WO1997010829A1

WO1997010829A1 - Pharmaceutical compositions containing an oligonucleotide and an anionic polysaccharide

Info

Publication number: WO1997010829A1
Application number: PCT/GB1996/002245
Authority: WO
Inventors: Paul Leslie Nicklin; Alan Steward
Original assignee: Novartis Ag
Priority date: 1995-09-19
Filing date: 1996-09-12
Publication date: 1997-03-27
Also published as: AU6937096A; GB9519109D0

Abstract

A pharmaceutical composition comprising (A) an oligonucleotide and (B) an anionic polysaccharide such as dextran sulphate which can be used in the treatment of mammalian cancer.

Description

Pharmaceutical Compositions Containing an Oligonucleotide and an Anionic Polysaccharide

This invention relates to oligonucleotide compositions, their preparation and their use.

Oligonucleotides have been employed as therapeutic moieties in the treatment of disease states in animals and man. For example, there have been identified antisense, triplex and other oligonucleotide compositions which are capable of modulating expression of genes implicated in viral, fungal and metabolic diseases.

Alterations in cellular genes which directly or indirectly control cell growth and differentiation are considered to be the main cause of cancer. There are some thirty families of genes, called oncogenes, which are implicated in human tumor formation. Members of one such family, the raf gene family, are frequently found to be mutated in human tumors. The raf family includes three highly conserved genes termed A-, B- and c-raf (also called raf -I), c- Raf, the best characterized member of the raf family, is the cellular homologue of v-raf, the transforming gene of the murine sarcoma virus 36H. Raf genes encode protein kinases that are thought to play important regulatory roles in signal transduction processes that regulate cell proliferation. Mutation of raf genes causing a truncation or other modification that leads to the expression of raf kinase without a functional negative regulatory domain at the amino-terminal end results in conversion to a form which is implicated in transformation of mammalian cells in culture, and tumor formation. A raf gene having an absent or inactive regulatory domain is said to be "activated." Activated (truncated) raf has been detected in a variety of human cancers including small-cell lung carcinoma, primary stomach cancer, renal cancer, breast cancer, laryngeal cancer, skin fibroblasts from members of a cancer- prone family (Li-Fraumeni syndrome), and in a human glioblastoma cell line. Abnormal expression of the normal (non-activated) c-raf protein is believed to play a role in abnormal cell proliferation since it has been reported that 60% of all lung carcinoma cell lines express unusually high levels of normal c-raf mRNA and protein. Rapp et al., The Oncogene Handbook, E.P. Reddy, A. M. Skalka and T. Curran, eds., Elsevier Science Publishers, New York, I988, pp. 2I3-253. There remains a need for compositions which can effectively inhibit abnormal raf gene expression, i.e. inhibit expression of the activated raf product or inhibit unusually high level of expression of the normal raf product.

There also remains a need for oligonucleotide compositions which show improved uptake by tumours.

It has now been found that uptake by tumours of an oligonucleotide, particularly an oligonucleotide targeted to mRNA encoding human raf, can be increased by administration of the oligonucleotide together with an anionic polysaccharide.

Accordingly, the present invention provides a pharmaceutical composition comprising (A) an oligonucleotide and (B) an anionic polysaccharide.

In the context of this invention, the term "oligonucleotide" refers to an oligomer or polymer of nucleotide or nucleoside monomers consisting of naturally occurring bases, sugars and intersugar (backbone) linkages. The term "oligonucleotide" also includes oligomers comprising non-naturally occurring monomers, or portions thereof, which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of properties such as, for example, enhanced cellular uptake and increased stability in the presence of nucleases. The oligonucleotide may comprise, for example, up to 50 nucleotides, preferably 8 to 50 nucleotides, especially 16 to 24 nucleotides.

