MODIFIED ANTISENSE NUCLEOTIDES COMPLEMENTARY TO A SECTION OF THE HUMAN Ha-Ras GENE

Abstract: The invention relates to a specific modified oligonucleotide complementary to a section of the human Ha-ras gene and mRNA, and its use to specifically regulate, modulate or inhibit expression of the Ha-ras gene, and its use as a pharmaceutical for the treatment of conditions arising from abnormal expression of the Ha-ras gene, in particular in combination with chemotherapy and radiotherapy. The modified oligodeoxynucleotide according to the invention has the sequence: 5'-TxAxTxTxCxCxGxTxCxAxT-3'-O-PO2-O-R, wherein X is o or s, with the proviso that x is 4 to 9 times equal to s and o means a phosphodiester internucleoside linkage, s means a phosphorothioate internucleoside linkage, R means a C8-C21 alkyl group, -(CH2-CH2O)n-(CH2)m-CH3 or -CH2-CH(OH)CH2O-(CH2)q-CH3 wherein n is an integer from 1 to 6, m is an integer from 0 to 20 and q is an integer from 7 to 20 and A is 2'-deoxyadenosine, G is 2'-deoxyguanosine, C is 2'-deoxycytidine and T is thymidine.

Description: Modified Antisense Nucleotides Complementary to a Section of the Human Haras Gene This invention relates to a specific modified oiigonucleotide complementary to a section of the human Ha-ras gene and mRNA, and its use to specifically regulate, modulate or inhibit expression of the Ha-ras gene, and its use as a pharmaceutical for the treatment of conditions arising from abnormal expression of the Ha-ras gene. Antisense oligonucleotides (AO) have proven to be specific inhibitors of gene expression in a large number of systems, both in vivo and in vitro. (Uhlmann and Peyman, Chem. Rev. 1 990, 90, 543). One of the major problems encountered when using unmodified oligonucleotides containing only phosphodiester intemucleoside linkages (PO-oligonucleotides) is the rapid degradation of this type of oiigonucleotide in cells and biological fluids, such as, for example, serum and cerebrospinal fluid, by a range of nucleolytic activities. A wide range of chemical modifications to oligonucleotides have been carried out in order to improve their nucleolytic stability (Uhlmann and Peyman, Chem. Res. 1 990, 90, 543) . These modifications include the modification or replacement of the phosphodiester intemucleoside linkage, the sugar unit, the nucleobase; or the sugar-phosphate backbone of the oligonucleotides. The most thoroughly investigated type of modification is alteration of the intemucleoside linkage, including phosphorothioate (PS), methylphosphonate (MeP) and phosphorodithioate (PSS) linkages. It should be stressed that modification of the oiigonucleotide alters not only its nuclease stability but also other characteristics of the oiigonucleotide, such as, for example, their cellular uptake, RNaseH activation, and the strength and specificity of binding to their target nucleic acid, and the like. It should be borne in mind that the stability of the modified oiigonucleotide in serum, frequently used to determine the nuclease stability of the oiigonucleotide, is not the sole determinant of intracellular activity (P.D. Cook in "Antisense Research and Applications", Crooke and Lebleu, Eds., CRC Press, Boca Raton, 1 993, Chap.9, p149 et seq.) . The phosphorothioate (PS) modified oligonucleotides are the most widely used type of modified oiigonucleotide. The following strategies have been developed for the positioning of PS linkages in antisense oligonucleotides: ( 1 ) Replacement of all phosphodiester intemucleoside linkages with phosphorothioate linkages. The resulting all-phosphorothioate oligonucleotides are much more stable to nucleases than PO-oligonucleotides (Monia et al. J. Biol. Chem. 1 996, 271 , 14533). For example, degradation of all-PS oligonucleotides by endonucleases is slowed down by a factor of 2-45 relative to a PO oiigonucleotide (Stein et al. Nucleic Acids Res. 1 988, 1 6, 3209). In Xenopus oocytes or embryos, the degradation of microinjected PO oligonucleotides proceeds with a half-life of 30 minutes, while all-PS oligonucleotides have a half-life of over 3 hours under the same conditions (Woolf et al. Nucleic Acids Res. 1 990, 1 8, 1 763) . All-PS oligonucleotides retain their ability to activate RNaseH. The major disadvantages of all-PS oligonucleotides are that their ability to form stable hybrids with their target nucleic acid is reduced, and that they frequently give rise to unspecific "non-antisense" effects (Monia et al., J. Biol. Chem. 1 996, 271 , 14533). (2) Oligonucleotides containing both phosphorothioate and phosphodiester intemucleoside linkages. In an effort to overcome the non-antisense effects observed with all-PS oligonucleotides, oligonucleotides containing both phosphorothioate and phosphodiester intemucleoside linkages have been synthesized and tested for stability and biological activity. Ghosh et al. (Anticancer Drug Design 1 993, 8, 1 5) describe a PS-PO oiigonucleotide containing various percentages of PS linkages. Their construction follows, for example, the pattern (PS-PO-PO-PO) n , (PO-PO-PS) n , (PS-PO) n , [(PO) 2 - (PS) 2 ] n , [PO-PS-PS] n . They teach that a PS linkage content of at least 50% is required for selective translation inhibition in vitro and that activity drops drastically when the PS content is less than 50% . More recently it has been demonstrated that an oiigonucleotide containing 50% PS-linkages arranged in the pattern (PS-PO) n showed no biological activity in an assay system where an all-PS oiigonucleotide and "end-capped" PO-PS oligonucleotides (see below) of the same sequence were highly active (Monia et al., J. Biol. Chem. 1 996, 271 , 14533) . (3) "End-capped" Oligonucleotides, where one, two or three intemucleoside bridges on the 5' and/or the 3' end of the oiigonucleotide are phosphorothioate modified, (also known as the "gap technique") This type of modification is designed primarily to protect the oiigonucleotide from degradation by exonucleases. In particular modifications at the 3'-end of the oiigonucleotide are desirable as they offer protection from 3'-exonucleases, which are the most abundant nucleases