HK1012000A - Stabilized oligonucleotids and the use thereof - Google Patents

Description

The invention relates to novel stabilised oligonucleotides modified in at least one non-terminal pyrimidine nucleoside and their use as a diagnostic or therapeutic agent for viral infections, cancer or diseases in which integrins or cell-cell adhesion receptors act.

Antisense oligonucleotides (AO) and triple helix forming oligonucleotides (TFO) have been shown to be specific inhibitors of gene expression in a wide variety of systems, both in vitro and in vivo [Uhlmann & Peyman, Chem. Rev. 1990, 90, 543; Milligan et al., J. Med. Chem. 1993, 36, 1923; Stein & Cheng, Science 1993, 261, 1004].

One of the main problems with the use of naturally occurring phosphodiester (PO) oligonucleotides is their rapid degradation by various nucleolytic activities both in cells and in the cell culture medium. Various chemical modifications have been used to stabilize oligonucleotides. An overview of the state of the art is given by Milligan et al., supra and Uhlmann & Peyman, supra. Stabilization against nucleolytic degradation can be done by modifying or replacing the phosphate bridge, the sugar structure, the nucleobase, or by replacing the sugar-phosphate backbone of the oligonucleotide.

Err1:Expecting ',' delimiter: line 1 column 634 (char 633)

The following strategies have been developed to determine the ideal positions in the oligonucleotide at which such modifications should be made (P. D. Cook (above); Uhlmann & Peyman (above); Milligan et al, (above)):

I) The replacement of all internucleoside bridges, e.g. to all PS oligonucleotides.

This exchange leads to highly stable oligonucleotides.For example, degradation by endonucleases (S1-nuclease) and by endo/exo-nuclease P1 is slowed by a factor of 2-45 compared to a PO oligonucleotide in an all-PS oligonucleotide [Stein et al., 1990, Nucl. Acids Res. 1988, 16, 1763]. All-PS oligonucleotides are also resistant in intact cells. In Xenopus oocytes or embryos, microinjected PO oligonucleotides are degraded with a half-life of thirty minutes, whereas all-PS oligonucleotides have a half-life of more than three hours under the same conditions [Woolf et al., 1990, Nucl. Acids Res. 18, 1763].

Err1:Expecting ',' delimiter: line 1 column 284 (char 283)

Other uniformly modified derivatives, such as all-2'-O-methyl derivatives or all α-2'-deoxyribo derivatives, are also generally characterised by a loss of RNase H activation capacity.

(ii) Copolymers of modified and unmodified phosphor ester bridges

Ghosh et al. [Anti-Cancer Drug Design 1993, 8, 15] describe a phosphorothioate phosphordiester oligonucleotide with different percentages of PS-bridges. These are, for example, constructed according to the scheme [PS-PO-PO-PO], [PO-PO-PS], [PS-PO], [(PO) 2-(PS) ], [PO-PS,PS] They teach that selective inhibition of the translation requires a content of at least 50% PS-bridges and that a sharp drop in effect occurs underneath them. The present invention shows that these results are not correct and that with the correct positioning of modifications of a content of well below 50% of the selective binding is achieved under positive selection (see also Ghosh et al., III. and that good binding is achieved under positive selection).

The alternating exchange of each second internucleoside bridge, e.g. against MeP bridges (Furdan et al., Nucl. Acids Res. 1989, 17, 9193), does not provide an advantage over uniformly modified MeP oligonucleotides.Oligonucleotides with alternating phosphate-O-ethyl or phosphate-O-isopropyl esters and alternating MeP oligonucleotides were also less active than all-MeP or all-PS oligonucleotides (Marcus-Secura et al., Nucl. Acids Res. 1987, 15, 5749).

(iii) The replacement of one, two or three internucleoside bridges at the 5' and 3' ends of the oligonucleotides (end-capping) and the replacement of one, two or three internucleoside bridges at the 5' and 3' ends of the oligonucleotides (gap technique).