In some preferred oligonucleotides (A), at least one nucleotide is modified at the 2' position of the sugar moiety. Certain preferred oligonucleotides (A) are chimeric oligonucleotides. "Chimeric oligonucleotides" or "chimeras", in the context of this invention, are oligonucleotides which contain two or more chemically distinct regions, each made up of at least one nucleotide. These oligonucleotides typically contain at least one region of modified nucleotides that confers one or more beneficial properties (such as, for example, increased nuclease resistance, increased uptake into cells, increased binding affinity for the RNA target) and a region that is a substrate for RNase H cleavage. In one preferred embodiment, a chimeric oligonucleotide comprises at least one region modified to increase target binding affinity and, usually, a region that acts as a substrate for RNAse H. Affinity of an oligonucleotide for its target (for example a nucleic acid encoding raf) is routinely determined by measuring the Tm of an oligonucleotide/target pair, which is the temperature at which the oligonucleotide and target dissociate; dissociation is detected spectrophotometrically. The higher the Tm, the greater the affinity of the oligonucleotide for the target. In a more preferred embodiment, the region of the oligonucleotide which is modified to increase mRNA binding affinity comprises at least one nucleotide modified at the 2' position of the sugar, particulariy a 2' - alkoxy, 2'-alkoxyalkoxy or 2'-fluoro-modified nucleotide. Such modifications are routinely incoφorated into oligonucleotides and these oligonucleotides have been shown to have a higher Tm (i.e., higher target binding affinity) than 2'-deoxyoligonucleotides against a given target. Where the target is raf, the effect of such increased affinity is to greatly enhance antisense oligonucleotide inhibition of raf gene expression. RNAse H is a cellular endonuclease that cleaves the RNA strand of RNA.DNA duplexes; activation of this enzyme therefore results in cleavage of the RNA target, and thus can greatly enhance the efficiency of antisense inhibition. Cleavage of the RNA target can be routinely demonstrated by gel electrophoresis. In another preferred embodiment, the chimeric oligonucleotide is also modified to enhance nuclease resistance. Cells contain a variety of exo- and endo-nucleases which can degrade nucleic acids. A number of nucleotide and nucleoside modifications have been shown to make the oligonucleotide into which they are incoφorated more resistant to nuclease digestion than the native oligodeoxynucleot.de. Nuclease resistance is routinely measured by incubating oligonucleotides with cellular extracts or isolated nuclease solutions and measuring the extent or isolated nuclease solutions and measuring the extent of intact oligonucleotide remaining over time, usually by gel electrophoresis. Oligonucleotides which have been modified to enhance their nuclease resistance survive intact for a longer time than unmodified oligonucleotides. A variety of oligonucleotide modifications have been demonstrated to enhance or confer nuclease resistance. Oligonucleotides which contain at least one phosphorothioate modification are presently more preferred. In some cases, oligonucleotide modifications which enhance target binding affinity are also, independently, able to enhance nuclease resistance.

Specific examples of some preferred oligonucleotides may contain phosphorothioate, phosphotriester, methyl phosphonate, short chain alkyl or cycloalkyl intersugar linkages, short chain heteroatomic or heterocyclic intersugar ("backbone") linkages or combinations thereof or combinations of one or more thereof with one or more phosphodiester backbone linkages. Most preferred are phosphorothioates and those with CH -NH-O-CH₂, CH₂- N(CH₃)-O-CH₂, CH₂-O-N(CH₃)-CH₂, CH₂-N(CH₃)-N(CH₃)-CH₂ and O-N(CH₃)-CH₂-CH₂ backbones or combinations thereof or combinations of one or more thereof with one or more phosphodiestr backbone linkages (where phosphodiester is O-P-O-CH ₂). Also preferred are oligonucleotides having morpholino backbone structures, for example as described in U.S. Patent No. 5, 034, 506. In other preferred embodiments, such as the protein-nucleic acid or peptide-nucleic acid (PNA) backbone, the phosphodiester backbone of the oligonucleotide may be replaced with a polyamide backbone, the bases being bound directly or indirectly to the aza nitrogen atoms of the polyamide backbone, as described by P.E. Nielsen, M. Egholm, R.H. Berg, O. Buchardt, Science 1991, 254, 1497. Other preferred oligonucleotides may contain substituted, e.g. alkyl and halogen-substituted, sugar moieties comprising one of the following at the 2' position: OH, SH, SCH ₃, F, OCN, OCH₂OCH₃, OCHzCHzOCHa. OCH₂O(CH₂)n CH₃ . O(CH₂)nNH₂ or O(CH₂)_nCH₃ where n is from I to about 10; Ci to Cio lower alkyl, substituted lower alkyl, alkaryl or aralkyl; Cl; Br; CN; CF₃; OCF₃; O-, S-, or N-alkyl; O-. S-. or N-alkenyl; SOCH₃; SO₂CH₃;ONO₂; NO₂; N₃; NH₂; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; an RNA cleaving group; a cholesteryl group; a conjugate; a reporter group; an intercalator; a group for improving the pharmacokinetic properties of an oligonucleotide; or a group for improving the pharmacodynamic properties of an oligonucleotide and other substituents having similar properties. Oligonucleotides may also have sugar mimetics such as cyclobutyls in place of the pentofuranosyl group. Other preferred embodiments may include at least one modified base form or "universal base" such as inosine.

