, a, b, c, d, e, f and g, equal or different from each other range from 0 to 10, a', b', c', d , e', f and g', equal or different from each

25 other range from 0 to 30, and a", b", c", d", e ', f" and g", equal or different from each other, range from 0 to 16, with the exception of the sequences 5'-TGTGTTTTTGTTTTGTTGGTTTTGTTT-3 (SEQ ID N° 1 ) and 5'-GTGTGTGTGTGTGTGTGTGT-3' (SEQ ID N° 11 ) Surprisingly, said phosphodiesteπc oiigonucleotides are able to exert a selective

30 cytotoxic activity on tumoural cells Further objects of the present invention are pharmaceutical compositions containing at least one of said phosphodiesteric oiigonucleotides and their use in the treatment of tumours

DETAILED DESCRIPTION OF THE INVENTION The features and the advantages of the new class of phosphodiesteric oiigonucleotides, of their therapeutic use as antitumoural agents and of the pharmaceutical compositions containing them, according to the present invention will be better descπbed in the following detailed description The above sequences of oligodeoxyπbonucleotides of formula (I) are able to act as specific and selective antitumoural agents In these oligonucleotidic sequences, N and N' can be T or G Moreover, said sequences

- contain a total number of nucleotides ranging from 10 to 60, preferably from 20 to 40

- contain a number of T nucleotides ranging from 10 to 40, preferably from 16 to 32,

- contain a number of G nucleotides ranging from 1 to 25, preferably from 2 to 10 Among the oiigonucleotides of formula (I), the sequence 5'- TGTGTTTTTGTTTTGTTGGTTTTGTTT-3' (SEQ ID N° 1 ), wherein N=N'=T, x=0, _a=b=c=d=f=1 , e=2, a'=1 , b'=5, c'=4, d'=2, e'=4, f=2 and a"=b"=c"=d"=e"=f"=1 , the remaining variables being 0, is already known in the state of the art Nevertheless it has been descπbed only the ability of said oligonucleotide to inhibit the production of a glycoprotein responsible for the drug-resistance in the MDR tumoural cell line, by inhibiting the transcription of the corresponding mRNA, with the mechanism of the molecular DNA triple-helix (Scaggiante B et al , reference cited above )

Also the sequence 5'-GTGTGTGTGTGTGTGTGTGT-3' (SEQ ID N° 1 1 ) corresponding to the formula ( I) wherein N=G, N'=T, x=1 , a=a'=1 , a"=8, b=b"=1 and b'=0 the remaining variables being 0, is already known in the state of the art, but no specific biological activity is reported (A Aharoni et al , reference cited above)

Among the phosphodiesteric oiigonucleotides of the invention, are particularly active the ones having the following sequences 5'-TGTTGTTGTTGTTGTTGTTGTTGTTGT-3' (SEQ ID N° 2), corresponding to the formula (I), wherein N=N'=T, x=0, a=1 , a'=2, a"=8, b=b"=1 and b'=0, the remaining variables being 0,

5'-TGTTTGTTTGTTTGTTTGTTTGTTTGT-3' (SEQ ID N° 3), corresponding to the > formula (I) wherein N=N'=T, x=0, a=1 , a'=3, a"=6, b=b"=1 and b'=0, the remaining variables being 0,

5'-TGTTTTGTTTTGTTTTGTTTTGTTTTGT-3' (SEQ ID N° 4) corresponding to the formula (I), wherein N=N'=T, x=0, a=1 , a'=4, a"=5 b=b"=1 and b'=0, the remaining variables being 0, I O 5'-TGTTTTTGTTTTTGTTTTTGTTTTTGT-3' (SEQ ID N° 5), corresponding to the formula (I), wherein N=N'=T, x=0, a=1 , a'=5, a"=4, b=b"=1 and b'=0, the remaining variables being 0,

5'-TTTGTTTTTTGTTTTTTGTTTTTTGTTTTTTGTTT-3' (SEQ ID N° 6), corresponding to the formula (I), wherein N=N'=T x=2, a=b=1 , a'=6, a"=4, b'=2 1 5 and b"=1 , the remaining variables being 0,

5'-TGTTTTTTTGTTTTTTTGTTTTTTTGTTTTTTTGT-3' (SEQ ID N° 7), corresponding to the formula (I), wherein N=N'=T, x=0, a=b=1 , a'=7, a"=4, b'=0 and b"=1 , the remaining variables being 0,

