WO1997032604A1 - Antiproliferative combinations, containing raf-targeted oligonucleotides and chemotherapeutic compounds - Google Patents

Antiproliferative combinations, containing raf-targeted oligonucleotides and chemotherapeutic compounds Download PDF

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Publication number
WO1997032604A1
WO1997032604A1 PCT/EP1997/000875 EP9700875W WO9732604A1 WO 1997032604 A1 WO1997032604 A1 WO 1997032604A1 EP 9700875 W EP9700875 W EP 9700875W WO 9732604 A1 WO9732604 A1 WO 9732604A1
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selected
present
oligonucleotide
oligonucleotide derivative
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PCT/EP1997/000875
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French (fr)
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Marcel Müller
Thomas Geiger
Karl-Heinz Altmann
Doriano Fabbro
Brett Monia
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Novartis Ag
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K45/00Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K31/00Medicinal preparations containing organic active ingredients
    • A61K31/70Carbohydrates; Sugars; Derivatives thereof
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K33/00Medicinal preparations containing inorganic active ingredients
    • A61K33/24Heavy metals; Compounds thereof

Abstract

The invention relates to combinations of raf-targeted (especially c-raf-targeted) deoxyribo- and ribo-oligonucleotides and derivatives thereof with other chemotherapeutic compounds, as well as to pharmaceutical preparations and/or therapies, in relation to disease states which respond to such oligonucleotides or oligonucleotide derivatives, especially to modulation of the activity of a regulatory protein. In particular, the invention relates to products or combinations comprising antisense oligonucleotides or oligonucleotide derivatives targeted to nucleic acids encoding raf and other (preferably standard) chemotherapeutics, either in fixed combination or for chronologically staggered or simultaneous administration, and the combined use of both classes of compounds, either in fixed combination or for chronologically staggered or simultaneous administration, for the treatment of proliferative diseases, especially tumor diseases, that can be treated by inhibition of raf activity, that is, where the antisense oligonucleotides or oligonucleotide derivatives are targeted to nucleic acids encoding the regulatory protein raf or active mutated derivatives thereof.

Description

ANTIPROLIFERATIVE COMBINATIONS, CONTAINING RAF-TARGETED OLIGONUCLEOTIDES AND CHEMOTHERAPEUTIC COMPOUNDS

Field of the Invention

This invention relates to combinations of raf-targeted (especially c-raf-targeted) deoxyribo- and ribo-oligonucleotides and derivatives thereof with other chemotherapeutic compounds, as well as to pharmaceutical preparations and/or therapies, in relation to disease states which respond to such oligonucleotides or oligonucleotide derivatives, especially to modulation of the activity of a regulatory protein. In particular, the invention relates to products or combinations comprising antisense oligonucleotides or oligonucleotide derivatives targeted to nucleic acids encoding (especially human) raf and other (preferably standard) chemotherapeutics, either in fixed combination or for chronologically staggered or simultaneous administration, and the combined use of both classes of compounds, either in fixed combination or for chronologically staggered or simultaneous administration, for the treatment of proliferative diseases, especially tumor diseases, that can be treated by inhibition of raf, especially c-raf, activity, that is, where the antisense oligonucleotides or oligonucleotide derivatives are targeted to nucleic acids encoding the regulatory protein raf, especially c-raf, or active mutated derivatives thereof.

Background of the Invention

The extensive information that has been accumulated over the past decade concerning the molecular basis of mammalian cell transformation has led to the unifying concept of growth regulation and its disorders in cancer cells. The fact that many products of "cancer genes" encode for proteins that regulate normal mitogenesis suggests that the carcinogenic pro¬ cess may be viewed as a multistep and progressive disorder of signal transduction. This conceptual framework has provided a basis for the development of novel anticancer strate¬ gies and therapeutic modalities. Almost every mitogenic signal is amplified and transduced inside cells by protein kinase (PK) cascades either by receptor activated tyrosine phospho- rylatioπ or by receptor coupling to GTP-binding proteins. Most mitogenic pathways utilize unique and/or overlapping parts of these protein kinase cascades. Accordingly mutant alleles of these PK genes or of oncogenes that signal through these PKs are able to perturb entire signaling networks leading to the deregulation of cell differentiation, division and/or apoptosis. The anticancer strategy is, consequently, based on the assumption that blocking deregulated mitogenic signal transduction at the level of PKs will cause cancer growth inhibition. This approach is likely to identify compounds with less side effects compared to standard chemotherapeutic agents.

The raf family of serine/threonine specific protein kinases comprises three members, A-raf, B-raf and c-raf (see Magnusson et al., Sem. Cancer Biol. 5, 247-53 (1994); Beck et al., Nucl. Acids Res. 15, 595-609 (1987) and Sithanandam et al., Oncogene 5, 1775-80 (1990)). The enzymes are expressed in a tissue specific manner and are important mediators of signal transduction involving cell growth, transformation and differentiation

Substantial evidence exists supporting a direct role for c-raf in the development and main¬ tenance of certain human malignancies. First, the MAP (= mitogen-activated protein) kinase signaling cascade has been shown to be essential for cellular proliferation and mediation of cellular transformation by protein kinases. Second, raf proteins have been shown to be direct downstream effectors of ras protein function within the MAP kinase signaling path¬ way. Since ras is present in a high proportion of human cancers, novel therapies directed against raf kinases are believed to prove useful in the treatment of ras-dependent tumors. Specific inhibitors of raf kinase have not been identified to date, mainly due to difficulties in the raf assay. The antisense approach represents a possibility to circumvent these difficul¬ ties. The antisense approach allows to knock-out target genes by a highly selective and sequence-specific mechanism. The identification of antisense raf kinase inhibitors has opened totally new approaches for the treatment of human cancer. In addition, these drugs, interfering with intracellular signaling, are expected to have far less unwanted side effects than the classical chemotherapeutic agents that are currently used.