In certain especially preferred embodiments of the invention, all nucleotides of the oligonucleotide (A) are 2'-deoxynucleotides and all backbone linkages are phosphorothioate linkages.

In certain other especially preferred embodiments, the oligonucleotide (A) is a chimeric oligonucleotide having one or more regions with 2'-deoxynucleotides and one or more regions with 2'-alkoxyalkoxynucleotides, particularly 2'-methoxyethoxynucleotides, the one or more, 2'-deoxynucleotide regions preferably having phosphorothioate backbone linkages and the one or more 2'-alkoxyalkoxynucleotide regions preferably having phosphodiester backbone linkages. These chimeric oligonucleotides preferably comprise a region of 2'- deoxynucleotides between two regions of 2'-alkoxyalkoxyπucleotides.

The relationship between an oligonucleotide and its complementary nucleic acid target to which it hybridises is commonly referred to as "antisense". Targetting an oligonucleotide to a chosen nucleic acid target may involve a multistep process. The process usually begins with identifying a nucleic acid sequence whose function is to be modulated. This may be, as examples, a cellular gene (or mRNA made from the gene) whose expression is associated with a particular disease state, or a foreign nucleic acid from an infectious agent. In preferred embodiments of the present invention, the target is a nucleic acid encoding raf; in other words, the raf gene or mRNA expressed from the raf gene. The targeting process also includes determination of a site or sites within the nucleic acid sequence for the oligonucleotide interaction to occur such that the desired effect - inhibition of abnormal raf gene expression- will result. Once the target site or sites have been identified, oligonucleotides are chosen which are sufficiently complementary to the target, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired inhibition.

Inhibition of abnormal raf gene expression can be measured in ways which are routine in the art, for example by Northern blot assay of mRNA expression or Western blot assay of protein expression. Effects on cell proliferation or tumor cell growth can also be measured, as described hereinafter in the Examples. "Hybridization," in the context of this invention, means hydrogen bonding, also known as Watson-Crick base pairing, between complementary bases, usually on opposite nucleic acid strands or two regions of a nucleic acid strand. Guanine and cytosine are examples of complementary bases which are known to form three hydrogen bonds between them. Adenine and thymine are examples of complementary bases which form two hydrogen bonds between them. "Specifically hybridizable" and "complementary" are terms which are used to indicate a sufficient degree of complementarity such that stable and specific binding occurs between the DNA or RNA target and the oligonucleotide. it is understood that an oligonucleotide need not be 100% complementary to its target nucleic acid sequence to be specifically hybridizable. An oligonucleotide is specifically hybridizable when binding of the oligonucleotide to the target interferes with the normal function of the target molecule to cause a loss of utility, and there is a sufficient degree of complementarity to avoid non-specific binding of the oligonucleotide to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, or, in the case of in vitro assays, under conditions in which the assays are conducted.