5'-GTTTGTTTGTTTGTTTGTTTGTTTGTG-3' (SEQ ID N° 8), corresponding to the 20 formula (I), wherein N=N'=G, x=3, a=b=1 , a'=3, a"=5, b'=b"=1 , the remaining variables being 0,

5'-TTTGTTGTTTTTGTTTTGTTTT-3' (SEQ ID N° 9), corresponding to the formula

(I), wherein N=N'=T, x=2, a=b=c=d=1 , a'=2, b'=5, c'=4, d'=3 and a"=b"=c"=d"=1 , the remaining variables being 0, 25 5'-TTTTTTTTGTTTTTTTTGTTTTTTTT-3' (SEQ ID N° 10) corresponding to the formula (I), wherein N=N'=T, x=7, a=b=1 , a'=8, b'=7, and a"=b"=1 , the remaining variables being 0,

5'-GGTTTGTTTGTTTGTTTGTTTGTTTGG-3' (SEQ ID N° 12), corresponding to the formula (I), wherein N=N'=G, a=1 , a'=3, a"=6 and b=b"=1 , the remaining 30 variables being 0, 5'-TGTTTGTTTGTTTGTTTGTTTGTTTGG-3' (SEQ ID N° 13), corresponding to the formula (I), wherein N=T N'=G, a=1 , a'=3, a"=6 and b=b"=1 the remaining variables being 0,

5'-TGGTTGGTTGGTTGGTTGGTTGGTTGGT-3' (SEQ ID NT 14) corresponding

=^; to the formula (I), wherein N=N'=T, a=2, a'=2, a"=6, b=2 and b"=1 the remaining variables being 0,

5'-TTTTTGTTTTTGTTTTTGTTTTTGTTTTTGTTTTT-3' (SEQ ID N° 15), corresponding to the formula (I), wherein N=N'=T, a=b=b"=1 , a'=5, a"=b'=4 and x=4, the remaining variables being 0,

I O 5'-TTTGTTTTGGTTGTTTTGTTT-3' (SEQ ID N° 16), corresponding to the formula (I), wherein N=N'=T, x=2, a=c=d=1 , b=2, a'=c'=4, b'=d'=2 and a"=b"=c '=d"=1 , the remaining variables being 0,

5 -TGTTTGTTTGTTTGTTTGT-3' (SEQ ID N° 17), corresponding to the formula (I), wherein N=N'=T, a=b= b"=1 and a'=a"=3, the remaining variables being 0,

1 5'-TGTTTGTTTGTTTGTTTGTTTGTTTGTTTGTTTGTTTGTTTGTTTGTTTGT-3' (SEQ ID N° 18), corresponding to the formula (I), wherein N=N'=T, a=1 , a'=3, a"=12 and b=b"=1 , the remaining variables being 0,

5'-TGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGT-3' (SEQ ID N° 19), corresponding to the formula (I), wherein N=N'=T, a=1 , a'=30, a"=1 and b=b"=1 ,

20 the remaining variables being 0

Moreover, the oiigonucleotides of the present invention can be modified on the iπterπucieosidic phosphatidic groups, on the terminal phosphate groups, on the bases and on the sugars, according to methods known in the state of the art, with the aim of increasing their resistance to the attack of the extra- and intra-cellular

25 nucleases In particular, the oligonucleotide sequences of the invention can be chemically modified on the terminal and/or internucleosidic phosphate groups to give methylphosphonates, phosphoroamidates, phosphorothioates, phosphorodithioates and phosphoroselenates Among the 3' and/or 5' phosphoroamidate analogs are preferred the deπvatizations with ι0 methoxyethylamine, dodecylamine and octadecylamine Furthermore, said sequences can be derivatized on the sugar mojeties to give L-desoxyπbose analogs, 2'-0-allyl- and 2'-O-methyl-desoxyπbose derivatives For illustative but not limitative purposes the following examples are reported

EXAMPLE 1

Preparation of oiigonucleotides of formula (I), according to the present invention.