Summary of the invention

Surprisingly, positive and preferably even highly synergistic effects between c-raf-targeted oligonucleotides or oligonucleotide derivatives (ODNs) and standard chemotherapeutic drugs have been observed in nude mouse xenograft models. It is thus reasonable to assume that the ODNs might be used not only as single agents, but also especially in combination therapy for the treatment of cancer diseases.

This combination offers a lot of advantages: In the first place, standard chemotherapeutics often display significant side effects up to really toxic effects, so that their use alone is often very difficult in order to obtain a responsible balance between therapeutic use and side ef¬ fects. In the new combinations decribed herein, however, it is possible to diminish the amount of standard chemotherapeutic needed and thus to alleviate side effects. Second, the ODNs have a very high toierability (up to 100 mg/kg have been found to be non-toxic in animals), thus allowing great flexibility in the treatment of cancer patients. Third, due to the fact that the c-raf-directed ODNs open up a totally new route of treatment, it is also possible to treat cancer types which have been very difficult to treat or even practically unaffected by therapy with standard chemotherapeutics, such as small cell lung carcinomas, large cell lung carcinomas, melanomas, prostate carcinomas and also lymphomas. Fourth, in a num¬ ber of cases it is even possible to bring about regression of tumors and complete cure. Most importantly, in none of the combination experiments antagonistic effects are observed.

Detailed Description of the Invention

The present invention preferably relates to combination preparations comprising a) at least one oligonucleotide or oligonucleotide derivative (ODN) targeted to nucleic acids encoding raf (especially human raf) with b) at least one other chemotherapeutic agent; or pharmaceutically acceptable salts of any component a), b) or a) and b) if at least one salt- forming group is present.

The invention also relates to a method for treating a proliferative disease that can be trea¬ ted by administration of an oligonucleotide or oligonucleotide derivative targeted to raf, especially c-raf, especially where the disease responds to modulation of raf activity, where a) at least one oligonucleotide or oligonucleotide derivative (ODN) targeted to nucleic acids encoding (especially human) raf and capable of modulating (especially human) raf expression and b) at least one other chemotherapeutic agent are administered to a mammal in combination in a quantity which is jointly therapeutically effective against proliferative diseases that can be treated by administration of an oligonucleotide or oligonucleotide derivative targeted to raf, especially c-raf, or that preferably depend on raf, especially c-raf, activity in order to treat them, where any component a) and/or b) can also be present in the form of a pharmaceutically acceptable salt, if at least one salt-forming group is present. The invention also relates to a product which comprises a) at least one oligonucleotide or oligonucleotide derivative (ODN) targeted to nucleic acids encoding raf, especially c-raf, and b) at least one other chemotherapeutic agent where any component a) and/or b).can also be present in the form of a pharmaceutically acceptable salt, if at least one salt-forming group is present, in the presence or absence of one or more pharmaceutically acceptable carrier materials, as a combination preparation for simultaneous or chronologically staggered use within a period of time which is small enough for the active compounds both of component a) and of component b) to mutually enhance antiproliferative activity against proliferating cells, especially in a patient, for treating a proliferative disease which responds to such active compounds.

The invention also relates to a pharmaceutical preparation which comprises a quantity, which is jointly effective for treating a proliferative disease that can be treated by administration of an oligonucleotide or oligonucleotide derivative targeted to raf, especially c-raf (preferably that can be treated by modulation of human raf, especially c-raf, activity) of a) at least one oligonucleotide or oligonucleotide derivative (ODN) targeted to nucleic acids encoding raf and b) at least one other chemotherapeutic agent, where any component a) and/or b) can also be present in the form of a pharmaceutically acceptable salt, if at least one salt-forming group is present, with one or more pharmaceutically acceptable carrier materials.

The invention also relates to the use of a combination of a) at least one oligonucleotide or oligonucleotide derivative (ODN) targeted to nucleic acids encoding raf, especially c-raf, and b) at least one other chemotherapeutic agent, where any component a) and/or b) can also be present in the form of a pharmaceutically acceptable salt, if at least one salt-forming group is present, for producing pharmaceutical preparations for use as compositions against a proliferative disease that can be treated by application of an oligonucleotide or oligonucleotide derivative - o

targeted to raf, especially human c-raf, preferably a proliferative disease that can be treated by modulation of raf (especially human c-raf) activity.

Included is use in a method of inhibiting hyperproliferation of cells comprising contacting hy- perproliferating cells with a pharmaceutical preparation or product as specified in the last two paragraphs, especially a method of treating a proliferative disease comprising contac¬ ting a subject, cells, tissues or a body fluid of said subject, suspected of having a hyperpro- liferative diesease with a pharmaceutical composition or product as specified in the last two paragraphs.

The term "at least one" taking reference to a) oligonucleotides or oligonucleotide derivatives or b) other chemotherapeutic agents refers to one or more, especially 1 to 5, members of each group a) or b), preferably to one compound of group a) and 1 or more, especially 1 to 5, most especially 1 or 2 compounds of group b).