in serum (Uhlmann and Peyman, Chem. Rev. 1 990, 90, 543). An interesting comparison of strategies is found in Hoke et al. (Nucleic Acids Res. 1 991 , 1 9, 5743) . The authors compare the activity of a range of antisense PS-oligonucleotides against HSV-1 in cell culture. Their findings confirm that 3' , or 3' + 5', end-capped oligonucleotides (the first three intemucleoside linkages being modified in each case), similarly to all-PS oligonucleotides are sufficiently protected against degradation by nucleases in serum. In contrast internally modified (three PS bridges) oligonucleotides and oligonucleotides in which only the 5 '-end has been capped (again, the first three intemucleoside linkages being modified) are degraded rapidly. In contrast, the authors found that neither 5' nor 3' end capping nor both are sufficient for activity within the cell, and they drew the conclusion that a uniform modification (all-PS) is required to achieve sufficient stability to nucleases in cells. More recently it has been discovered that pyrimidine nucleosides are the most nuclease susceptible points in oligonucleotides (Peyman, A. and Uhlmann, E., Biol. Chem. Hoppe-Seyler 1 996, 377, 67; EP 0 653 439 A2) . It was found that a combination of end-capping and PS protection of the pyrimidine positions of oligonucleotides (the so called "minimal modification" approach) is sufficient to make them highly resistant to nuclease degradation. The biological activity (against Herpes simplex virus) of an oiigonucleotide with this type of PS modification pattern was comparible to that of an all-PS oiigonucleotide. One of the major problems encountered when using AOs, whether or not they are stabilized against degradation, is their poor cellular uptake. Many approaches have been tried to attempt to ameliorate this problem. Most of these approaches involve the attachment of a variety of substances to the oiigonucleotide. Modifications include: the attachment of peptides to oligonucleotides ( Lemaitre, M. et al. Proc. Natl. Acad. Sci. USA 1 987, 84, 648) and the attachment of lipophilic residues, such as alkyl chains or cholesterol, to oligonucleotides (Saison-Behmoaras, T. et al. EMBO J. 1 991 , 10, 1 1 1 1 -1 1 1 8; Will, D.W. and Brown, T. Tetrahedron Lett. 1 992, 33, 2729). It has been found, however, that in many cases the introduction of a lipophilic group causes biological effects which are independent of the sequence of the oiigonucleotide. Non-specific effects have been reported for cholesterol-oligonucleotide conjugates (Henderson, G.B. and Stein, CA. Nucleic Acids Res. 1 995, 23, 3726.), and for oligonucleotides attached to alkyl chains (Shea, R.G. et al. Nucleic Acids Res. 1 990, 1 8, 3777) . Saison-Behmoaras et al (EMBO J. 1 991 , 1 0, 1 1 1 1 -1 1 1 8; WO 96/34008) have reported that a 9mer all-PO oiigonucleotide derivatized with a 3'-dodecanol moiety and a 5'-acridine crosslinking agent, and antisense to mutated Ha-ras inhibited T24 human bladder carcinoma cell proliferation. No comparison of the antiproliferative activity of this oiigonucleotide with that of the corresponding oiigonucleotide without a dodecanol conjugate was made. The cellular uptake of the dodecanol oiigonucleotide was reported to be 4 times that of the unmodified oiigonucleotide in T24 cells. The acridine-dodecanol modified oiigonucleotide had no effect on the proliferation of a human mammary cell line carrying an unmutated Ha-ras gene. This effect can either be attributed to the sequence specificity of the antisense oiigonucleotide or to insufficient cellular uptake. Since the uptake of the acridine-dodecanol oiigonucleotide was not determined in the human mammary cell line, the effect may be entirely , or at least partly due to the fact that the oiigonucleotide was not taken-up by the human mammary cell line, and thus did not have an opportunity to exhibit non- sequence-specific effects on cell proliferation. About 20% of human tumors have a mutation in one of the three ras genes (Haras, Ki-ras, and N-ras) leading to over-expression of p21 protein which plays an important role in the transformed phenotype (Bos, T.L.1 988 Mutation Res. 1 988, 1 95, 255). It has been reported that inhibition of different ras genes in different cell lines can either have no effect, or inhibit cell proliferation depending on which of the ras genes is controlling the cell proliferation (Chen et al. J. Biol. Chem., 1 996, 271 , 28259-65) . It was suggested that differential modulation of individual ras genes may be an approach to inhibit tumor growth while minimising effects on normal cell growth. One approach to devising successful therapy of Ha-ras-induced tumors is to regulate, modulate or inhibit expression of the Ha-ras gene. Antisense oligodeoxynucleotides (oligonucleotides) have been shown to act as specific inhibitors of ras mRNA expression in cell-free systems, in transformed cells in culture (T. Saison-Behmoaras et al. EMBO J. 1 991 , 1 0, 1 1 1 1 -1 1 1 8; Monia et al. J. Biol. Chem. 1 996, 271 , 1 4533), and in ras-activated tumors in vivo (Gray, G.D. et al. Cancer Research, 1 973, 53, 577) . In order to modulate the expression of the ras-gene in wild type or in mutated form Brown et al. (Oncogene Res. 1 989, 4, 243-252) propose the use of anti- ras oligodeoxy ribonucleoside methylphosphonates which are complementary to the initiation codon region of Balb-ras P21 mRNA (a mouse version of the human Ha-ras Gene) . However, it is well known that methyl phosphonates show some major disadvantages compared to phosphorothioates, among other their poor cellular uptake. Monia et al. (J. Biol. Chem. 267, 1 9954-1 9962, 1 992; WO 92/22651 ) disclose all-PS antisense oligonucleotides and antisense oligonucleotides containing various percentages of PS linkages (WO 94/08003) directed to the translation initiation site or to codon 1 2 of the human Ha-ras gene, which however, have the above-mentioned disadvantages concerning the cellular uptake and their poor stability. Pirollo et al. (Biochem. Biophys. Research Comm. 230, 1 96-201 , 1 997) propose the use of anti-ras all-PS oligodeoxynucleotides