^ The phosphodiesteπc oiigonucleotides according to the present invention have been synthesized by means of a DNA automated synthesizer by Applied Biosystem model 380 B, by using the phosphoroamidite method, according to a 1 μM standard procedure The oiigonucleotides thus obtained were then deprotected by heating at 56°C overnight 0 The oligonucletoides were then purified by FPLC on a MONO Q HR 5/5 column by using an ammoniun bicarbonate gradient The puπfied oiigonucleotides were free∑e-dπed and then suspended in 300 μl of NaCl (0 9%w) Their concentration was spectrophotochemically determined at the wavelength of 260 nm, at the temperature of 60°C ^ The purity of the thus obtained oiigonucleotides mesured by electrophoresis on a 15% polyacrylamide gel in 0 1 M acetic acιd/7M urea, under denaturating conditions, tumed out to be in the range of 80 to 90% The yield ranged from 30 to 60% Finally, the oiigonucleotides were sterilized by filtration on membranes with a 0 porosity of 0 2 μm EXAMPLE 2

Preparation of phosphorothioate derivatives of the oiigonucleotides of formula (I), according to the present invention. Fully phosphorothioate modified oiigonucleotides and 3'-phosphorothιoate ^ modified oiigonucleotides were synthesized by using the phosphoroamidite method according to a 1 μM standard procedure as descπbed in Example 1 The derivatized oiigonucleotides were then purified by gel permeation chromatography on Sephadex G50 fine resin using 0 05M ammoniun bicarbonate The eluted fractions were monitored by UV absorbance, at 254 nm The purified derivatized O oiigonucleotides were freeze-dried and then suspended in 300 μl of 0 9% NaCl Their concentration was spectrophotochemically determined by UV absorbance at the wavelength of 260 nm at the temperature of 60°C The purity of the thus obtained derivatized oiigonucleotides was mesured by gel electrophoresis, under denaturating conditions, as described in Example 1 The purity of the samples turned out to be of about 80% The reaction yield ranged from 50 to 60% Finally, the derivatized oiigonucleotides were sterilized by filtration on membranes with a porosity of 0 2 μm

BIOLOGICAL ACTIVITY

The oligonucleotide sequences according to the present invention exhibited the ability to exert, even only after one single administration, a significant and specific I O cytotoxic activity in human tumoural lines Moreover, the selectivity of the cytotoxic action of siad sequences for tumoural ceils was proved by the lack of effects on human healthy cells

In particular, toxicity tests were performed on several human cellular lines

The following lines were used 15 - CCRF-CEM lymphoblastic line, for a liquid tumour model,

- epithelial line of LoVo 1 09 colon adenocarcinoma, for a solid tumour model,

- U937 monocyte line, for a solid tumour line, in particular lymphoma

The results of the different experimental modeis showed that the oligonucleotidic sequences according to the present invention are very active both on liquid 20 (lymphoblastic) tumours and on solid (lymphoid) tumours, and exhibit an effective action on epithelial solid tumours

EXAMPLE 3

Evaluation of the cytotoxicity of the oiigonucleotides according to the present invention on CCRF-CEM tumoural cells. 25 CCRF-CEM tumoural cells were cultured in RPMI 1640 medium containing 10%w fetal calf serum, 20 mM Hepes, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 M

L-GIn

The cells were then seeded at the density of 5x10^ cells/ml in a 96 wells microtiter

(100 μl cell suspension, equal to 5x10^ total cells for each well) 30 After 24 hours of incubation, the oiigonucleotides having sequences SEQ ID N° 1 to SEQ ID N° 10, SEQ ID N° 12 to SEQ ID N° 16, SEQ ID N° 18 and SEQ ID N° 19 according to the present invention, were added directly to the culture medium in concentrations ranging from 2 5 to 30 μM

The effect of the administration of the above oiigonucleotides on the cellular growth and viability was evaluated after 24, 48 and 72 hours, by incorporation of

3-(4 5-dιmethyltιazol-2-yl)-2 5-dιphenyltetrazolιumbromιde (MTT) which was added at the concentration of 0 5 mg/ml

After 4 hours of incubation in the presence of the dye, the cells were centrifuged for 8 minutes at 400xg The medium was then removed by suction, the cells were disrupted and the dye was solubilized in 200 μl DMSO

The absorbance was read spectrophotometrically at the weavelengths of 540 and

690 nm

The percentage of cellular growth was measured assuming as 100% the cellular growth of untreated cells

The thus obtained results are reported in Table 1

Table 1

Cytotoxic effect of the oiigonucleotides according to the present invention on CCRF-CEM cells, after 72 hours from the administration of the sequences.