Err1:Expecting ',' delimiter: line 1 column 1488 (char 1487)

Giles et al. [Anti-Cancer Drug Design 1993, 8, 33] describe methylphosphonate-phosphodiester chimeric oligonucleotides in which the gap at unmodified PO bridges was continuously reduced from eight to two bridges, showing a trend towards improved cell uptake at shortened gap, but the oligonucleotides were not studied for their antisense effect.

An interesting comparison of different strategies is provided by Hoke et al. [Nucl. Acids Res. 1991, 19, 5743], who compare the effects of different PS-modified anti-antisense oligonucleotides against HSV-1 in cell culture and their results confirm that 3' and 3' + 5' end-capping oligonucleotides (each modifying the first three internucleoside bridges) in serum, comparable to all-PS oligonucleotides, provide sufficient protection against nuclease degradation.On the other hand, internally modified (3 PS bridges) and only 5' endcapping oligonucleotides (the first three internucleoside bridges are also modified) are rapidly degraded, but they found that neither 5' nor 3' endcapping or both is sufficient for the effect in the cell and concluded that a uniform modification (all-PS) is necessary to achieve sufficient stability against nucleases in cells.

Surprisingly, pyrimidine nucleosides in oligonucleotides were found to be vulnerable to nuclease resistance, and when these sites were protected by modifications that increased the nuclease resistance, this in turn led to a significant improvement in stability and efficacy.

The invention is therefore based on oligonucleotides of the formulawith at least one non-terminal pyrimidine nucleoside modified.

Oligonucleotides with 2 to 10 modified, in particular 3 to 6 non-terminal pyrimidine nucleosides, are preferred, with no more than 8 consecutive nucleotides modified.In particular, oligonucleotides with additionally modified 5'- and/or 3'-ends are preferred, in particular those with the first 1-5 and in particular 1-3 nucleotides, especially 2-3 nucleotides at the 5'- and/or 3'-ends, preferably linked by phosphorothioate bridges, phosphorororothioate bridges and/or methylphosphonate bridges.For example, Table 1 shows the anti-HSV-1 oligonucleotide 01 double-spotted with PS at the 5' and 3' ends.The introduction of three PS bridges at 5' and 3' ends results in an increase in activity to 9 μM, the same effect is achieved by introducing a single additional PS 3' bridge to a cytosine residue C (antisense oligonucleotide No 03). The introduction of two PS bridges 5' and 3' respectively to T and C (antisense oligonucleotide No 05) or the introduction of four PS bridges 5' and 3' respectively to T and C (antisense oligonucleotides No 06) results in further increases in MHK-W activity (minimum inhibitory concentration) of 3 μM and 1 μM respectively.OtherThus, protecting the pyrimidine nucleoside allowed an increase in stability and activity comparable to that of the all-modified oligonucleotide, without having to face the disadvantages of such a drastic change described above.