in order to reverse radio resistance of cancer cells. However, these antisense oligonucleotides show the disadvantages (poor cellular uptake, unstability) as mentioned above. Therefore, this invention aims to provide an oiigonucleotide, modified to improve its stability and cell uptake, complementary to Ha-ras mRNA which specifically regulates, modulates or inhibits expression of the Ha-ras gene in the form of its wild type as well as in its mutated forms, and which can be used to inhibit the proliferation of cancer cells, to reverse radio-resistance in cancer cells, and to treat conditions arising from abnormal expression of the Ha-ras gene. According to the invention, this problem is solved by providing a modified oligodeoxynucleotide of the sequence SEQ ID NO. 1 5'-TxAxTxTxCxCxGxTxCxAxT-3'-O-PO 2 -O-R wherein x is o or s A is 2'-deoxyadenosine, G is 2'-deoxyguanosine, C is 2'-deoxycytidine and T is thymidine. The modified oligodeoxynucleotide according to the invention is particularly characterized in that it has one of the following sequences (a) 5'-TsAsToToCsCoGoTsCsAsT-3'-O-PO 2 -O-R, (b) 5'-TsAsToToCsCoGsToCsAsT-3'-O-PO 2 -O-R, (c) 5'-TsAoTsToCsCoGoTsCsAsT-3'-O-PO 2 -O-R, (d) 5'-TsAoTsTsCsCoGoTsCsAsT-3'-O-PO 2 -O-R, (e) 5'-TsAoTsToCsCsGoTsCsAsT-3'-O-PO 2 -O-R, (f) 5'-TsAoTsTsCsCsGoTsCsAsT-3'-O-PO 2 -O-R, (g) 5'-TsAsToToCsCsGsToCsAsT-3'-O-PO 2 -O-R, (h) 5'-TsAoTsTsCsCsGoTsCsAsT-3'-O- PO 2 -O-R, (i) 5'-TsAsTsTsCsCsGoTsCsAsT-3'-O- PO 2 -O-R, (j) 5'-TsAsToToCoCsGoTsCsAsT-3'-O- PO 2 -O-R, (k) 5'-TsAsTsToCsCsGoTsCsAsT-3'-O- PO 2 -O-R, (I) 5'-TsAsTsToCsCoGoTsCsAsT-3'-O- PO 2 -O-R, (m) 5'-TsAsToTsCsCoGoTsCsAsT-3'-O- PO 2 -O-R, (n) 5'-TsAsToToCsCsGoTsCoAsT-3'-O- PO 2 -O-R, (o) 5'-TsAsToTsCsCoGoTsCoAsT-3'-O- PO 2 -O-R, (p) 5'-TsAoTsTsCoCsGoTsCoAsT-3'-O- PO 2 -O-R, (q) 5'-TsAsTsToCsCsGoToCsAsT-3'-O- PO 2 -O-R, (r) 5'-TsAsTsToCoCsGoToCsAsT-3'-O- PO 2 -O-R, (s) 5'-ToAoTsToCsCoGoToCsAsT-3'-O- PO 2 -O-R or (t) 5'-TsAsTsTsCsCsGoTsCsAsT-3'-O- PO 2 -O-R, wherein all the variables o, s, R, m, n and q and A, G, C and T have the above- mentioned meanings. The modified oiigonucleotide according to the present invention is complementary to the DNA or RNA deriving from the human Ha-ras gene. The oiigonucleotide is complementary to the translation initiation region of Ha-ras mRNA. Therefore it inhibits both wild-type and mutant ras expression resulting in inhibition of tumor cell proliferation. The oiigonucleotide is modified to improve its stability and cell uptake characteristics. The oiigonucleotide contains four to nine phosphorothioate linkages at certain positions which are especially vulnerable to attack by nucleases. The oiigonucleotide is modified at the 3'-end with a C 8 -C 21 -alkyl or with a C C 21 -alkyl, preferably C 16 -alkyl chain, covalently attached through either a phosphodiester bridge, an oligoethyleneglycol phosphodiester linkage, or a glyceryl ether phosphodiester linkage. A particularly preferred embodiment of the present invention is a modified oligodeoxynucleotide which has the sequence 5'-TsAsToToCsCoGoTsCsAsT-3'-O-PO 2 -O-R containing six phosphorothioate linkages, wherein the variables o, s, R, n, m and q and A, G, C and T have the above-mentioned meanings. R is particularly preferably -CH 2 CH(OH)CH 2 O(CH 2 ) 15 CH 3 , -(CH 2 ) 15 CH 3 or -(CH 2 CH 2 O) n -(CH 2 ) 15 CH 3 , wherein n is an integer from 1 to 6; preferably, n is 1 , 2 or 3. The invention further relates to the preparation of a modified oiigonucleotide. Preferably the oiigonucleotide is synthesized using standard phosphoramidite chamistry as outlind in examples 1 and 2. Phosphorothioate linkages are for example introduced by sulfurization using the Beaucage reagent. Surprisingly it was found that particularly the glycerol alkyl ether, i.e. the group -CH 2 -CH(OH)CH 2 O-(CH 2 ) q -CH 3 , q being an integer from 7 to 20, in particular the group -CH 2 CH(OH)CH 2 O(CH 2 ) 15 CH 3 , linked by a phosphodiester linkage to the 3'-end of the oiigonucleotide renders an oiigonucleotide which no longer depends on the mixing with uptake enhancers for optimal biological activity. This is the first time that an oiigonucleotide has been found to exhibit the same biological activity with and without the addition of an uptake enhancer. The invention further relates to the preparation of a modified oiigonucleotide. Preferably the oiigonucleotide is synthesized using standard phosphoramidite chemistry as outlined in examples 1 and 2. Phosphorsthioate linkages are for example introduced sulfuization using the Beaucage reagent. The modified oligodeoxynucleotide according to the present invention as described above is particularly suitable for use as a pharmaceutical. Therefore, a further object of the present invention is the modified olidodeoxynucleotide as described above for use as a pharmaceutical, in particular a pharmaceutical formulation which contains an effective amount of at least one modified oligodeoxynucleotide according to the invention. More particularly, the oligodeoxynucleotide according to the present invention is suitable for preparing a pharmaceutical for the treatment of a disease arising from the overexpression and/or mutation of the Ha-ras gene or diseases arising from a hyperproliferative disorder. Such diseases are in particular cancer, restenosis, or psoriasis. Transforming activation of ras occurs in approximately 1 0-20% of human tumors by amplification of the ras gene at the DNA level or by overexpression of the ras protein at the mRNA level. Ras genes can be activated by point mutation at codon 1 2, 1 3, or 61 (Barbacid, M. Ann. Rev. Biochem. 