Oligonucleotide % Cellular growth reduction with oligonucleotide concentrations of 5μM 7 5μM 15μM

SEQ ID N° 1 62±17 79±1 3 92±6

SEQ !D N° 2 42±10 62±6 79±4

SEQ ID N° 3 59+19 72±1 1 85±7

SEQ 1D N° 4 43±12 57±7 78±2

SEQ ID N° 5 46+7 65±5 79±3

SEQ ID N° 6 50±14 66±1 1 87±4

SEQ ID N° 7 58±14 75±6 83±4

SEQ ID N° 8 51 ±12 67±6 83±5

SEQ ID N° 9 53±14 67±9 79±5

SEQ ID N° 10 36±17 48±15 76+1 1

SEQ ID N° 12 46±13 66±12 82±8

SEQ ID N° 13 41 ±12 66±1 1 83±8

SEQ ID N° 14 36±18 47±17 70±15

SEQ ID N° 15 59±12 67±13 87+9

SEQ ID N° 16 36±5 48±10 71 +1 1

SEQ ID N° 18 55±20 69±1 1 93±3

SEQ ID N° 19 34±22 46±21 71 ±10

The above data outline a significant reduction of the cellular growth, which is detectable 48 hours after the administration of the oligonucleotide of the invention

Moreover, the cytotoxic effects of the sequence SEQ ID N° 3 were evaluated for concentrations of 7 5 μM, on CCRF-CEM cells, after 24, 48 or 72 hours from the administration of the sequence. A decrease of 5% in cellular growth was detected after 24 hours and a decrease of 31 % was noted after 48 hours.

EXAMPLE 4

Evaluation of the relevance of the repeating unit (GT) in the specificity of the cytotoxic action of the oiigonucleotides of the present invention.

In order to check the relevance of the repeating unit (GT_n) in the sequences according to the present invention, was calculated the cytotoxic activity of oligonucleotidic sequences containing (CT_n), (AT_n), (GC_n) and (GA_n) as repeating unit, and more specifically of the following oligonucleotide sequences 5'-TCTTTCTTTCTTTCTTTCTTTCTTTCT-3', which will be indicated as (CT), 5'-TATTTATTTATTTATTTATTTATTTAT-3', which will be indicated as (AT), 5'-CGCCCGCCCGCCCGCCCGCCCGCCCGC-3'. which will be indicated as (GC), and

5'-AGAAAGAAAGAAAGAAAGAAAGAAAGA-3', which will be indicated as (GA) These sequences, which were synthesized and purified as described in Example 1 , were administered to CCRF-CEM cells at concentrations of 5, 7.5 and 15μM, according to the procedure described in Example 3. The results, detected after 72 hours from the administration of the sequences, are reported in Table 2. Table 2

Cytotoxic effect of oiigonucleotides with different repeating units on the growth of CCRF-CEM cells, after 72 hours from the administration of the sequences.

Oligonucleotide % Cellular growth reduction with oligonucleotide concentrations of 5μM 7.5μM 15μM

(CT) 4±10 9±9 11+1 1

(AT) 13±1 1 17±11 25±8

(GC) 12±11 23±13 28±19

(GA) 20±11 26±6 52±10 The results reported above prove that the oiigonucleotides having (CT), (AT) and (GC) repeating units cannot significantly alter the cellular growth, while the (GA) oligonucleotide is poorly toxic only if used at high concentrations ( 15μM), showing a cellular growth inhibition of 52% EXAMPLE 5

Evaluation of the relevance of the features of formula (I) to the cytotoxic activity exerted by the oiigonucleotides of the present invention. In order to check the relevance of the specific features of the sequences of formula (I) according to the present invention, was evaluated the cytotoxic activity of oligonucleotidic sequences containing the repeating unit (GT_π), but having the following characteristics with respect to formula (I)

A) N and/or N' are different from G and T

More specifically, the following oligonucleotide sequences were tested. 5'-AGTTTGTTTGTTTGTTTGTTTGTTTGA-3', which will be indicated as SEQ A1 ; 5'-CGTTTGTTTGTTTGTTTGTTTGTTTGC-3', which will be indicated as SEQ A2, 5'-TGTTTGTTTGTTTGTTTGTTTGTTTGC-3', which will be indicated as SEQ A3

B) The sequence has flanking fragments containing C and T bases. More specifically, the following oligonucleotide sequences were tested. 5'-CTTTTCTTTCTTTGTGTGTGTGTTTCTTTCTTTTC-3', which will be indicated as SEQ B1 ;

5'-TTTCTTTCTTTGTTGTTGTTGTTGTTTCTTTCT-3\ which will be indicated as

SEQ B2;

5'-TCTTTCTTTGTTTGTTTGTTTGTTTGTTTCTTTCT-3', which will be indicated as SEQ B3,

5'-TTTCTTTGTTTTGTTTTGTTTTGTTTTGTTTTCTTT-3', which will be indicated as SEQ B4,

5'-TCTTTGTTTTTGTTTTTGTTTTTGTTTTTGTTTTTGTTTCT-3', which will be indicated as SEQ B5. C) The sequence has a number of nucleotides lower than 10.