The stabilisations at the pyrimidine positions and at the 5' and/or 3' ends may be independently performed as follows:(a) replacement of the 3'- and/or 5'-diester bridge of phosphoric acid, for example by a phosphorothioate, phosphorodiethylate, NR1 R2-phosphoramidate, borano-phosphorophosphate, phosphate- ((C1-C21) -O-alkylester, phosphate- ((C6-C12) -C1-C21) -O-alkyl]ester, 2,2,2-trichloro-dimethylethylphosphate, (C1-C8) -alkylphosphonate, (C6-C12) -arylphosphorophosphate bridge. The preferred replacement by a phenophorothioate, phosphorodiethylate, (C1-C12) -orano-phosphorophosphate, (C1-C21) -O-alkylester, (C6-C12) -orano-phosphorophosphate, (C1-C12) -orano-phosphorophosphate, (C1-C12) -orano-phosphorophosphorophosphate, (C1-C1-C12) -O-alkylphosphorophosphorophosphorophosphate, (C1-C1-C1-C1-C12) -orophosphorophosphorophosphorophosphorophosphorophosphorophosphate, (C1-O-O-O-O-O-O-phosphorophosphorophosphorophosphorophosphorophosphorophosphorophosphorophosphorophosphorophosphorophosphorophosphorophosphorophosphor), (C1-O-O-O-O-O-O-O-O-O-O-phosphorophosphorophosphorophosphorophosphorophosphorophosphorophosphorophosphorophosphor), (O-O-O-O-O-O-O-O-O-O-O-phosphorphorphorphorophosThe substitution of a phosphorothiate bridge is particularly desirable.R1 and R2 stand for hydrogen independently of each other or for C1-C18 alkyl, C6-C20 aryl, (C6-C14) aryl- ((C1-C8) alkyl, - ((CH2) c-[NH(CH2) c-d-NR3R3, where c is an integer from 2 to 6 and d is an integer from 0 to 6, and R3 is independent of each other hydrogen,Err1:Expecting ',' delimiter: line 1 column 497 (char 496)Err1:Expecting ',' delimiter: line 1 column 104 (char 103)The following substances are to be classified in the same category as the active substance:The preferred substitution is 5-C1-C6-alkyl-uracil, 5-C2-C6-alkenyl-uracil, 5-C2-C6-alkinyl-uracil, 5-C1-C6-alkyl-cytosine, 5-C2-C6-alkenyl-cytosine, 5-C2-C6-alkinyl-cytosine, 5-fluoruracil, 5-fluoruracil, 5-chloruracil, 5-chloruracil, 5-bromuracil, 5-bromurasacil, 5-bromurasacil, which are the only substitutions that are used.The substitution of 5-C3-C6-alkyl-uracil, 5-C2-C6-alkenyl-uracil, 5-C2-C6-alkynyl-uracil, 5-C1-C6-alkylyl-cytosine, 5-C2-C6-alkenyl-cytosine, 5-C2-C6-alkylyl-cytosine is particularly preferred.The most preferred substitution is 5-pentinylcytosine, 5-hexinyluracil, 5-hexinylcytosine.

Of the above modifications, the modifications from groups a), b), c) and d) are preferred.

In addition, the oligonucleotides of the invention may be conjugated, for example, at the 3'- and/or 5'-ends with molecules that have a beneficial effect on the properties of antisense oligonucleotides or triple helix oligonucleotides (such as cell penetration, nuclease degradation, affinity for target RNA/DNA, pharmacokinetics), e.g. conjugates with poly-lysine, with intercalators such as pyrene, acridine, phenazine, anthracin, with fluorescent compounds such as fluorescein, with cross-linkers such as polyester, azidoproflaidin, with polyphenol derivatives such as C12kyl-Pyrosyl-Pyrosyl-Pyrosyl-CH20-OH, or their conjugates such as lipophosphate, such as C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-C12-OtherOtherOtherThe following is a list of the active substances that may be used in the preparation of the active substance:OtherOtherOther

Err1:Expecting property name enclosed in double quotes: line 1 column 223 (char 222)

Furthermore, the oligonucleotides of the invention may be carried at the 3' and/or 5' end in 3'-3' and 5'-5' inversions (described, for example, in M. Koga et al., J. Org. Chem. 56 (1991) 3757).

The invention also relates to processes for the production of the compounds of the invention by means of processes known to the professional, in particular chemical synthesis, the use of the compounds of the invention for the manufacture of a medicinal product and a process for the manufacture of a medicinal product characterized by mixing the oligonucleotides of the invention with a physiologically acceptable carrier and, where appropriate, suitable additives and/or excipients.

More generally, the present invention also covers the use of therapeutically active oligonucleotides to produce a medicinal product in which at least one non-complete pyrimidine nucleoside is modified. Therapeutically active oligonucleotides are generally understood to include antisense oligonucleotides, triple helix-forming oligonucleotides, aptamers (RNA or DNA molecules that can bind to specific target molecules, e.g. proteins or receptors (e.g. L.C. Bock et al., 1992, Nature 355, 564) or ribozymes (catalytic RNA, e.g. Castanetto et al., Critical Eukaryotic Gene Expr. Rev. 2, Rev. 331, 1992, particularly antisense oligonucleotides).

In addition, another subject of the present invention is the use of oligonucleotides with at least one non-terminal and modified pyrimidine nucleoside as a diagnostic agent, for example to detect the presence or absence or amount of a specific double-stranded or single-stranded nucleic acid molecule in a biological sample.