1 987, 56, 779-827; Bos, supra; Lowy, supra). Point mutation of the Ha-ras gene at codon 1 2 converts the normally regulated protein to one that is continually active. It is thought that the loss of regulation of normal ras protein function is responsible for the transformation from normal to malignant cell growth. The modified oligodeoxynucleotide according to the invention is particularly suitable for the regulation, modulation, or inhibition of Ha-ras gene expression. The oligodeoxynucleotide is complementary to the translation initiation region of Ha-ras mRNA. Therefore, it inhibits both wild-type and mutant ras expression resulting in inhibition of tumor cell proliferation. The oligodeoxynucleotide according to the invention is further modified to improve its stability and cell uptake characteristics. The oligodeoxynucleotide contains four to nine, preferably six, phosphorothioate linkages at certain positions which are especially vulnerable to attack by nucleases. The cellular uptake of the oligodeoxynucleotide is further improved by a C 8 -C 21 -alkyl group or by an C^C^- alkyl group covalently attached through either a phosphodiester bridge, an oligoethyleneglycol phosphodiester linkage, or a glyceryl ether phosphodiester linkage. Due to the improved stability and to the improved cellular uptake the oligodeoxynucleotide according to the present invention depends no longer on the mixing with uptake enhancers and is rendered for optimal biological activity. Therefore, the oligodeoxynucleotide according to the present invention is particularly suitable for preparing a pharmaceutical which is directed to the regulation modulation or inhibition of Ha-ras gene expression of both, of the Haras wild type gene and its mutants. In case that the disease resulting from mutation of the Ha-ras gene is cancer, the oiigonucleotide according to the invention is particularly suitable for preparing a pharmaceutical which inhibits the proliferation of cancer cells. However, on the other hand the oligodeoxy nucleotide is advantageously used for preparing a pharmaceutical against cancer which is used in combination with chemotherapy, in particular in cases where the cancer cells have become resistant to chemotherapy, since the oligodeoxynucleotide according to the invention reverses chemoresistance in cancer cells. Therefore, a further object of the present invention is the use of the modified oligodeoxynucleotide as described above for preparing a pharmaceutical which reverses the chemoresistance in cancer cells. In particular due to this particular effect the oiigonucleotide according to the present invention is suitable for preparing a pharmaceutical formulation which contains an effective amount of at least one further chemotherapeutically effective agent. Such chemotherapeutically effective agents are for example cis- platinum and its derivatives, preferably cis-platinum, N-lost derivatives, preferably cyclophosphamide, trofosfamide and ifosfamide, aziridine derivatives, preferably thiothepa, N-nitrosourea derivatives, folic acid antagonists, preferably methotrexate, analogues of purine and pyrimidine bases, preferably 5-fluoro- uracil, cytostatically effective antibiotics, preferably adriamycine, mitomycine and daunorubicine, estrogene antagonists, preferably tamoxifene, and nucleoside derivatives, preferably MDL 101 , 731 ((E)-2'-deoxy-2'-(fluoromethylene)cytidine; Cancer Research 54, 1 485-1 490, 1 994) . Ras genes are known to be involved in signal-transduction of various factors for growth, differentiation and oncogenesis. Recent studies have implicated the raf- 1 oncogene, which is downstream from ras in the signal transduction pathway, in the expression of the radiation resistance phenotype. It can be shown that the antisense oiigonucleotide according to the present invention which is directed to the initiation codon of Ha-ras reverses the radiation resistance level of cancer cells. Therefore, a further object of the present invention is the use of the modified oligodeoxynucleotide according to the invention for preparing a pharmaceutical for the treatment of cancer in combination with radiotherapy. Particularly, the modified oligodeoxynucleotide according to the present invention is suitable for preparing a pharmaceutical which reverses the radioresistance in cancer cells. The modified oiigonucleotide according to the invention is prepared using an 1 - alkanol and oligoethyleneglycol monoalkyl ether, respectively, in conjunction with the 3'-derivatization method described in EP 0 552 767 A2 and EP 0 552 766 A2; or a glyceryl ether phosphodiester linkage, using the solid-support described in Example 1 . Examples: Table 1 : List of Oligonucleotides used in Examples. Example 1 Synthesis of solid support for the synthesis of oligonucleotides functionalised at the 3'-end with a glycerol hexadecyl ether linked by a phosphodiester linkage. DL-a-Hexadecylglycerol ( 1 mmol) was dried by coevaporation with 3x5ml anhydrous pyridine, then dissolved in anhydrous pyridine (3ml). The solution was cooled to 0°C and 4,4'-dimethoxytrityl chloride ( 1 .1 5 mmol) was added. The reaction was stirred overnight. The reaction was quenched by the addition of water (1 00ml) and evaporated to dryness. The residue was taken-up in dichloromethane (20ml) and extracted 3 times with 1 0ml 0.1 M phosphate buffer pH7. The organic phase was dried over sodium sulfate, filtered and evaporated. The residue was dried by coevaporation with 2x5ml anhydrous pyridine, then dissolved in anhydrous pyridine (2 ml) . 4,4-Dimethylaminopyridine (1 .4 mmol), and succinic anhydride (2.1 mmol) were added. The reaction was stirred overnight. The reaction solution was evaporated to dryness and coevaporated twice with toluene:dichloromethane/1 : 1 . The residue was taken-up in dichloromethane (20ml), extracted with 1 0% citric acid ( 1 1 ml) and washed 3 times with water (1 1 ml) . Two drops of triethylamine were added to the organic phase which was then dried over sodium sulfate, filtered and evaporated. The product was purified by silica gel chromatography using a gradient of 1 -4% methanol, 1 % triethylamine in dichloromethane. The product was obtained as a clear oil in 51 0mg (62%) yield. The product (0.056mmol), N-ethylmorpholine (0.07mmol) and TBTU (0.056mmol) were dissolved in DMF (2ml) and added to aminopropyl controlled-pore glass (CPG; 500mg; 50θA, Fluka). The mixture was shaken for 4h and the CPG was filtered-off and washed with methanol and dichloromethane. The CPG was capped for 1 h using acetic anhydride/ N-methyl imidazole in THF, filtered and washed with