In especially preferred embodiments of this invention, the oligonucleotide (A) is 8 to 50 nucleotides in length, especially 18 to 24 nucleotides in length, and is targeted to mRNA encoding human raf, particulariy c-raf or A-raf. In accordance with this invention, persons of ordinary skill in the art will understand that mRNA includes not only the coding region which carries the information to encode a protein using the three letter genetic code, but also associated ribonucleotides which form a region known to such persons as the 5'- untranslated region, the 3'-untranslated region, the 5' cap region, intron regions and intron/exon or splice junction ribonucleotides. Thus, oligonucleotides may be formulated in accordance with this invention which are targeted wholly or in part to these associated ribonucleotides as well as to the coding ribonucleotides. In preferred embodiments, the oligonucleotide is targeted to a translation initiation site (AUG codon) or sequences in the 5' - or 3' -untranslated region of the human c- raf mRNA. The functions of messenger RNA to be interfered with include all vital functions such as translocation of the RNA to the site for protein translation, actual translation of protein from the RNA, splicing or maturation of the RNA and possibly even independent catalytic activity which may be engaged in by the RNA. The overall effect of such interference with the RNA function is to cause interference with raf protein expression.

Oligonucleotides targeted to mRNA encoding human A-raf and, especially, human c-raf are presently preferred; however, compositions for modulating expression of other forms of raf are also believed to have utility and are comprehended by this invention.

The oligonucleotides used as component (A) of the composition of the invention may be conveniently and routinely made using well-known techniques such as solid phase synthesis. Equipment for such synthesis is available commercially from various sources including Applied Biosystems. The use of such techniques to prepare oligonucleotides such as the phosphorothioates and alkylated derivatives is well known. It is also well known to use similar techniques and commercially available modified amidites and controlled-pore glass (CPG) products such as biotin, fluorescein, acridine or psoralen-modified amidites and or CPG (available from Glen Research, Sterling VA) to synthesize fluorescently labeled, biotinylated or other modified oligonucleotides such as cholesterol-modified oligonucleotides.

Specific especially preferred oligonucleotides, for which the nucleotide sequences and preparation have been described in WO 95/32987, are referred to hereinafter as oligonucleotide Nos. ON1 to ON43. These include the following: No. Sequence (5' → 3"> Site SEQ ID NO:

ON1 GCTCCATTGATGCAGCTTAA AUG 1

ON2 GATGCAGCTTAAACAATTCT 5'UTR 2

ON3 TCCCGCCTGTGACATGCATT 3'UTR 3

ON4 GTCTGGCGCTGCACCACTCT 3'UTR 4

ON5 CGCTCCTCCTCCCCGCGGCG 5'UTR 5

ON6 TCCTCCTCCCCGCGGCGGGT 5'UTR 6

ON7 CTCGCCCGCTCCTCCTCCCC 5'UTR 7

ON8 CTGGCTTCTCCTCCTCCCCT 3'UTR 8

ON9 CGGGAGGCGGTCACATTCGG 55'' U UTTRR 9

ON10 TCTGGCGCTGCACCACTCTC 3'UTR 10

ON1 to ON10 are oligodeoxynucleotides with phosphorothioate backbones desgined using the Genbank c-raf sequence HUMRAFR (Genbank listing x 03484), synthesised and tested for inhibition of c-raf mRNA expression in T24 bladder carcinoma cells using a Northern blot assay.

Other specific especially preferred oligonucleotides include:

No. Seouence Site SEQ ID NO:

ON11 CGGGAGGCGGTCACATTCGG 5'UTR 9

ON12 GATGCAGCTTAAACAATTCT 5'UTR 2

ON13 GCTCCATTGATGCAGCTTAA AUG 1

ON14 CGCTCCTCCTCCCCGCGGCG 5'UTR 5

ON 15 CGGGAGGCGGTCACATTCGG 5' UTR 9

ON11 , ON12 and ON13 are oligonucleotides synthesised with phosphorothioate backbones and uniformly substituted at the 2' position of the sugar moiety by a methoxy group. ON14 is synthesized with a phosphodiester backbone and is uniformly substituted by a propoxy group at the 2' position of the sugar moiety. ON15 is synthesized with a phosphorothioate backbone and is uniformly substituted by fluoro at the 2' position of the sugar moiety. Specifically especially preferred chimeric oligonucleotides include:

No. Seguence Target Site SEQ ID NO:

ON16 TCCTCCTCCCCGCGGCGGGT 5'UTR 6

ON17 CTCGCCCGCTCCTCCTCCCC 5'UTR 7

ON18 TTCTCGCCCGCTCCTCCTCC 5'UTR 11

ON19 TTCTCCTCCTCCCCTGGCAG 3OTR 12

ON20 CTGGCTTCTCCTCCTCCCCT 3'UTR 8

ON21 CCTGCTGGCTTCTCCTCCTC 3'UTR 13

ON22 TCCCGCCTGTGACATGCATT 3'UTR 3

ON23 TCCCGCCTGTGACATGCATT 3'UTR 3

ON24 TCCCGCCTGTGACATGCATT 3'UTR 3

ON25 TCTGGCGCTGCACCACTCTC 3'UTR 10

ON16 to ON25 are chimeric oligonucleotides with uniform phosphorothiate backbones, the nucleotides shown underlined being substituted by methoxy at the 2' position of the sugar moiety.

Other specific especially preferred chimeric oligonucleotides include:

No. Seguence Target Site SEQ ID NO: ON26 TCCCGCCTGTGACATGCATT 3'UTR 3

ON27 TCCCGCCTGTGACATGCATT 3'UTR 3

ON28 TCTGGCGCTGCACCACTCTC 3'UTR 10

ON26, ON27 and ON28 are chimeric oligonucleotides with uniform phosphorothioate backbones, the nucleotides shown underlined being substituted at the 2' position of the sugar moiety, in ON26 by propoxy and in ON27 and ON28 by fluoro.

Specific preferred chimeric oligonucleotides with 2' modifications and chimeric phosphorothiote/phosphodiester backbones include: No. Seguence Target Site SEQ ID NO:

ON29 TCCCGCCTGTGACATGCATT 3'UTR 3

ON30 TCTGGCGCTGCACCACTCTC 3'UTR IO

ON31 TCCCGCCTGTGACATGCATT 3'UTR 3

ON29 and ON30 have regions, shown underlined, which have both 2'-propoxy substituents and phosphodiester backbones. ON31 has regions, shown underlined, which have both 2'- methoxyethoxy substituents and phosphodiester backbones.

It is believed that certain oligonucleotides targeted to portions of the A-raf mRNA and which inhibit A-raf expression will be useful for interfering with cell hyperproliferation.

Specific phosphorthioate deoxyoligonucieotides of this kind, designed and synthesised using the Genbank A-raf sequence HUMARAFIR (Genbank listing x 04790), include the following:

No. Seguence Target Site SEQ ID NO:

ON32 CCA TCC CGG ACA GTC ACC AC Coding 15

ON33 ATG AGC TCC TCG CCA TCC AG Coding 16

ON34 AAT GCT GGT GGA ACT TGT AG Coding 17

ON35 CCG GTA CCC CAG GTT CTT CA Coding 18

ON36 CTG GGC AGT CTG CCG GGC CA Coding 19

ON37 CAC CTC AGC TGC CAT CCA CA Coding 20

ON38 GAG ATT TTG CTG AGG TCC GG Coding 21

ON39 GCA CTC CGC TCA ATC TTG GG Coding 22

ON40 CTA AGG CAC AAG GCG GGC TG Stop 23

ON41 ACG AAC ATT GAT TGG CTG GT 3'UTR 24

ON42 GTA TCC CCA AAG CCA AGA GG 3^*UTR 25

ON43 GTC AAG ATG GGC TGA GGT GG 5'UTR 14 In other embodiments of the invention, the oligonucleotide (A) is 5 to 50 nucleotides in length and is specifically hybridizable with DNA or RNA derived from the gene encoding protein kinase C, particulariy such an oligonucleotide as described in WO 95/02069. Preferred such oligonucleotides are those described in Examples 1 to 18 of WO 95/02069.

The anionic polysaccharide (B) may be, for example, an anionic dextran, polyinosinic acid, polyguanylic acid, polyxanthinylic acid or fucoidin.