More specifically, the following oligonucleotide sequence was tested 5'-TGTTTGT-3 which will be indicated as SEQ C1

D) The oligonucleotide has at least a long stretch of sequence containing only G bases More specifically, the following oligonucleotide sequence was tested

5'-TTGGTTGTTTTTGGGGGGGGGGGTGG-3', which will be indicated as SEQ

D1

These sequences, which were synthesized and purified as descπbed in Example

1 were administered to CCRF-CEM cells at concentrations of 5, 7 5 and 15μM, according to the procedure described in Example 3 The results, detected after 72 hours from the administration of the sequences, are reported in Table 3

Table 3

Cytotoxic effect of oiigonucleotides with different sequence features on the growth of CCRF-CEM cells, after 72 hours from the administration of the same oiigonucleotides.

SEQ A1 12+1 1 17±10 23±15

SEQ A2 5±10 15±9 24±20

SEQ A3 1 1 +10 14±6 23±1 1

SEQ B1 9+10 7±8 9±9

SEQ B2 9±9 1 1 +9 26±18

SEQ B3 8±8 13+10 19+13

SEQ B4 15±4 22±8 26+16

SEQ B5 1 3+1 1 23±8 26±19

SEQ C1 2±5 7±5 15±14

SEQ D1 6±8 9±9 17±6

The results reported above demonstrate that

A) when N and/or N' in the sequences of formula (I) are other than G and T, the oiigonucleotides do not show any significant cytotoxic effects,

B) oiigonucleotides having flanking sequences containing C and/or T bases do not exert significant cytotoxic activities,

C) sequences having a number of nucleotides lower than 10 are not toxic with respect to cellular growth,

D) sequences with a number of adjacent G bases of 1 1 or more do not inhibit cellular growth

EXAMPLE 6

Selectivity of the cytotoxic activity of the oiigonucleotides of the present invention. Experimental trials were performed in order to evaluate the selectivity of the cytotoxic activity of the oiigonucleotides of formula (I) and the lack of cytotoxic effects on healthy human cells

The following cultures were used

- primary cultures of lymphocytes obtained from peripheral blood, seeded at 5x1 θ5 cells/ml (5x10^ cells/well), these lymphocytes were used both during the resting phase and after activation with 10 μg/ml of lectin, 24 hours before the addition of the oiigonucleotides,

- primary cultures of fibroblasts from human skin

The above cells were treated with the phosphodiesteric oligonucleotide SEQ ID N° 3 of the invention and with the (CT) sequence described above, according to the procedure descπbed in Example 3 The obtained results are reported in Table 4 Table 4.

Lack of cytotoxic effects of the oiigonucleotides according to the present invention in cultures of healthy human cells, 72 hours after the administration of the sequences.

Oligonucleotide (%) Cellular growth reduction with oligonucleotide concentrations of 7 5μM 15μM 30uM 50μM

Activated Lymphocytes n d 17±9 8±12 15+13

SEQ ID N° 3 n d 12±7 2±6 7±9 (CT) Sequence

Resting Lymphocytes

SEQ ID N° 3 n d 9+3 13+1 3 20±5

(CT) Sequence n d 2±2 4±5 4±5

Fibroblasts

SEQ ID N° 3 2±9 5±6 3±9 n d (CT) Sequence 3±7 14±7 10+7 n d The results reported above show that the sequences of the invention have no cytotoxic effects on the growth and viability of primary cultures of healthy cells, thus indicating a highly selective activity of said oiigonucleotides toward tumoural cells

EXAMPLE 7

Evaluation of the cytotoxicity of the oiigonucleotides according to the present invention in drug-sensitive and drug-resistant human tumoural lines. At the light of the enormous importance that drug-resistance plays in the treatment of many human tumourals at a primary onset or after partial remission experimental tests of cytotoxicity were performed on the following tumoural lines

- CEM-VLB100 drug-resistant lymphoblastic line,

- drug-sensitive epithelial cells from LoVo 109 colon adenocarcinoma,

- drug-resistant epithelial cells from LoVo Dx colon adenocarcinoma, - monocytes cell lines from the U937 lymphoma

These cells were treated with the phosphodiesteric oiigonucleotides SEQ ID N° 1 to SEQ ID N° 7, SEQ ID N° 9 and SEQ ID N° 12 to SEQ ID N° 14 of the present invention according to the procedure described in Example 3 For comparative purposes, the cells were also treated, under the same operating conditions, with the oiigonucleotides (CT), as described in Example 4, SEQ A1 , SEQ A3 and SEQ D1 , as described in Example 5

Again for comparative purposes, the above tumoural cellular lines were also treated, under the same operating conditions, with the 3'-phosphorothιoate derivative of SEQ ID N° 3, prepared as descπbed in Example 2 (hereinafter referred to as SEQ ID N° 3-3'-ρhosphorothιoate) The obtained results are reported in Table 5 Table 5.