The oligonucleotides have a length of about 6-100 for use according to the invention, preferably about 10-40 and especially about 12-25 nucleotides.

The medicinal products of the present invention may be used, for example, to treat diseases caused by viruses, such as HIV, HSV-1, HSV-2, influenza, VSV, hepatitis B or papilloma viruses.

Antisense oligonucleotides of the invention that are effective against such targets include:The drugs of the present invention are, for example, also suitable for the treatment of cancer, for example by using oligonucleotide sequences directed against targets responsible for the development or growth of cancer, such targets being:1) Nuclear oncoproteins such as c-myc, N-myc, c-myb, c-fos, c-fos/jun, PCNA, p1202) Cytoplasmic/membrane-associated oncoproteins such as EJ-ras, c-Ha-ras, N-ras, rrg, bcl-2, cdc-2, c-raf-1, c-mos, c-src, c-abl3) Cellular receptors such as EGF receptor, c-erbA, retinoid receptor, protein kinase regulatory subunit, c-fms4) Cytokines, growth factors, extracellular matrix such as ILF-1, ILF-1, CSF-1, IL-1a, ILB-2, ILF-4, blastocyst, fibroblast, are effective against such targets as mycobacterium, which are effective against mycobacterium, mycobacterium, mycobacterium, and mycobacterium.For example, the drugs of the present invention are also suitable for the treatment of diseases affected by integrins or cell-cell adhesion receptors, such as VLA-4, VLA-2, ICAM or ELAM.

Antisense oligonucleotides of the invention, which are effective against such targets, are, for example:The medicinal products may be used, for example, as pharmaceutical preparations which can be administered orally, e.g. as tablets, dragees, hard or soft gelatine capsules, solutions, emulsions or suspensions.They can also be administered rectally, e.g. in the form of suppositories, or parenterally, e.g. in the form of solutions for injection. For the manufacture of pharmaceutical preparations, these compounds can be processed into therapeutically inert organic and inorganic media. Examples of such media for tablets, dragees and hard gelatine capsules are lactose, cornstarch or derivatives thereof, tallow and stearic acid or their salts.Polyoles, sucrose, invert sugar and glucose. Suitable carriers for solutions for injection are water, alcohols, polyoles, glycerol and vegetable oils. Suitable carriers for suppositories are vegetable and cured oils, waxes, fats and semi-liquid polyoles. Pharmaceutical preparations may also contain preservatives, solvents, stabilizers, net agents, emulsifiers, sweeteners, dyes, flavourings, salts to alter osmotic pressure, buffers, emulsifiers, antioxidants, and other therapeutic agents, if appropriate.A preferred form of administration is injection, where the antisense oligonucleotides are formulated in a liquid solution, preferably in a physiologically acceptable buffer such as Hank's solution or Ringer's solution, but the antisense oligonucleotides can also be formulated in a solid form and dissolved or suspended before use.The doses preferred for systemic administration are approximately 0.01 mg/ kg body weight to approximately 50 mg/ kg body weight per day.

The following examples are intended to illustrate the invention:

Example 1Oligonucleotide synthesis

Modified oligonucleotides were synthesised on an automatic DNA synthesizer (Applied Biosystems Model 380B or 394) using standard phosphoramidite chemistry and iodine oxidation. For introduction of phosphorothiate bridges into mixed phosphorothiates and phosphodiester oligonucleotides were oxidised with TETD (tetraethyl disulfide) instead of iodine (Applied Biosystems User Bulletin 65). After cleavage from the solid carrier (CPG or tentagel) and removal of the protective groups with NH3 at 55oC for 18h, the oligonucleotides were obtained first by precipitation of butanol (Sawadogo, Vanke, Dyucl.

The introduction of the [4- ((1-pyrenyl) butanyl]phosphodiester at the 5' end is as described in J. S. Mann et al. Bioconj. Chem. 3 (1992) 554.