methanol, dichloromethane, THF and diethyl ether. After drying in vacuo the loading of dimethoxytrityl groups was determined to be 80 mmolg 1 . Example 2 Oiigonucleotide Synthesis The oligonucleotides were synthesized using an Applied Biosystems 394 DNA synthesizer (Applied Biosystems, Inc., Foster City, USA) and standard phosphoramidite chemistry. After coupling, phosphorothioate linkages were introduced where required by sulfurization using the Beaucage reagent (Iyer et al., 1 990) followed by capping with acetic anhydride and N-methylimidazole in tetrahydrofuran. After cleavage from the solid support and final deprotection by treatment with cone, ammonia, the oligonucleotides were purified by gel electrophoresis. For the synthesis of the hexadecyl-modified oligonucleotides the solid support described in Example 1 above was employed. All oligonucleotides were analyzed by negative ion electrospray mass spectroscopy (Fisons Bio-Q) which in all cases confirmed the calculated mass. Example 3 Inhibition of in vitro translation of Ha-ras mRNA. 0.06 mg RNA was annealed with the appropriate oiigonucleotide by heating them together in 1 0 mM Tris-HCI, pH 7.5, 5 mM MgCl2, 25 mM KCI and 1 mM DTT at 90 °C for 2 minutes and cooling slowly to room temperature. The translation reaction mixture (10 μl) consisted of 0.06 mg of RNA, varying amounts (1 , 5, 10 or 20 μM) of oiigonucleotide, 8 U of RNasin Ribonuclease Inhibitors (40 U/μl) (Promega Biotec), 20 μM of amino acid mixture minus methionine (Promega Biotec), 0.8 mCi of 35s-methionine (>1000 mCi/μM, Amersham), 3-6 μl of nuclease-treated Rabbit Reticulocyte Lysate (Promega Biotec), and 1 μM dithiothreitol (DTT). Translation proceeded for 1 hour at 30°C. After translation, 10 μl of RIPA buffer (PBS, 1 % NP40, 0.5% sodium deoxycholeate, 0.1 % SDS) with fresh inhibitors (0.1 mg/μl PMSF, 1 mM sodium orthovanadate, 30 ml/μl aprotinin, Sigma, Cat. #A6279) was added to each sample, and the mixture incubated on ice for 20 minutes. 7 μl of 4 x SDS sample buffer containing 1.25 M Tris-HCI pH 6.8, 6% SDS, 40% glycerol, 12% 2-mercaptoethanol and 0.001 % bromphenol blue was added, and the sample was boiled for 5 minutes. 10 μl of each sample was electrophoresed in a 5-12.5% polyacrylamide gel (PAGE). The completed gel was fixed, dryed, and exposed to Kodak XAR-5 film with an intensifying screen at -80°C. Results: The claimed antisense oiigonucleotide 5'-TsAsT T CsC G TsCsAsT-(PO 2 -O- CH 2 CH(OH)CH 2 O(CH 2 ) 15 CH 3 )-3' (PPS-C16) inhibited translation of ras p21 protein by approximately 10, 40, 50 and 80% at concentrations of 1 , 5, 10, and 20μM respectively. Controls using the sense oiigonucleotide 5'- AsTsG A CsG G AsAsTsA- (PO 2 -O-CH 2 CH(OH)CH 2 O(CH 2 ) 15 CH 3 )-3' (PPS-C16-S) showed no inhibition of translation until 20 μM at which point an 11 % inhibition was observed. This strongly suggests that the C16 alkyl chain does not give rise to non-sequence specific effects in this system. Example 4 Inhibition of p21 ras protein expression in cell culture Cell Lines: NIH3T3 mouse fibroblasts (Chang et al. Biochemistry 30 (1991 ) 8283-86) transformed by the human Ha-ras (RS485) were used as an experimental cell line to evaluate the sequence-specific inhibition of cell growth and p21 ras protein expression by oligonucleotides directed to the first eleven nucleotides of the initiation codon region of Ha-ras mRNA. RS485 cells were grown in Dulbecco's Modified Eagle's Medium (DMEM, Flow Labs) supplemented with 10% heat inactivated (30 min. at 65DC) fetal calf serum (FCS), antibiotics (50 U/μl each of penicillin, streptomycin, and neomycin ), and 4 mM of L- glutamine. Oiigonucleotide Treatment of RS485 Cells in Culture RS485 cells were seeded in 6-well plates (105 cells per well) and, after 20-24 hours (40-60% confluency), were treated with oligonucleotides. Medium was removed from the wells, 1 ml of fresh medium with oligonucleotides at various concentrations was added to each well, and the plates were incubated at 37 °C in an humidified atmosphere containing 5% CO2/95% air for 24-48 hours. Oiigonucleotide Lipofection of RS485 Cells in Culture RS485 cells (40-60% confluency) were transfected with oligonucleotides using Lipofectin™ Reagent and the supplier's protocol (from Life Technologies). Briefly, varying concentrations of oligonucleotides were diluted in 100 μl of serum-free medium. In a separate tube Lipofectin was added to serum-free medium at a concentration of 7μl/100μl. After 30 minutes at room temperature, 100 μl of Lipofectin solution was mixed with 100 μl of oiigonucleotide solution, incubated for an additional 15 minutes at room temperature, and mixed with 800 μl of serum-free medium. Cells were washed two times with serum-free medium, and the total solution (1 ml) containing the oligonucleotide-Lipofectin complex was overlayed onto the cells. After 6 hours, 1 ml of fresh medium containing 8 mM L-glutamine and 20% calf serum was added, and the cells were incubated for an additional 38 hours. Treated cells were washed with PBS, harvested with trypsin, and cellular proteins were extracted. Results: The effect of PPS and PPS-C16 oligonucleotides on p21 expression in RS485 cells was compared in the absence or presence of Lipofectin (supra). After treatment total protein was extracted and p21 levels were determined by Western Blot analysis (infra). Lipofectin alone had no effect on p21 expression. Both the PPS- and PPS-C16- Lipofectin complexes almost completely inhibited p21 expression in RS485 cells at 1 μM concentration. Surprisingly the PPS-C16 oiigonucleotide without Lipofectin completely inhibited p21 expression at 5μM concentration. The PPS oiigonucleotide without a C16 modification inhibited p21 expression by only 50% at the same concentration. A comparison was then made of the effect of PPS-Lipofectin complex and PPS-C16 alone on p21 expression in RS485 cells. The p21 level was decreased by 30% and 43% at 0.25μM and 0.5μM PPS-Lipofectin concentrations respectively. Remarkably, the PPS-C16 oiigonucleotide without any added Lipofectin showed approximately the same level of inhibition, namely 