Cytotoxicity of oiigonucleotides according to the present invention and of modified oiigonucleotides in drug-sensitive and drug-resistant human tumoural lines, after 72 hours from the administration of the sequences.

Oligonucleotide (%) Cellular growth reduction with ol gonucleotide concentrations of

5μM 75μM 15μM 30μM

CEM-VLB100

SEQ ID N° 1 57±18 66+9 73±6 nd

SEQ ID N°3 55±14 69±5 78±10 nd

SEQ ID N°3-3'- 52±14 nd nd nd phosphor 30±7 41 ±6 60±8 nd

SEQ ID N°9 9±4 20±6 37±12 nd

(CT) 0±9 4±12 6±12 nd SEQ D1

U937

SEQ ID N°2 58±16 66±11 86±7 nd

SEQ IDN°3 50±11 63±7 74+8 nd

SEQ IDN°4 45+18 66±12 78±15 nd

SEQ ID N°5 44+21 59±21 76±12 nd

SEQ ID N°6 53±21 65±19 76±112 nd

SEQ IDN°7 45±21 59±19 76±13 nd SEQ ID N° 12 59±9 69±3 79±5 nd SEQ IDN° 13 49±16 64±14 80^9 nd SEQ ID N° 14 67±16 73±10 86±6 nd (CT) 0±15 nd 1+19 1+13 SEQ A3 8+7 nd 21±5 34±8

LoVo 109

SEQ IDN°3 nd 17±2 26±2 32±2

LoVo Dx

SEQ ID N°3 nd 17±9 27±9 44±3

The data reported above prove that the phosphodiesteπc oiigonucleotides according to the present invention are able to exert very significant cytotoxic effects also on drug-resistant tumoural lines, especially on those of lymphoblastic origin On the contrary, oiigonucleotides with repeating units other than (GT_n) as well as with different sequence features with respect to the sequence of formula (I) do not significantly inhibit the cellular growth of drug-resistant tumoural lines

Furthermore the above data demonstrate that the cytotoxic activity of the 3'- phosphorothioate oiigonucleotides is comparable to the one exerted by the corresponding unmodified phosphodiesteric oiigonucleotides The oiigonucleotides according to the present invention can be profitably used in the treatment of tumours both of liquid type, in particular of lymphoblastic origin, and of solid type, in particular lymphomas Said oiigonucleotides are taken up by cells by receptor-mediated pynocytosis and endocytosis mechanisms According to an hypothesis of mechanism non-limitative of the present invention the phosphodiesteric oiigonucleotides corresponding to formula (I) can selectively bind and sequester some proteins which are essential to the viability and growth of tumoural lines, and in particular some nuclear proteins which could be expressed only in transformed cells In this case, said oiigonucleotides, contrary to other cytoxic compounds, would specifically and selectively block proteins essential to tumoural proliferation by meanwhile maintaining the viability of healthy cells Moreover the oligonucleotide-protein interaction could protect the nucleic acid from the intracellular degradation thus allowing the achievement of specific pharmacologic effects at doses lower than those used in the antisense or tπple- helix systems

Further objects of the present invention are pharmaceutical compositions containing as the active principle a therapeutically effective amount of at least a phosphodiesteric oligonucleotide having a sequence corresponding to formula (i) Said compositions can be systemically administered both orally and parenterally, as well as topically and transdermally Among the parenteral administrations, the intravenous, the intramuscular, the rectal and the intravaginal routes are preferred The therapeutically effective dose depends on the seriousness of the pathology, on the administration route and on the application conditions furthermore, it depends on the the age, the weight and the general health state of the patient The compositions of the invention include all the formulations with pharmaceutically acceptable excipients, useful for the administration of the active compound in the form which is more suitable to the pathology and which can render the oiigonucleotides of the invention remarkably bioavailable Said formulations can advantageously comprise the oiigonucleotides according to the present invention in association with carriers or ingredients able to increase their cellular uptake and to stabilize them to degradation