An oligonucleotide or oligonucleotide derivative (ODN) targeted to nucleic acids encoding (especially human) raf is primarily characterized as follows: The relationship between an such an ODN and its complementary nucleic acid target to which it hybridizes is commonly referred to as "antisense". "Targeting" an oligonucleotide to a chosen nuclei acid target, in the context of this invention, is a multistep process. The process usually begins with identifying a nucleic acid sequence of which the function is to be modulated. This may be, as examples, a cellular gene or especially a mRNA made from that gene the expression of which is associated with a particular disease state, or for a foreign nucleic acid from an infectious agent. In the present invention, the target is a nucleic acid encoding raf, that is, the raf gene or preferably the mRNA expressed from the raf gene. The targeting process also includes determination of the site or sites within the nucleic acid sequence for the oligonucleotide interaction to occur in such a way that the desired effect-modulation of gene-expression will result. Once the target site or target sites have been identified, oligonucleotides are selected which are sufficiently complementary to the target, i.e., that hybridize sufficiently well and show sufficiently specific hybridization to provide the desired modulation.

By the term "raf there is meant any isoenzyme of raf, preferably human raf, especially human c-raf. ln the context of this invention, the term "modulation" means stimulation, but preferably in¬ hibition. Inhibition of raf, especially c-raf gene expression is the preferred form of modula¬ tion. This modulation can be measured in ways which are routine in the art, for example by Northern blot assay of mRNA expression or Western blot assay (see, for example, Sam- brook, Fritsch and Maniatis, "Molecular Cloning: A Laboratory Manual", 2nd edition, Cold Spring Harbor Laboratory Press, 1989; see also International Application WO 95/32987). Effects on tumor growth can be measured in analogy to or in accordance with the proces¬ ses taught in the examples of the present application. "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 or adenine and uracil are examples of complementary bases which form two hydrogen bonds between them. "Specifically hybrid izable" and "complementary" are terms used to indicate a sufficient degree of complementarity such that stable and specific binding occurs between the DNA and RNA target and the ODN. It is understood that an ODN need not be 100 % comple¬ mentary to its target nucleic acid sequence to be specifically hybridizable. An oligonucleo¬ tide is specifically hybridizable when binding of the oligonucleotide to the target interferes with the previously uninfluenced function of the target molecule to cause a loss of its effectiveness, and there is a sufficient degree of complementarity to avoid non-specific binding of the oligonucleotide to non-target sequences under conditions in wnich specific binding is desired, i.e. under physiological conditions in the case of in vivo application or therapeutic treatment (or, in the case of in vitro assays, under conditions in which the assays are conducted).

In vitro hybridization conditions are known in the art and can be found, inter alia, in the re¬ ference given below for Northern blotting or in Maniatis et al., "Molecular Cloning - A Labo¬ ratory Manual", second edition, Cold Spring Harbor Laboratory Press, 1989, vol. 2, 9.47 to 9.58. For example, the respective raf cDNA can be immobilized on nitrocellulose filters. Prehybridization (to reduce nonspecific background binding) is possible as a first step, for example using a mixture comprising 6X SSC (prepared by dilution of 20 X SSC = 175.3 g of NaCl and 88.2 g of sodium citrate in 800 ml of H2O, pH adjusted by a few drops of 10 M NaOH to 7.0, volume adjusted with H2O to 11)/ 5X Denhardt's reagent (prepared by tenfold dilution with 6X SSC of a solution of 5 g of Ficoll (Type 400, Pharmacia, Sweden), 5 g of polyvinylpyrrohdone, 5 g of bovine serum albumin (Pentex Fraction V) and H2O to 500ml)/ 0 5 % sodium dodecylsulfate (= SDS)/ 10 mg denatured, fragmented salmon sperm DNA (Sigma type III sodium salt (10 mg/ml) is dissolved in water, if necessary, stirring with a magnetic stirrer at room temperature; the solution is adjusted to 0 1 M NaCl and extracted once with phenol and once with phenol/chloroform; the aqueous phase is recovered and the DNA is sheared by passing it 12 times rapidly through a 17-gauge hypodermic needle, the DNA is precipitated by adding two volumes of ice-cold ethanol; it is then recovered by centrifugation and redissolved at a concentration of 10 mg/ml in water; the OD260 is deter¬ mined and the exact concentration of the DNA is calculated; the solution is then boiled for 10 minutes and stored at -20 °C in small ahquois; just before use, the solution is heated for 5 mm in a boiling-water bath and then chilled quickly in ice water; denatured, fragmented salmon sperm DNA is preferably used at a concentration of 100 mg/ml hybridization solu¬ tion) and, if desired, 50 % formamide. The prehybridization solution is preferably filtered through a 0.45 micrometer disposable cellulose acetate filter (e.g. Schieicher and Schuell Uniflow syringe filter No. 57240 or equivalent).