33% at 0.25μM and 40% at 0.5μM. Complete (100%) inhibition of p21 expression was observed at 1 μM concentration, indicating that Lipofectin delivery does not augment PPS-C16-mediated inhibition. Finally, a comparison of the inhibition of p21 expression in RS485 cell by PPS and PPS-C16 in the absence of Lipofectin was made. PPS alone gave an inhibition of approximately 50% at 5μM. However, at a concentration of only 0.75μM the PPS- C16 oiigonucleotide exhibited nearly 60% inhibition of p21 expression in RS485 cells. Example 5 Inhibition of Cell Growth and p21 ras Protein Synthesis in Cell Culture Cell Growth Inhibition Studies Cells treated with antisense oligonucleotides, or cells transfected with the oligonucleotide-Lipofectin complexes were harvested and lysed in RIPA buffer. Total protein levels in each well were determined spectrophotometrically. Inhibition of cell growth was estimated as the ratio of protein (mg) in the oligonucleotide-treated cells compared with that in the control. The control was either protein from cells treated with Lipofectin only, or protein from untreated cells. Western Blot Analysis of ras Protein Total cellular protein was prepared by lysing cells in RIPA buffer with freshly added inhibitors (PMSF, aprotinin, sodium orthovanadate) and homogenizing through 21- gauge needles according to the protocol provided by Santa Cruz Biotechnology, Inc. The extracts were cleaned by centrifugation at 15,000 xg for 20 min at 4 °C and the protein concentration of the supernatant was determined. 40 μg of protein was size fractionated in 5-12.5% PAGE and electroblotted onto a nitrocellulose membrane. The membrane was incubated with primary antibody against Ha-r?as (C-20) (Santa Cruz Biotechnology, Inc.) and then with Horseradish Peroxidase-conjugated goat anti-rabbit immunoglobulin G. The ECL chemo-luminescent Western system (Amersham, Arlington Heights, III.) was used to detect secondary probes. Results: A series of experiments were performed to study the sequence-sequence toxic effect of the PPS and PPS-C16 oligonucleotides on RS485 cells. Figure 6A shows that treatment of RS485 cells with 1-5μM of PPS and PPS-C16 in the presence of Lipofectin (supra) reduces cell growth by approximately 35-50% over a 2 day period. Lipofectin alone has a slight toxic effect (ca. 15%) on cells. Comparison of the Western blot analysis of p21 expression inside RS485 cells (supra) with the cell growth inhibition by PPS and PPS-C16 demonstrates that a 1μM oligonucleotide/Lipofectin complex concentration totally inhibits p21 expression, but inhibits cell growth by only 35%. Figure 6B shows that PPS-C16 without Lipofectin inhibits cell growth by 11 % at 0.75μM while inhibiting p21 expression by 60% (supra). PPS inhibits cell growth by 22% at a concentration of 5μM, a concentration at which it inhibits p21 expression by 50%. Figure 6C shows that Lipofectin exerts a toxic effect when used with PPS at concentrations higher than 1 μM and that the PPS-C16 oiigonucleotide without Lipofectin is, in the concentration range 0.25-1 μM, just as effective as the PPS oiigonucleotide with Lipofectin. Example 6 Reversal of Radioresistance - Animal Study Antitumor Effects of antisense oligonucleotides in vivo RS504 cells are NIH 3T3 cells transformed by the Ha-ras oncogene isolated from EJ/T24, a human bladder carcinoma. Tumor were induced by injecting 5x10 6 RS504 cells subcutaneous in female athymic NCr-nu mice. Forty-eight hours later, when tumors were evident (-6mm 3 ), partially phosphorothioated C16-modified [PPS-C16] antisense [AS] or sense [S] oligonucleorides were injected directly into the tumors at a concentration of 50 μl of 5 μM solution (250 pmol) per tumor. After 24 hours, tumors were irradiated with a 2.0 Gy dose (thin arrows, see Fig. 1). Irradiation was repeated every 48 hours until an accumulated dose of 20 Gy had been delivered. Oiigonucleotide injections were repeated at 48 hours [50 μl], 192 hours [50 μl), and 240 hours [100 μl] following the initial injection (bold arrows, see Fig. 1). Tumor volume was recorded in mm 3 . Controls consisted of tumors that were not injected with oligonucleotides and not irradiated, tumors that were not injected with oligonucleotides but were irradiated; and tumors injected with antisense PPS-C16 oligonucleotides but not irradiated. Tumor Growth Ras genes are known to be involved in signal-transduction of various factors for growth, differentiation and oncogenesis. Recent studies have implicated the raf-1 oncogene, which is downstream from ras in the signal transduction pathway, in the expression of the radiation resistence phenotype. It can be shown that the antisense oiigonucleotide according to the invention reverses the radiation resistance level of the cells. To determine if there were differences in the effects of radiation on ras- induced tumors following treatment with oligonucleotides, RS 504-induced tumors in nude mice were treated with antisense and sense PPS-C16 oligonucleotides. Figure 1 shows that antisense PPS-C16 oiigonucleotide treatment in conjunction with radiation was most effective in inhibiting tumor growth during the 17 days of observation and treatment. Example 7 Reversion of radioresistance in cell culture Reversion of radioresistance phenotype in cell culture by treatment with PPS-C16 Bladder (T24) carcinoma cell lines (obtained from ATCC, Rockville, MD) were maintained in McCoy's 5A medium supplemented with 10 % fetal bovine serum 50 μg/ml each of penicillin, streptomycin anfd neomycin and 2 mM L-glutamine. For oiigonucleotide treatment, the cells were plated at 1 x 10 5 cells/well in 6-well tissue culture plates. Twenty-four hours later, at approximately 40-60 % confluency, the cells were transfected with oligonucleotides, facilitated by Lipofectin Reagent, using essentially the protocol supplied by the manufacturer, Life Technologies, Inc. After 6 hours, the lipofection solution was removed and the monolayer washed with fresh medium containing 8 mM L-glutamine and 20 % serum. The cells were then incubated for an additional 16 - 18 hours in 1 ml of this medium. Cellular