The anionic polysaccharide (B) is preferably an anionic dextran or fucoidin, more preferably a dextran sulphate having a molecular weight of 5 to 500 kD, e.g. 5 to 50 kD, especially 5 to 10 kD.

The weight ratio of anionic polysaccharide to oligonucleotide may be generally from 1 :1 to 1000:1, for example from 1:1 to 100:1, preferably from 3:1 to 300:1, for example from 3:1 to 30:1, especially from 5:1 to 150:1, for example from 5:1 to 15:1 or from 10:1 to 100:1.

A composition of the invention may be prepared simply by mixing the oligonucleotide and anionic polysaccharide together, for example before dissolution in a medium such as water, phosphate buffered saline or other pharmaceutically acceptable aqueous medium for parenteral administration or by simultaneous or separate dissolution in such a medium.

The invention includes a method of inhibiting the expression of human raf which comprises contacting tissues or cells which express human raf with a composition of the invention as hereinbefore described in which (A) is targeted to m RNA encoding human raf. The invention also includes a method of treating mammalian cancer which comprises administering a composition of the invention as hereinbefore described to a mammal, particularly a human, in need of such treatment.

The composition of the invention may be administered by pulmonary delivery or, preferably, parenterally, for example intravenously, subcutaneously, intraperitoneally or intramuscularly. The dosage depends principally on the method of administration and on the severity and responsiveness of the condition to be treated. Individual doses and the administration regime can best be determined by individual judgement of a particular case of illness. Diseases which may be treated with the composition include mammalian cancer, particularly human cancer such as lung cancer, stomach cancer, renal cancer, breast cancer, laryngeal cancer, pancreatic cancer, colorectal cancer and malignant melanoma. The invention is illustrated by the following Examples.

Example 1

Oligonucleotide ON3 as hereinbefore defined (15 μg) is mixed with dextran sulphate having a molecular weight of 8kD (150μg). The resulting mixture is dissolved in phosphate buffered saline (0.2ml) to give a solution which can be administered by intravenous injection.

Example 2

Example 1 is repeated, using 15 μg of the dextran sulphate instead of the amount used in Example 1 , to give a solution which can be administered by intravenous injection.

Example 3

Example 1 is repeated, using 1500 μg of the dextran sulphate instead of the amount used in Example 1 , to give a solution which can be administered by intravenous injection.

Example 4

Example 1 is repeated, using oligonucleotide ON31 in place of ON3, to give a solution which can be administered by intravenous injection.

Example 5

Example 3 is repeated using oligonucleotide Isis # 3521 as described in WO 95/02069 in place of ON3, to give a solution which can be administered by intravenous injection. Example 6

Oligonucleotide ON3 as hereinbefore defined in tritiated form (15 μg) is mixed with dextran sulphate having a molecular weight of 8 kD (1500 μg). A solution of the resulting mixture in water (0.2ml) is administered by intravenous injection to nude mice having tumours formed by innocuiation 10 days previously with human lung adenocarcinoma A549 cells implanted subcutaneously under the dorsal outer skin. The oligonucleotide dosage is 0.6 mg kg. Two hours after the injection, the mice are sacrificed and the content of the radioactively labelled oligonucleotide in the tumours is determined. The above test procedure is repeated using a solution of oligonucleotide ON3 alone, at the same dosage, instead of the mixture with dextran sulphate, in water. The content of the oligonucleotide in the tumour is 0.351 μg/g following injection with the oligonucleotide-dextran sulphate mixture, but only 0.249 μg/g following injection with the oligonucleotide alone (average of 6 replicates in each case).

Claims

1. A pharmaceutical composition comprising (A) an oligonucleotide and (B) an anionic polysaccharide.

2. A composition according to claim I, in which at least one nucleotide of the oligonucleotide (A) is modified at the 2' position of the sugar moiety.

3. A composition according to claim I, in which the oligonucleotide (A) is a chimeric oligonucleotide which contains a first region having at least one nucleotide modified to enhance target affinity and a second region which is a substrate for RNAse H.