The nitrocellulose filter is then floated on the surface of a tray of 6 X SSC until it is tho¬ roughly wetted and is then submerged for 2 mm. The wet filter is slipped in a heat-sealable bag (e.g. Sears Seal-A-Meal or equivalent). 2 ml of prehybridization solution/cm2 filter are added Air is squeezed from the filter, and the open end of the bag is heat-sealed. The bag is incubated for 1 - 2 h at 68 °C (for aqueous solvents) or 42 °C (in the presence of 50 % formamide). The bag containing the filter is removed from the water bath and opened by cutting off one corner with scissors. The labeled probe (e.g. a 32P-labeled nucleotide or preferably nucleotide derivative according to the invention) is added to the prehybridization solution and air is removed by squeezing. The bag is resealed (entrapping as few bubbles as possible). The bag is submerged in water bath at the required temperature for the requi¬ red time of hybridization. Then the bag is opened at a corner After removal of the hybridi¬ zation solution, the filter is removed and submerged in several hundred ml of 2X SSC and 0.5 % SDS at room temperature for some minutes not allowing the filter to dry; this treat¬ ment is repeated with fresh solution. Then the filter is incubated in 0.1 X SSC/0.5 % SDS for 30 to 60 mm at 37°C with gentle agitation. The solution is replaced by a fresh aliquot and in¬ cubation at 68 °C follows for 30 to 60 mm The amount of radioactivity can then be determi¬ ned on X-ray films (e g Kodak XAR-2 or equivalents) to obtain an autoradiographic image Preferably, an ODN is employed which is targeted to human mRNA encoding c-raf (prefer¬ ably corresponding to the sequence given in Bonner et al., Nucl.Acids Res. t4, 1009-1015 (1986)). Persons of ordinary skill in the art will be aware 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 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 preferably be formulated which are targeted wholly or in part to these associated ribonucleotides. In pre¬ ferred embodiments, the oligonucleotide is targeted to a translation initiation site (AUG co¬ don) or sequences in the 5'- or 3'-untranslated region of the human c-raf mRNA. The func¬ tions of messenger RNA to be interfered with include ail vital functions such as translocation of the RNA to the site for protein translation, actual translation of protein from the RNA, spli¬ cing 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. More preferred among the ODNs are those that correspond to the 3'-untranslated region of the human c-raf mRNA.

The term "correspond" means that the given compound has base pairing characteristics comparable to the nucleic acid sequence referred to, that is, comparable hybridization characteristics.

Antisense oligonucleotides or oligonucleotide derivatives for combination according to the invention comprising nucleotide units or analogues/derivatives thereof sufficient in number and identity to allow hybridization preferably have a length that allows specific binding to the target sequence, especially a length corresponding to 5 to 50 nucleotide units, preferably to 10 to 35 nucleotide units, more preferably to 15 to 22 nucleotide units, and most preferably to 18 to 21 nucleotide units.

In order to allow also for the inclusion of allelic variants of the (especiallyhuman) raf, especially c-raf, gene and for hybridizable oligonucleotides or oligonucleotide analogues that show minor numbers of mispairing that still allow hybridization, the sequences can vary from those corresponding to the human c-raf mRNA (preferably corresponding to the sequence given in Bonner et al., Nucl.Acids Res. 14, 1009-1015 (1986)) by some nude- otides or nucleotide analogues; preferably, up to 3 nucleotides or nucleotide analogues can differ in the sequence of a given oligonucleotide or oligonucleotide derivative with respect to the corresponding raf cDNA, more preferably in the sense of conservative mutations.

Preferred is an oligonucleotide or oligonucleotide derivative as published in International Application WO 95/32987, especially in the examples thereof, or a salt thereof.

Most preferred is an oligonucleotide or oligonucleotide derivative that corresponds to the following sequence, or a salt thereof:

5'- TCC CGC CTG TGA CAT GCA TT -3' SEQ.-ID NO: 1.

It is evident that also shortened and/or shifted versions of this sequence are encompassed, especially those which show hybridization as defined above, e.g. derivatives with 15 to 20 of the abovementioned building blocks. "Shortened" means preferably that the sequence is shortened terminally on one or both ends.

Generally, oligonucleotide derivatives, especially oligo-2-'deoxynucleotide derivatives, are preferred over oligonucleotides as such.

The oligonucleotides or oligonucleotide derivatives used in a combination according to the invention can be designed to selectively inhibit a given isozyme or particular set of iso- zymes, or to inhibit all members of a given family of isozymes of human raf, especially human c-raf.

The oligonucleotides or their derivatives can be isomerically pure or they can be present in isomeric mixtures. Thus, if asymmetric phosphorus atoms are present, the compounds can be present as diastereomeric mixtures or as pure diastereomeres.

Some of the oligonucleotides or oligonucleotide derivatives can be present in different tau- tomeric forms, depending inter alia on the solvent and the ioπization status of ionizable groups. Thus, for example, the central group in phosphorothioates [O-(P-SH)(=O)-O] being tautomerizable to [O-(P=S)(-OH)-O] with the more stable form depending, among others, on the solvent and the state of ionization. Within the present specification, the term oligonucle- otide derivatives is also to be understood to encompass these tautomeric forms, the presen¬ ce of which is known to the person skilled in the art.

The prefix "lower" denotes a radical with up to and including 7 carbon atoms, preferably up to and including 4, and most preferably with up to and including 2 carbon atomsl, if not indicated otherwise.

A nucleotide unit is a base-sugar or base-sugar analogue combination suitably bound to an adjacent nucleotide unit through phosphodiester or other bonds.

In the context of this invention, the term "oligonucleotide" refers to an oligonucleotide for¬ med from naturally occurring base radicals and pentofuranosyl (ribosyl or (preferably) 2'-de- oxyribosyl) groups or modified forms thereof joined by native phosphodiester bonds, that is which comprises building blocks of the following formulae I and/or I* wherein Q is H or OH:

Figure imgf000012_0001
wherein B is a radical of a base selected from thymine, uracil, 5-propynyluracil, cytosine, 5- methylcytsine, 5-propynylcytosine, adenine, 2-aminoadenine and guanine. The term "oligonucleotide derivative" refers to synthetic species derived from naturally occurring nucleotide subunits or their close homologues and may also refer to moieties which function similarly to naturally occurring oligonucleotides but which have non-naturally occurring portions, for example at least one building block that differs from the building blocks of a natural oligonucleotide. Thus, oligonucleotides with regard to their backbone may have altered sugar moieties and/or inter-sugar linkages, and, with regard to the bases, altered bases may be present.