response to radiation was evaluated by the colony survival assay. Exponentially growing monolayer cultures of each cell line were treated with the oligonucleotides as described above, the cells were harvested 24 - 48 hours later, suspended in fresh medium and irradiated at room temperature with graded doses of 137 Cs γ rays at a dose of approximately 36 Gy/minute in a J.L. Shephard and Associates Mark I irradiator. Afterward, the cells were diluted and plated at a concentration of 300 to 5000 cells per well in a 6-well tissue culture plate. Two to three days after plating, the cells were supplemented with 0,5 ml of serum plus 5 μg/ml hydrocortisone. Approximately 7 - 14 days later, the cells were stained with 1 % crystal violet and colonies (comprising 50 or more cells of normal appearance) were scored. The Devalue (radiation dose required to reduce survival of cells to 10 %) for antisense- treated (PPS-C16) T24 cells drops from the highly resistant level of 5.5 (untreated or sense-treated PPS-C16-S) to 4.5 Gy, a value dose to what is considered to be radiosensitive. These results are shown in Figure 2. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: Hoechst Marion Roussel Deutschland GmbH & Co. KG (B) STREET: - (C) CITY: Frankfurt (D) STATE: - (E) COUNTRY: Germany (F) POSTAL CODE (ZIP) : 65926 (G) TELEPHONE: 069-306-7072 (H) TELEFAX: 069-35-7175 (I) TELEX: - (ii) TITLE OF INVENTION: Modified Antisense Nucleotides Complementary to a Section of the Human Ha-ras Gene (iii) NUMBER OF SEQUENCES: 5 (iv) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentln Release #1.0, Version #1.30 (EPO) (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS : single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (ix) FEATURE: (A) NAME/KEY: exon (B) LOCATION: 1..21 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: TxAxTxTxCx CxGxTxCxAx T-3-0-P0 2 -0-R 21 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) ( ix ) FEATURE : (A) NAME/KEY: exon (B) LOCATION:!..21 (xι) SEQUENCE DESCRIPTION: SEQ ID NO: 2: TsAsToToCs CoGoTsCsAs T 21 (2) INFORMATION FOR SEQ ID NO: 3: (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (ix) FEATURE: (A) NAME/KEY: exon (B) LOCATION: 1..21 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: AsTsGoAoCs GoGoAsAsTs A 21 (2) INFORMATION FOR SEQ ID NO: 4: (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (n) MOLECULE TYPE: DNA (synthetic) (ix) FEATURE: (A) NAME/KEY: exon (B) LOCATION: 1..21 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: AsTsGoAoCsGoGoAsAsTs A- (P0 2 -0-CH 2 CH (OH) CH 2 0 (CH 2 ) _ 5 CH 3 ) 21 (2) INFORMATION FOR SEQ ID NO: 5: (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (synthetic) (ix) FEATURE: (A) NAME/KEY: exon (B) LOCATION:!..21 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: TsAsToToCsCoGoTsCsAs T- ( P0 2 -0-CH 2 CH (OH) CH 2 0 (CH, ) 15 CH 3 ) 21

Claims: Claims: 1. A modified oligodeoxynucleotide of the sequence SEQ ID NO. 1 5'-TxAxTxTxCxCxGxTxCxAxT-3'-O-PO 2 -O-R wherein x is o or s, with the proviso that x is 4 to 9 times equal to s and o means a phosphodiester intemucleoside linkage, s means a phosphorothioate intemucleoside linkage, R means a C 8 -C 21 alkyl group, -(CH 2 -CH 2 O) n -(CH 2 ) m -CH 3 or -CH 2 -CH(OH)CH 2 O-(CH 2 ) q -CH 3 wherein n is an integer from 1 to 6, m is an integer from 0 to 20 and q is an integer from 7 to 20 and A is 2'-deoxyadenosine, G is 2'-deoxyguanosine, C is 2'-deoxycytidine and T is thymidine. 2. A modified oligodeoxynucleotide as claimed in claim 1 , characterized in that it has one of the following sequences (a) 5'-TsAsToToCsCoGoTsCsAsT-3'-O-PO 2 -O-R, (b) 5'-TsAsToToCsCoGsToCsAsT-3'-O-PO 2 -O-R, (c) 5'-TsAoTsToCsCoGoTsCsAsT-3'-O-PO 2 -O-R, (d) 5'-TsAoTsTsCsCoGoTsCsAsT-3'-O-PO 2 -O-R, (e) 5'-TsAoTsToCsCsGoTsCsAsT-3'-O-PO 2 -O-R, (f) 5'-TsAoTsTsCsCsGoTsCsAsT-3'-O-PO 2 -O-R, (g) 5'-TsAsToToCsCsGsToCsAsT-3'-O-PO 2 -O-R, (h) 5'-TsAoTsTsCsCsGoTsCsAsT-3'-O- PO 2 -O-R, (i) 5'-TsAsTsTsCsCsGoTsCsAsT-3'-O- PO 2 -O-R, G) 5'-TsAsToToCoCsGoTsCsAsT-3'-O- PO 2 -O-R, (k) 5'-TsAsTsToCsCsGoTsCsAsT-3'-O- PO 2 -O-R, (I) 5'-TsAsTsToCsCoGoTsCsAsT-3'-O- PO 2 -O-R, (m) 5'-TsAsToTsCsCoGoTsCsAsT-3'-O- PO 2 -O-R, (n) 5'-TsAsToToCsCsGoTsCoAsT-3'-O- PO 2 -O-R, (o) 5'-TsAsToTsCsCoGoTsCoAsT-3'-O- PO 2 -O-R, (p) 5'-TsAoTsTsCoCsGoTsCoAsT-3'-O- PO 2 -O-R, (q) 5'-TsAsTsToCsCsGoToCsAsT-3'-O- PO 2 -O-R, (r) 5'-TsAsTsToCoCsGoToCsAsT-3'-O- PO 2 -O-R, (s) 5'-ToAoTsToCsCoGoToCsAsT-3'-O- PO 2 -O-R or (t) 5'-TsAsTsTsCsCsGoTsCsAsT-3'-O- PO 2 -O-R, wherein o means a phosphodiester intemucleoside linkage, s means a phosphorothioate intemucleoside linkage, R means a C 8 -C 21 alkyl group, -(CH 2 -CH 2 O) n -(CH 2 ) m -CH 3 or -CH 2 -CH(OH)CH 2 O-(CH 2 ) q -CH 3 wherein n is an integer from 1 to 6, m is an integer from 0 to 20 and q is an integer from 7 to 20 and A is 2'-deoxyadenosine, G is 2'-deoxyguanosine, C is 2'-deoxycytidine and T is thymidine. 3. A modified oligodeoxynucleotide as claimed in claims 1 or 2, characterized in that it has the sequence 5'-TsAsToToCsCoGoTsCsAsT-3'-O-PO 2 -O-R wherein o means a phosphodiester intemucleoside linkage, s means a phosphorothioate intemucleoside linkage, R means a C 8 -C 21 alkyl group, -(CH 2 -CH 2 O) n -(CH 2 ) m -CH 3 or -CH 2 -CH(OH)CH 2 O-(CH 2 ) q -CH 3 wherein n is an integer from 1 to 6, m is an integer from 0 to 20 and q is an integer from 7 to 20 and A is 2'-deoxyadenosine, G is 2'-deoxyguanosine, C is 2'-deoxycytidine and T is thymidine. 4. A modified oligodeoxynucleotide as claimed in one or more of claims 1 to 3 wherein R is -CH 2 CH(OH)CH 2 O(CH 2 ) 15 CH 3 . 5. A modified oligodeoxynucleotide as claimed in one or more of claims 1 to 3 wherein R is -(CH 2 ) 15 CH 3 . 6. A modified oligodeoxynucleotide as claimed in one or more of claims 1 to 3 wherein R is -(CH 2 CH 2 O) n -(CH 2 ) 15 CH 3 and n is an integer from 1 to 6. 7. A modified oligodeoxynucleotide as claimed in claim 6 wherein n is 1. 8. A modified oligodeoxynucleotide as claimed in claim 6 wherein n is 2. 9. A modified oligodeoxynucleotide as claimed in claim 6 wherein n is 3. 