The analysis of oligonucleotides was carried out by:(a) Analytical gel electrophoresis in 20% acrylamide, 8M urea and/or HPLC: Waters GenPak FAX, Gradient CH3CN (400 ml), H2O (1.6 l), NaH2PO4 (3.1 g), NaCl (11.7 g), pH6.8 (0.1 M in NaCl) after CH3CN (400 ml), H2O (1.6 l), NaH2PO4 (3.1 g), NaCl (175.3 g), pH6.8 (1.5 M in NaCl) and/or) Capillary gel electrophoresis Beckmann Capillary eCAPTM, U100P Column, 65 cm length, 100 mm I.D., 15 cm from one end, Buffer 140 μM Tris, 360 mm boric acid, 7Mn and 7Mn/day) The microscopy of the test specimen showed that these were of greater than 90% purity.

The structures of the synthesised oligonucleotides are shown in Table 1.

Example 2Testing of antiviral activity of test substances against herpes viruses in vitro

The antiviral activity of the test substances against various human pathogenic herpes viruses is investigated in the cell culture test system.

For the test, monkey kidney cells (Vero, 2x105/ml) are sown in serum Dulbecco's MEM (5% fetal calf serum FCS) in 96-pot microtiter plates and incubated for 24 h at 37°C and 5% CO2.

The test substances are pre-diluted in H2O to a concentration of 600 μM and stored at -18 °C. Further dilution steps are performed in Dulbecco's Minimal Essential Medium (MEM) for the test. Each 100 μl of the individual test substance dilutions is added to the rinsed cells together with 100 μl of serum-free Dulbecco's MEM (-FCS).

After 3 h incubation at 37°C and 5% CO2, the cells are infected with herpes simplex virus type 1 (ATCC VR733, HSV-1 F strain) or herpes simplex virus type 2 (ATCC VR734, HSV-2 G strain) at concentrations where the cell walls are completely destroyed within 3 days. In HSV-1, the infection rate is 500 plaque-forming units (PFU) per nap, in HSV-2, 350 PFU/nap. The test pieces then contain test substance at concentrations of 80 μM to 0.04 μM in MEM, supplemented by 100 U/ml penicillin G and 100 mg/l streptomycin. All tests are performed as a double determination with eight controls, except for the one panel.

The test pieces are incubated for 17 h at 37°C and 5% CO2.The cytotoxicity of the test substances is determined after 20 h total incubation time by microscopic examination of the cell cultures.The maximum tolerated dose (DTM) is the highest concentration of the product which under the specified test conditions does not cause microscopically detectable cell damage.

FCS is then added to a final concentration of 4% with further incubation for 55 h at 37°C and 5% CO2.After microscopic examination of the cell cultures, they are then stained with neutral red according to the Finter (1966) vitalising method.The antiviral activity of a test substance is defined as the minimum inhibitory concentration (MIC) needed to protect 30-60% of cells from the viral cytopathogenic effect.Other

Example 3Testing of antiproliferative activity of test substances in smooth muscle cells

The following oligonucleotides were tested for their ability to inhibit smooth muscle proliferation. The test was performed as described in S. Biro et al. [Proc. Natl. Acad. Sci. USA 90 (1993) 654]. All oligonucleotides were effective in the range 5 to 20 μM.$\text{P=S}$) substituted phosphodiester bonds are marked with * in the sequence.

Claims (16)

1. Oligonucleotides of the formula characterised by the modification of at least one non-terminal pyrimidine nucleoside.
2. Oligonucleotide as claimed 1, characterised by modification of 2 to 10 non-terminal pyrimidine nucleosides.
3. Oligonucleotides as claimed 1 or 2, characterised by the addition of modifications to the 5' and/or 3' ends of the oligonucleotides.
4. Oligonucleotides as defined in claims 1 to 3, characterised by the absence of modification of one or more groups of at least 1 to 4 interconnected nucleotides.
5. Oligonucleotide according to one of claims 1 to 4, characterised by the modification being defined asOther

   (a) replacement of at least one 3' and/or 5' phosphoric acid diester bond of adjacent nucleotides and/or