Such oligonucleotide derivatives are best described as being functionally interchangeable with natural oligonucleotides (or synthesized oligonucleotides along natural lines), but having one or more differences from natural structure. All such oligonucleotides are com¬ prehended within this invention so long as they function effectively to show the hybridization properties to DNA or RNA deriving from the c-raf gene, preferably to mRNA.

With regard to the backbone, that is to the altered sugar moieties and/or inter-sugar linkages (intemucleosidic bridges), preferred among these are the following types:

- Species derived from naturally occurring nucleotide subunits or their close homologues of formula I or I* wherein B is a base radical as defined below and Q is H, OH, SH, SCH3, F, N3, CN, OCN, OCH3, O(CH2)zNH2 or O(CH2)zCH3 where z is from 1 to about 10, O(CH2CH2O)vCH3 wherein v is from 0 to 12, especially 1 or 3, CH2CH(CH3)OCH3 or CH2CH(OH)CH2OH, or in a broader sense another substituent having similar properties, for example selected from Cl, Br, CF3, ONO2, NO2, NH2 and O-, S- or NH-lower alkyl, most especially Q being hydroxy, F, methoxy, (preferably) 2'-(2-methoxy)ethoxy, or (more prefer¬ ably) hydrogen;

- phosphorothioate and in a broader sense other species such as phosphorodithioate, sul¬ fate, sulfonate, sulfonamide, sulfone, sulfite, sulfoxide, sulfide, formacetal, 3'-thιoformace- tal, 5'-thioether, hydroxylamine (with CH2-NH-O-CH2 instead of the phosphodiester bond O-[(HO-)P(=O)]-O-CH2), methylene(methyiimino) (with CH2-N(CH3)-O-CH3 instead of the phosphodiester bond); methyleneoxy(methylimino) (with CH2-O-N(CH3)-CH2 instead of the phosphodiester bond), methylene-((methylimino)-methylιmino) (with CH2-N(CH3)-N(CH3)- CH2 instead of the phosphodiester bond), carbonate, 5'-N-carbamate, amide (with CH2- (C=O)-NH-CH2 instead of the phosphodiester bond, see International Application WO 92/20823), morpholino-carbamate (see Summerton, J.E. and Weller, D.D., U.S Patent No: 5,034,506) or peptide nucleic acid (see P.E. Nielsen, M. Egholm, R.H. Berg, O. Buchardt, Science 254. 1497 (1991)) which are known for use in the art (for reviews with references concerning these modified nucleotides, see Milligan et al., J. Med. Chem. 36(14), 1923-37 (1993), and Uhlmann et al., Chemical Reviews 90(4), 543-84 (1990)). In accordance with some preferred embodiments, at least one of the phosphodiester bonds of the oligonucleo¬ tide has been substituted with a structure which functions to enhance the ability of the com¬ positions to penetrate into the region of cells where the RNA or DNA whose activity to be modulated is located and in order to avoid extensive degradation of the oligonucleotide de¬ rivative due to nucleases that would result in ineffective cleavage products. It is preferred that such substitutions comprise phosphorothioate bonds, phosphorodithioate bonds, methyl phosphonate bonds, phosphoramidate bonds, amide bonds, boranophosphate bonds, phosphotriester bonds, short chain alkyl or cycloalkyi structures, or heteroatom- substituted short chain alkyl structures, and most especially phosphorothioate bonds or amide bonds.

Preferred of these are oligonucleotide derivatives which (in their nucleotide/nucleotide deri¬ vative sequence) comprise at least one of the following units (bivalent radicals) of the for¬ mulae given hereinafter, wherein B is a base radical as defined below; Q is H, OH, SH, SCH3, F, N3, CN, OCN, OCH3, O(CH2)zNH2 or O(CH2)zCH3 where z is from 1 to about 10, O(CH2CH2O)vCH3 wherein v is from 0 to 12, especially 1 or 3, CH2CH(CH3)OCH3 or CH2CH(OH)CH2OH, or in a broader sense another substituent having similar properties, for example selected from Cl, Br, CF3, ONO2, NO2, NH2 and O-, S- or NH-lower alkyl, most especially Q being hydroxy, methoxy, F or (preferably) -O-CH2CH2OCH3 or (most preferably) hydrogen; and the other moieties have the meanings given behind the respective formula:

(lla - llf)

Figure imgf000014_0001
/

O

X— P=Y

Figure imgf000015_0001

Radical of formula type lla), phosphorothioate X = SH Y = O

Ha*)

Mb), phosphorodithioate X = SH Y = S lib*) lie), methylphosphonate X = CH3 Y = O lie*) lid), phosphoramidate X = NH-R Y = O lid*)

He), boranophosphate X = BH3 Y = O

He*) llf), phosphotriester X = O-R Y = O

(IH*) wherein R is lower alkyl;

Figure imgf000015_0002
Figure imgf000016_0001

Radical of formula type n X' Y Ilia), sulfate 2 O S

O CH2

O NH

NH CH2

CH2 CH2

O O

CH2 CH2

Figure imgf000016_0003
CH2 CH2 llh*)

Figure imgf000016_0002
Figure imgf000017_0001

Radical of formula type X" Y"

(IVa), (IVa*) formacetal O O (IVb), (IVb*) 3'-thioformacetal S O (IVc), (IVc*) 5'-thioformacetal O S (IVd), (IVd*) thioether CH2 S

Figure imgf000017_0002

/

CH,

X*

\

Figure imgf000017_0003

Radical of formula type Y*

(Va), (Va*) hydroxylamine N-H O (Vb), (Vb*) methylene(methyl- N-CH3 O imino)

(Vc), (Vc*) methyleneoxy(methyl- O N-CH3 imino)