10. The modified oligodeoxynucleotide as claimed in one or more of claims 1 to 9 for use as a pharmaceutical. 11. A pharmaceutical formulation which contains an effective amount of at least one modified oligodeoxynucleotide as claimed in one or more of claims 1 to 9. 12. The use of the modified oligodeoxynucleotide as claimed in one or more of claims 1 to 9 for preparing a pharmaceutical for the treatment of a disease arising from the overexpression and/or mutation of the Ha-ras gene. 13. The use of the modified oligodeoxynucleotide as claimed in one or more of claims 1 to 9 for preparing a pharmaceutical for the treatment of a disease arising from a hyperprol iterative disorder. 14. The use as claimed in claim 13 wherein the disease is cancer. 15. The use as claimed in claim 14 in combination with radiotherapy. 16. The use as claimed in claim 14 in combination with chemotherapy. 17. The use as claimed in claim 13 wherein the disease is restenosis. 18. The use as claimed in claim 13 wherein the disease is psoriasis. 19. The use of the modified oligodeoxynucleotide as claimed in one or more of claims 1 to 9 for preparing a pharmaceutical which is directed to the regulation, modulation or inhibition of Ha-ras gene expression. 20. The use of the modified oligodeoxynucleotide as claimed in one or more of claims 1 to 9 for preparing a pharmaceutical which inhibits the proliferation of cancer cells. 21. The use of the modified oligodeoxynucleotide as claimed in one or more of claims 1 to 9 for preparing a pharmaceutical which reverses the radio- resistance in cancer cells. 22. The use of the modified oligodeoxynucleotide as claimed in one or more of claims 1 to 9 for preparing a pharmaceutical which reverses the chemoresistance in cancer cells. 23. A pharmaceutical formulation as claimed in claim 11 which contains an effective amount of at least one further chemotherapeutically effective agent. 24. A pharmaceutical formulation as claimed in claim 23 wherein at least one of the further chemotherapeutically effective agents is selected from the group of cis-platinum and its derivatives. 25. A pharmaceutical formulation as claimed in claim 24 wherein at least one of the further chemotherapeutically effective agents is cis-platinum. 26. A pharmaceutical formulation as claimed in claim 23 wherein at least one of the further chemotherapeutically effective agents is selected from the group of N-lost derivatives. 27. A pharmaceutical formulation as claimed in claim 26 wherein at least one of the further chemotherapeutically effective agents is cyclophosphamide. 28. A pharmaceutical formulation as claimed in claim 26 wherein at least one of the further chemotherapeutically effective agents is trofosfamide. 29. A pharmaceutical formulation as claimed in claim 26 wherein at least one of the further chemotherapeutically effective agents is ifosfamide. 30. A pharmaceutical formulation as claimed in claim 23 wherein at least one of the further chemotherapeutically effective agents is selected from the group of aziridine derivatives. 31. A pharmaceutical formulation as claimed in claim 30 wherein at least one of the further chemotherapeutically effective agents is thiothepa. 32. A pharmaceutical formulation as claimed in claim 23 wherein at least one of the further chemotherapeutically effective agents is selected from the group of N-nitroso-urea derivatives. 33. A pharmaceutical formulation as claimed in claim 23 wherein at least one of the further chemotherapeutically effective agents is selected from the group of folic acid antagonists. 34. A pharmaceutical formulation as claimed in claim 33 wherein at least one of the further chemotherapeutically effective agents is methotrexate. 35. A pharmaceutical formulation as claimed in claim 23 wherein at least one of the further chemotherapeutically effective agents is selected from the group of analogs of purine and pyrimidine bases. 36. A pharmaceutical formulation as claimed in claim 35 wherein at least one of the further chemotherapeutically effective agents is 5-fluoro-uracil. 37. A pharmaceutical formulation as claimed in claim 23 wherein at least one of the further chemotherapeutically effective agents is selected from the group of cytostatically effective antibiotics. 38. A pharmaceutical formulation as claimed in claim 37 wherein at least one of the further chemotherapeutically effective agents is adriamycine. 39. A pharmaceutical formulation as claimed in claim 37 wherein at least one of the further chemotherapeutically effective agents is mitomycine. 40. A pharmaceutical formulation as claimed in claim 37 wherein at least one of the further chemotherapeutically effective agents is daunorubicine. 41. A pharmaceutical formulation as claimed in claim 23 wherein at least one of the further chemotherapeutically effective agents is selected from the group of estrogene antagonists. 42. A pharmaceutical formulation as claimed in claim 41 wherein at least one of the further chemotherapeutically effective agents is tamoxifene. 43. A pharmaceutical formulation as claimed in claim 23 wherein at least one of the further chemotherapeutically effective agents is selected from the group of nucleoside derivatives. 44. A pharmaceutical formulation as claimed in claim 43 wherein at least one of the further chemotherapeutically effective agents is MDL101.731. 45. A method of preparing an oiigonucleotide as claimed in one or more of claims 1 to 9, wherein phosphorothioate linkages were introduced by sulfurization using Beaucage reagent.

Inventors: CHANG ESTHER, PEYMAN ANUSCHIRWAN, PIROLLO KATHLEEN, RAIT ANTONINA, UHLMANN EUGEN, WILL DAVID WILLIAM, CHANG, ESTHER, PEYMAN, ANUSCHIRWAN, PIROLLO, KATHLEEN, RAIT, ANTONINA, UHLMANN, EUGEN, WILL, DAVID, WILLIAM
Applicants: CHANG ESTHER, HOECHST MARION ROUSSEL DE GMBH, PEYMAN ANUSCHIRWAN, PIROLLO KATHLEEN, RAIT ANTONINA, UHLMANN EUGEN, WILL DAVID WILLIAM, CHANG, ESTHER, HOECHST MARION ROUSSEL DEUTSCHLAND GMBH, PEYMAN, ANUSCHIRWAN, PIROLLO, KATHLEEN, RAIT, ANTONINA, UHLMANN, EUGEN, WILL, DAVID, WILLIAM
Application year: 1998
Main classification: C12N15
IPC codes: A61K31, A61K33, A61K38, A61K45, A61P35, C07H21, C12N15