In particular, injectable solutions or suspensions can be advantageously used, comprising said oiigonucleotides in salted buffer, in physiological solution, in Ringer solution or in the solutions commonly used in the state of the art said injectable solutions and suspensions are particularly suitable for general endovenous subcutaneous and intramuscular administrations Solid or semi-solid formulations are also suitable, in the form of inserts, gels or ointments for topical administration In particular, the oiigonucleotides of the invention can be advantageously prepared in the form of powder, tablet or freeze- dried solid to be dissolved in a solution immediately before the parenteral use Liposomal formulations commonly used in the state of the art, both for parenteral and topical use, can also be particularly advantageous For oral administration, granules, tablets, pills and capsules are preferred, controlled-release formulations, known in the state of the art, are also suitable such as micro- or nano-spheres based on lipids and/or polysaccharides For dermal or transdermal administration, creams, ointments or gels, where the active principle can be entrapped in slow-release microspheres, are preferred

SEQUENCE LISTING

1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: SAICOM S.r.l.

(B) STREET: Padriciano, 99

(C) CITY: Trieste

(D) STATE: Trieste

(E) COUNTRY: ITALY

(F) POSTAL CODE ( ZIP) : 34012

(G) TELEPHONE: 040/3756611 (H) TELEFAX: 040/7797091

( ii ) TITLE OF INVENTION: A new class of phosphodiesteric oiigonucleotides, therapeutically useful as antitumoural agents.

(iii) NUMBER OF SEQUENCES: 19

( iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.25 (EPO)

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(iii) HYPOTHETICAL: NO

( ix) FEATURE:

(D) OTHER INFORMATION: Cytotoxic oligonucleotide sequence

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

TGTGTTTTTG TTTTGTTGGT TTTGTTT 27

(2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

( ii ) MOLECULE TYPE: DNA

(iii) HYPOTHETICAL: NO

( ix) FEATURE:

(D) OTHER INFORMATION: Cytotoxic oligonucleotide sequence

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

TGTTGTTGTT GTTGTTGTTG TTGTTGT 27

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(iii) HYPOTHETICAL: NO

( i ) FEATURE:

(D) OTHER INFORMATION: Cytotoxic oligonucleotide sequence

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

TGTTTGTTTG TTTGTTTGTT TGTTTGT 27

(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 28 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (iii) HYPOTHETICAL: NO ( ix ) FEATURE :

(D) OTHER INFORMATION: Cytotoxic oligonucleotide sequence

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

TGTTTTGTTT TGTTTTGTTT TGTTTTGT 28

(2) INFORMATION FOR SEQ ID NO: 5:

( i ) SEQUENCE CHARACTERIS ICS:

(A) LENGTH: 27 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(iii) HYPOTHETICAL: NO

( ix) FEATURE:

(D) OTHER INFORMATION: Cytotoxic oligonucleotide sequence

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

TGTTTTTGTT TTTGTTTTTG TTTTTGT 27

(2) INFORMATION FOR SEQ ID NO: 6:

( i ) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 35 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(iii) HYPOTHETICAL: NO

( ix) FEATURE:

(D) OTHER INFORMATION: Cytotoxic oligonucleotide sequence

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

TTTGTTTTTT GTTTTTTGTT TTTTGTTTTT TGTTT 35

(2) INFORMATION FOR SEQ ID NO: 7: ( l ) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 35 base pairs

(B ) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: DNA

(m) HYPOTHETICAL: NO

( x ) FEATURE:

(D) OTHER INFORMATION: Cytotoxic oligonucleotide sequence

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:

TGTTTTTTTG TTTTTTTGTT TTTTTGTTTT TTTGT 35

(2) INFORMATION FOR SEQ ID NO: 8:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 base pairs

(B ) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: DNA

(in) HYPOTHETICAL: NO

( x) FEATURE:

(D) OTHER INFORMATION: Cytotoxic oligonucleotide sequence

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

GTTTGTTTGT TTGTTTGTTT GTTTGTG 27

(2) INFORMATION FOR SEQ ID NO: 9:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ll) MOLECULE TYPE: DNA (ill) HYPOTHETICAL: NO ( ix ) FEATURE :

(D) OTHER INFORMATION: Cytotoxic oligonucleotide sequence

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

TTTGTTGTTT TTGTTTTGTT TT 22

(2) INFORMATION FOR SEQ ID NO: 10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

( ii ) MOLECULE TYPE: DNA

(iii) HYPOTHETICAL: NO

( ix ) FEATURE:

(D) OTHER INFORMATION: Cytotoxic oligonucleotide sequence

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

TTTTTTTTGT TTTTTTTGTT TTTTTT 26

(2) INFORMATION FOR SEQ ID NO: 11:

( i ) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

( B ) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(iii) HYPOTHETICAL: NO

(ix) FEATURE:

(D) OTHER INFORMATION: Cytotoxic oligonucleotide sequence

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

GTGTGTGTGT GTGTGTGTGT 20

Claims

1 1 A phosphodiesteric oligonucleotide having a sequence corresponding to

2 formula (I)

. N-T_x-(G_aT_a a"-(G_bT_b '- (G_cT_c.)c"-(G_dT_d')_d-(GeTe e"-(GfTf )f'-(GgT_g g-N' (I)

^ with orientation 5'-3' or 3'-5\ where N and N', equal or different from each other, b are T or G, x ranges from 0 to 8, a, b, c, d, e, f and g, equal or different from each

7 other, range from 0 to 10, a', b', c', d', e', f and g , equal or different from each

5 other, range from 0 to 30, a" b", c", d", e", f" and g", equal or different from each

0 other, range from 0 to 16, with the exclusion of the oiigonucleotides having the sequences SEQ ID N° 1 and 1 SEQ ID N° 1 1

1 2 The oligonucleotide according to claim 2, characterized by having at least a

2 derivatization on the internucleosidic phosphatidic groups, on the terminal

3 phosphate groups, on the bases and/or on the sugars.

1 3 The oligonucleotide according to claim 1 , characterized by the fact that said

2 derivatization is selected from the group consisting of methylphosphonate, ^"5 phosphoroamidate, phosphorothioate, phosphorodithioate, phosphoroselenate, L-

4 desoxyπbose, 2'-0-allyl- and 2'-0-methyl-desoxyπbose.

1 4 The oligonucleotide according to claim 1 or 2, characterized by containing a

2 number of nucleotides ranging from 10 to 60

1 5 The oligonucleotide according to claim 4, characterized by the fact that said

2 number of nucleotides ranges from 20 to 40

! 6 The oligonucleotide according to claim 1 or 2, characterized by containing a

2 number of T nucleotides ranging from 10 to 40

1 7 The oligonucleotide according to claim 6, characterized by the fact that said

2 number of T nucleotides ranges from 16 to 32

1 8 The oligonucleotide according to claim 1 or 2, characterized by containing a

2 number of G nucleotides ranging from 1 to 25

1 9 The oligonucleotide according to claim 8, characterized by the fact that said

2 number of G nucleotides ranges from 2 to 10 1 0 The oligonucleotide according to claim 1 , selected from the group consisting of the following sequences SEQ ID N° 2. SEQ ID N° 3, SEQ ID N° 4, SEQ ID N° 5, SEQ ID N° 6 SEQ ID N° 7, SEQ ID N° 8, SEQ ID N° 9, SEQ ID N° 10, SEQ ID N° 12. SEQ ID N° 13, SEQ ID N° 14, SEQ ID N° 15, SEQ ID N° 16 SEQ ID N° 1 7 SEQ ID N° 18 and SEQ ID N° 19

1 1 An oligonucleotide as described in anyone of claims 1 to 10, including the sequences SEQ ID N° 1 and SEQ ID N° 1 1 , for use as a medicament

12 The oligonucleotide according to claim 1 1 , for treating tumours

13 The oligonucleotide according to claim 12, characteπzed by the fact that said tumours are liquid tumours

14 The oligonucleotide according to claim 12, characterized by the fact that said tumours are solid tumours

15 The oligonucleotide according to claim 14, characterized by the fact that said solid tumours are lymphomas

16 A pharmaceutical composition containing as the active principle a therapeutically effective amount of at least an oligonucleotide as described in anyone of claims 1 to 10, including the sequences SEQ ID N° 1 and SEQ ID N° 1 1 in combination with suitable excipients and/or diluents

1 7 The pharmaceutical composition according to claim 16, characterized by being orally, parenterally, topically or transdermally administered

18 The pharmaceutical composition according to claim 16 characterized by being in the form of injectable solution or suspension

19 The pharmaceutical composition according to claim 16, characterized by being in the form of granules, tablets, pills, capsules, liposomes freeze-dried solids, micro- or nano-spheres based on lipids and/or polysaccharides

20 The pharmaceutical composition according to claim 16, characterized by being in the form of cream, ointment or gel