Figure imgf000018_0001

/

X** o=c

Figure imgf000018_0002

Figure imgf000018_0003
wherein A is H, methyl or phenyl, preferably H;
Figure imgf000019_0001

Radical of formula type X Y,-

(VII), (VII*) amide III NH CH2

Figure imgf000019_0002
O-

Figure imgf000020_0001

Figure imgf000020_0003

Figure imgf000020_0002

Radical of formula type IX, IX* morpholino-earbamate

Figure imgf000021_0001

(X*)

Figure imgf000021_0002

The oligonucleotide derivatives can be composed of a combination of these units, or they can preferably (i) be chimeric oligonucleotides or (ii) comprise only one type of these units with regard to the backbone (sugar moieties and/or inter-sugar linkages) which is present throughout the chain of the respective oligonucleotide derivative, preferably oligo-2'deoxy- nucleotide derivative, most preferably of the 2'-deoxyribose-phosphorothioate type. At the 5'- and 3'-termini of the respective oligonucleotide derivative molecules, the free valency of the radicals of any of the above formulae I, I*, II to X and II* to X* is bonded preferably to hydrogen if the terminal atom is selected from N, O and S and to hydroxy or an analogue thereof, such as halogen, for example Cl, Br or I, mercapto (SH) or azido (N3), if the terminal atom is C, more preferably to one of the following residues, but may also (in a broader aspect of the invention) be bound to other conjugated moieties as described below forming conjugates. Both groups (i) and (ii) are also preferred independently as separate group.

The "chimeric oligonucleotides" or "chimerae", in the context of this invention, are oligonucleotides which contain two or more chemically distinct regions, each made up of at least one oligonucleotide or oligonucleotide derivative. 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, diminished probability for sequence independent side effects), the so-called wing, and a region that premits RNase H mediated cleavage of the target complement, the so-called RNase H-window. In one embodiment, a chimeric oligonucleotide comprises at least one region modified to increase target binding affinity and, usually, a region that permits RNase H mediated cleavage of the target com¬ plement. Affinity of an oligonucleotide or an oligonucleotide derivative for its target is rou¬ tinely determined by measuring the Tm of an oligonucleotide/target pair, which is the tempe¬ rature at which the oligonucleotide or its derivative and the target dissociate. Dissociation is detected spectrophotometrically. The higher the Tm, the greater the affinity of the oligonu¬ cleotide for the target. Methods for Tm measurement are known in the art (see, e.g., Sambrook, Fritsch and Maniatis, "Molecular Cloning - A Laboratory Manual", 2nd edition, Cold Spring Harbor Laboratory Press, 1989). Such modified regions M are routinely in¬ corporated into oligonucleotides and these oligonucleotides have been shown to have a higher Tm (i.e., a higher target binding affinity) than 2'-deoxyoligonucleotides against a given target. The effect of such increased affinity is to greatly enhance antisense oligo¬ nucleotide 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 can thus greatly enhance the efficiency of antisense inhibition. Cleavage of the RNA target can be routinely demonstrated by gel electropho- resis. In another 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 incorporated more resistant to nuclease digestion than the native oligodeoxynucleotide. Nuclease resistance is routinely measured by incubating oligonucleotides or derivatives thereof with cellular extracts or isolated nu¬ clease 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. In some cases, oligonucleotide modifications which enhance target binding affinity are also, independently, able to enhance nuclease resistance. Especially preferred is the 2'-O-CH2CH2OCH3 (2'-(2-methoxy)ethoxy) modification or an F at the 2' position of at least one oligonucleotide. This modification has been shown to increase both the affinity for its target and nuclease resistance of the oligonucleotide. Chimeric oligonucleotides preferably show, on the one hand, M regions of between 3 and 20 (preferably 5 to 15) modified building blocks (either with phosphodiester or with phos¬ phorothioate backbone), especially with 2'-modification, especially 2'-(2-methoxy)ethoxy or 2'-F modified building blocks, which are in succeeding order; and, on the other hand, U regions of 4 to 20 2'-deoxy building blocks with phosphorothioate structure that are otherwise unmodified. Preferably, the M and U regions can be in one of the following arrangements:

5'-M-U--M-3'

5'-M-U-3' or

5'-U-M-3'.

In compounds with a terminal moiety of any one of the formulae I, Ha - llh, Ilia, lllc, lllf, IVa - IVd, Va - Vc, Via - Vlc, IX and X*, the 5' terminus is preferably bonded to a terminal OH group, and the 3'-terminus to a hydrogen.

In compounds with a terminal moiety of any one of the formulae I*, Ha* - llh*, Ilia* - lllh*, IVa* - IVc*, Va* - Vc*, Via* - Vld, VII*, VIII* and X, the 5'-terminus is preferably bonded to a terminal hydrogen, and the 3'-terminus to a OH group.

In compounds with a terminal moiety of the formula Vie, both the 5' and the 3' terminus are preferably bonded to a terminal hydrogen.

In compounds with a terminal moiety of any one of the formulae IX*, the 5'-terminus is pre¬ ferably bonded to a terminal OH group which is bonded replacing the terminal -(C=O)-O, and the 3'-terminus to a hydrogen atom.

In compounds with a terminal moiety of any one of the formulae lllb, Hid, llle, llle*, lllg, lllg*, lllh, lllh*, IVd*, Vld, VII, VIII and VIII*, the 5'-terminus is preferably bonded to a terminal OH group and the 3'-terminus is preferably bonded to an OH group.