   (b) Dephosphorus bonding of neighbouring nucleotides and/or

   (c) substitution of at least one sugar residue and/or

   (d) Replacement of at least one naturally occurring nucleoside.
6. Oligonucleotide as claimed 5, characterised by the modification being defined asOther

   (a) a phosphorothioate, phosphorodithioate, NR1R2−phosphoramidate, borano phosphate, phosphate- ((C1-C21) -O-alkylester, phosphate- ((C6-C12) -O-alkylester, 2,2,2-trichlorodimethylethylphosphonate, (C1-C8) -alkylphosphate, (C6-C12) -arylphosphate bond of neighbouring nucleotides, whereby the following is added:R1 and R2 are independently hydrogen, or C1-C18 alkyl, C6-C20 aryl, (C6-C14) aryl- ((C1-C8) alkyl, - ((CH2) c-[NH(CH2) c-d-NR3R3, where c is an integer from 2 to 6 and d is an integer from 0 to 6, and R3 is independently hydrogen, or C1-C6-alkyl or C1-C4-alkoxy-C1-C6-alkyl, is, orR1 and R2 together with the nitrogen atom they carry form a 5- to 6-membered heterocyclic ring, which may contain an additional heteroatom from the O, S, N series, if appropriate,

   (b) a formacetal, 3'-thioformacetal, methylhydroxylamine, oxime, methyldimethylhydrazine, dimethylensulfone, silyl bond of a neighbouring nucleotide,

   Err1:Expecting ',' delimiter: line 1 column 89 (char 88)

   (d) the substitution of β-D-2'-deoxyribose by α-D-2'-deoxyribose, L-2'-deoxyribose, 2'-F-2'-deoxyribose, 2'-O- ((C1-C6) alkyl-ribose, 2'-O- ((C2-C6) alkenyl-ribose, 2'-NH2-2'-deoxyribose, β-D-xylofuranose, α-arabinofuranose, 2,4-deoxy-β-D-erythro-hexo-pyranoses, carbocyclic or open sugar analogues or bicyclo-sugar analogues;

   (e) Replacement of the natural nucleoside bases by 5-hydroxymethyl) uridine, 5-aminouridine, pseudouridine, dihydrouridine, 5-C1-C6-alkyl-uridine, 5-C1-C6-alkenyl-uridine, 5-C1-C6-alkynyl-uridine, 5-C1-C6-alkylyl-cytidine, 5-C1-C6-alkenyl-cytidine, 5-C1-C6-alkynyl-cytidine, 5-Fluoruridine, 5-Fluorcytidromine, 5-Cloruridine, 5-Cloruridine, 5-Buridine or 5-Bromcytidine.
7. Oligonucleotide according to one of claims 1 to 6, characterised by the oligonucleotide carrying a lipophilic residue at the 5' and/or 3' end, preferably a farnesyl, phytyl, hexadecyl, cholesterol, hexamethyl entetraamine, vitamin E or bile acid residue.
8. Process for the production of oligonucleotides according to claims 1 to 7, characterised by their chemical synthesis.
9. A process for the manufacture of a medicinal product characterised by the mixing of at least one oligonucleotide according to one of claims 1 to 7 with a physiologically acceptable carrier and, where appropriate, with appropriate additives and/or excipients.
10. Use of oligonucleotides according to claims 1 to 7 to manufacture a medicinal product.
11. Use of oligonucleotides in the manufacture of a medicinal product or diagnostic device, characterised by modification of at least one incomplete pyrimidine nucleoside.
12. Use according to claim 11, characterised by modification of 2 to 10 non-terminal pyrimidine nucleosides.
13. Use according to claim 11 or 12, characterised by addition of modification of the oligonucleotides at the 5' and/or 3' ends.
14. Use according to one of claims 11 to 13, characterised by the absence of modification of one or more groups of at least 1 to 4 interconnected nucleotides.
15. Use according to one of claims 11 to 14 characterised by the modification being defined according to claim 5 or 6.
16. Use according to one of claims 11 - 15, characterised by oligonucleotides approximately 6 - 100 nucleotides in length.