4. A composition according to claim 3, in which a nucleotide modified to enhance target affinity is modified at the 2' position of the sugar moiety.

5. A composition according to claim 2 or 4, in which the modified nucleotide has an alkoxy, alkoxyalkoxy or fluoro substituent at the 2' position.

6. A composition according to claim 3, 4 or 5, in which the oligonucleotide (A) is a chimeric oligonucleotide and the region which is a substrate for RNAse H comprises at least one 2'-deoxynucieotide.

7. A composition according to any of the preceding claims, in which the oligonucleotide (A) has at least one phosphorothioate linkage.

8. A composition according to claim 1 , in which, in the oligonucleotide (A), all nucleotides are 2'-deoxynucleotides and all backbone linkages are phosphorothioate linkages.

9. A composition according to any of claims 1 to 7, in which the oligonucleotide (A) is a chimeric oligonucleotide having one or more regions with 2'-deoxynucleotides and one or more regions with 2'-alkoxyalkoxynucleotides.

10. A composition according to claim 9, in which the 2'-alkoxyalkoxynucleotides are 2'- methoxyethoxynucleotides.

11. A composition according to claim 9 or 10, in which the one or more regions with 2'- deoxynucleotides have phosphorothioate backbone linkages and the one or more regions with 2'-alkoxyaikoxynucleotides have phosphodiester backbone linkages.

12. A composition according to any of claims 9 to 11 , in which the oligonucleotide (A) comprises a region of 2'-deoxynucleotides between two regions of _'- alkoxyalkoxynucieotides.

13. A composition according to any of the preceding claims, in which the oligonucleotide (A) is 8 to 50 nucleotides in length and is targeted to mRNA encoding human raf.

14. A composition according to claim 13, in which the oligonucleotide (A) is targeted to mRNA encoding human c-raf.

15. A composition according to claim 14. in which the oligonucleotide (A) is targeted to a translation initiation site.3' untranslated region or 5' untranslated region of mRNA encoding human c-raf.

16. A composition according to any of claims 1 to 12. in which the oligonucleotide (A) comprises a nucleotide sequence

GCTCCATTGATGCAGCTTAA or GATGCAGCTTAAACAATTCT or TCCCGCCTGTGACATGCATT or GTCTGGCGCTGCACCACTCT or CGCTCCTCCTCCCCGCGGCG or TCCTCCTCCCCGCGGCGGGT or CTCGCCCGCTCCTCCTCCCC or CTGGCTTCTCCTCCTCCCCT or CGGGAGGCGGTCACATTCGG or TCTGGCGCTGCACCACTCTC or TTCTCGCCCGCTCCTCCTCC or TTCTCCTCCTCCCCTGGCAG or CCTGCTGGCTTCTCCTCCTC

17. A composition according to any of claims 1 to 16, in which the anionic polysaccharide is an anionic dextran or fucoidin.

18. A composition according to claim 17, in which the anionic polysaccharide is a dextran sulphate.

19. A composition according to claim 18, in which the dextran sulphate has a molecular weight of 5 to 500 kD.

20. A composition according to claim 19, in which the dextran sulphate has a molecular weight of 5 to IO kD.

21. A composition according to any of the preceding claims, in which the weight ratio of anionic polysaccharide to oligonucleotide is from 1 :1 to 1000:1.

22. A composition according to claim 21. in which said weight ratio is from 1 :1 to 100:1.

23. A composition according to claim 21 , in which said weight ratio is from 3:1 to 300:1.

24. A composition according to claim 21 , in which said weight ratio is from 5:1 to 150:1.

25. Use of a composition according to any of the preceding ciaims in the preparation of a medicament for the treatment of mammalian cancer.

26. A method of treating mammalian cancer which comprises administering a composition according to any of claims 1 to 24 to a mammal in need of such treatment

27. A method of inhibiting the expression of human raf which comprises contacting tissues or cells which express human raf with a composition according to any of ciaims 13 to 24 in which the oligonucleotide (A) is targeted to mRNA encoding human raf.