In order to allow for modified and improved pharmacokinetic properties, such as enhanced uptake into cells or the oligonucleotides or oligonucleotide derivatives in combinations ac- cording to the invention can also be conjugated to one or more (then identical or different) additional moieties, for example selected from: A group forming micelles, an antibody, a carbohydrate, a receptor-binding group, a steroid, such as cholesterol, a polypeptide, an intercalating agent, such as an acridine derivative, a long-chain alcohol, a phospholipid and other lipophilic groups.

The very most preferred of the oligonucleotide (preferably oligo-2'-deoxyoligonucleotide) derivatives are those of the phosphorothioate type.

B in any of the formulae (Ii) to (Xi) and (Ii*) to (Xi*) ("i" standing for the respective indices in the formulae above or below, such as, for example, "a", "b" or no index if none is required) is a base radical and is selected from the group comprising a purine radical or an analogue thereof and a pyrimidine radical or an analogue thereof.

If B is a purine radical or an analogue thereof, it may be a radical of formula XI, Xla, Xlb, Xlc, Xld, Xle or Xlf

Figure imgf000024_0001
Figure imgf000025_0001
wherein

Rb1 is H, Cl, Br, OH or -O-d-Cι_alkyl, and b2, R 3 and Rb5 are each independently of the others H, OH, SH, NH2, NHNH2, NHOH,

NHO-Cι-C12alkyl, -N=CH-N(C C12alkyl)2, F, Cl, Br, d-dzalkyl, hydroxy-C,-C12alkyl, amino- d-C12alkyl, Cι-C,2alkoxy, benzyloxy or Cι-Ci2alkylthio, the hydroxy and amino groups being present as such or substituted by a protecting group; or phenyl, benzyl, primary amino having from 1 to 20 carbon atoms or secondary amino having from 2 to 30 carbon atoms,

Rb is hydrogen, CN or -C-C-Rb7, and

Rbe and Rb7 are hydrogen or d-C4alkyl.

Protecting groups and processes for derivatising amino groups (as well as imino groups, "amino" groups in the following paragraphs that refer to amino protecting groups also, if possible, meaning imino) having such protecting groups are generally known in sugar, amino acid and nucleotide chemistry and are described, for example, in standard text books (see J. F. W. McOmie, "Protective Groups in Organic Chemistry", Plenum Press, London and New York 1973; Th. W. Greene, "Protective Groups in Organic Synthesis", Wiley, New York 1981 , in "The Peptides", Volume 3 (E. Gross and J. Meienhofer, eds.), Academic Press, London and New York 1981 ; Greene, B.T., "Protective Groups in Organic Synthesis", Wiley Interscience, New York (1991), Sonveaux, E., Bioorganic Chemistry 14, 274-325 (1986), Beaucage, S.L., et al., Tetrahedron 48, 222, "Methoden der organischen Chemie", Houben-Weyl, 4th edition, Volume 15/1, Georg Thieme Verlag, Stuttgart 1974; and H.-D. Jakubke and H. Jescheit, "Aminosauren, Peptide, Proteine" ("Amino acids, peptides, proteins"), Verlag Chemie, Weinheim, Deerfield Beach and Basle 1982). Examples of such protecting groups are: benzyl, methylbenzyl, dimethylbenzyl, methoxybenzyl, dimethoxy- benzyl, bromobenzyl, 2,4-dichlorobenzyl; diphenylmethyl, di(methylphenyl)methyl, di(di- methylphenyl)methyl, di(methoxyphenyl)methyl, di(dimethoxyphenyl)methyl, triphenylmethyl, tris-4,4',4"-tert-butylphenylmethyl, di-p-anisylphenylmethyl, tri(methylphenyl)methyl, tri (dimethyl phenyl) methyl, methoxyphenyl(diphenyl)methyl, di(methoxyphenyl)phenylmethyl, tri(methoxyphenyl)methyl, tri(dimethoxyphenyl)methyl; triphenylsilyl, alkyldiphenylsilyl, di- alkylphenylsilyl and trialkylsilyl having from 1 to 20, preferably from 1 to 12, and especially from 1 to 8, carbon atoms in the alkyl groups, for example trimethylsilyl, triethylsilyl, tri-n- propylsilyl, isopropyl-dimethylsilyt, tert-butyl-dimethylsilyl, tert-butyl-diphenylsilyl, n-octyl- dimethylsilyl, (1 ,1 ,2,2-tetramethylethyl)-dimethylsilyl; -(d-CaalkyOzSi-O-S Ci-Cβalky r, wherein alkyl is, for example, methyl, ethyl, n- and iso-propyl or n-, iso- or tert-butyl; C2-C12. acyl, especially C2-Cβacyl, such as acetyl, propanoyl, butanoyl, pentanoyl, hexanoyl, ben- zoyl, methylbenzoyl, methoxybenzoyl, chlorobenzoyl and bromobenzoyl; RSι-SO2- wherein Rsι is d-C12alkyl, especially C C6alkyl, C5- or C6-cycloalkyl, phenyl, benzyl, C C12alkyl- phenyl, especially Cι-C4alkyl-phenyl, or d-C12alkyl-benzyl, especially Cι-C alkyl-benzyl, or halophenyl or halobenzyl, for example methyl-, ethyl-, propyl-, butyl-, phenyl-, benzyl-, p- bromo-, p-methoxy- and p-methylphenyl-sulfonyl; d-d2alkoxycarbonyl, preferably CrC8. alkoxycarbonyl, that is unsubstituted or substituted by F, Cl, Br, Cι-C4alkoxy, tri(d-C4alkyl)- silyl or by C -C4alkylsulfonyl, for example methoxy-, ethoxy-, n- or iso-propoxy- or n-, iso- or tert-butoxycarbonyl, 2-trimethylsilylethoxycarbonyl, 2-methyisulfonylethoxycarbonyl, allyioxy- carbonyl, or phenoxycarbonyl or benzyloxycarbonyl that is unsubstituted or is substituted as for alkoxycarbonyl, for example methyl- or methoxy- or chloro-phenoxycarbonyl or methyl- or methoxy- or chloro-benzyloxycarbonyl, and 9-fluorenylmethoxycarbonyl. If a hydroxy protecting group is alkyl, this moiety may be substituted by fluorine, chlorine, bromine, d- dalkoxy, phenoxy, chlorophenoxy, methoxyphenoxy, benzyloxy, methoxybenzyloxy or by chlorophenoxy. If more than one hydroxy group is protected in the respective oligonucleotide or its derivative, the protecting groups may be identical or different.

A protected amino group may be protected, for example, in the form of an acylamino, aryl- methylamino, etherified mercaptoamino, 2-acyl-lower alk-1-enylamino, silylamino or N-lower alkylpyrrolidinylidene group or in the form of an azido group.

In a corresponding acylamino group, acyl is, for example, the acyl radical of an organic carboxylic acid having, for example, up to 18 carbon atoms, especially an unsubstituted or substituted, for example halo- or aryl-substituted, lower alkanecarboxylic acid or an unsub¬ stituted or substituted, for example halo-, lower alkoxy- or nitro-substituted, benzoic acid, or, preferably, of a carbonic acid semiester. Such acyl groups are, for example, lower alkanoyl, such as formyl, acetyl, propionyl, isobutyryl or oivaloyl, halo-lower alkanoyl, for example 2- haloacetyl, such as 2-chloro-, 2-bromo-, 2-iodo-, 2,2,2-trifluoro- or 2,2,2-trichloro-acetyl, phenyoxy- or (lower alkoxy)phenoxy-lower alkyl, such as phenoxyacetyl or 4-tert- butylphenoxyacetyl, unsubstituted or substituted, for example halo-, lower alkoxy- or nitro- substituted, benzoyl, such as benzoyl, 4-chlorobenzoyl, 4-methoxybenzoyl or 4-nitro- benzoyl, lower alkoxycarbonyl, preferably lower alkoxycarbonyl that is branched in the 1- position of the lower alkyl radical or suitably substituted in the 1- or 2-position, for example tert-lower alkoxycarbonyl, such as tert-butoxycarbonyl, arylmethoxycarbonyl having one, two or three aryl radicals which are phenyl that is unsubstituted or mono- or poly-substitu¬ ted, for example, by lower alkyl, especially tert-lower alkyl, such as tert-butyl, lower alkoxy, such as methoxy, hydroxy, halogen, such as chlorine, and/or by nitro, for example benzyl- oxycarbonyl, 4-nitrobenzyloxycarbonyl, diphenylmethoxycarbonyl, 9-fluorenylmethoxy- carbonyl or di(4-methoxyphenyl)methoxycarbonyl, aroylmethoxycarbonyl wherein the aroyl group is preferably benzoyl that is unsubstituted or substituted, for example, by halogen, such as bromine, for example phenacyloxycarbonyl, 2-halo-lower alkoxycarbonyl, for exam¬ ple 2,2,2-trichloroethoxycarbonyl, 2-bromoethoxycarbonyl or 2-iodoethoxycarbonyl, 2-(tri- substituted silyl)-lower alkoxycarbonyl, for example 2-tri-lower alkylsilyl-lower alkoxycar¬ bonyl, such as 2-trimethylsilylethoxycarbonyl or 2-(di-n-butyl-methyl-silyl)-ethoxycarbonyl, triarylsilyl-lower alkoxycarbonyl, for example 2-triphenylsilylethoxycarbonyl, or N,N-di-iower alkylformamidinyl, such as N,N-dimethylformamidinyl.

In an arylmethylamino group, for example a mono-, di- or especially tri-arylmethyiamino group, the aryl radicals are especially unsubstituted or substituted phenyl radicals. Such groups are, for example, benzyl-, diphenylmethyl- or especially trityl-amino.

In an etherified mercaptoamino group the mercapto group is especially in the form of sub¬ stituted arylthio or aryl-lower alkylthio, wherein aryl is, for example, phenyl that is unsubsti¬ tuted or substituted, for example, by lower alkyl, such as methyl or tert-butyl, lower alkoxy, such as methoxy, halogen, such as chlorine, and/or by nitro, for example 4-nitrophenylthio.

In a 2-acyl-lower alk-1-enyl radical that can be used as an amino-protecting group, acyl is, for example, the corresponding radical of a lower alkanecarboxylic acid, of a benzoic acid that is unsubstituted or substituted, for example, by lower alkyl, such as methyl or tert-butyl, lower alkoxy, such as methoxy, halogen, such as chlorine, and/or by nitro, or especially of a carbonic acid semiester, such as a carbonic acid lower alkyl semiester. Corresponding pro¬ tecting groups are especially 1 -lower alkanoyl-iower alk-1 -en-2-yl, for example 1 -lower alkanoylprop-1 -en-2-yl, such as 1 -acetylprop-1 -en-2-yl, or lower alkoxycarbonyl-lower alk-1- en-2-yl, for example lower alkoxycarbonylprop-1 -en-2-yl, such as 1-ethoxycarbonylprop-1 - en-2-yl.

A silylamino group is, for example, a tri-lower alkylsilylamino group, for example trimethyl- silylamino or tert-butyl-dimethylsilylamino. The silicon atom of the silylamino group can also be substituted by only two lower alkyl groups, for example methyl groups, and by the amino group or carboxy group of a second molecule of a compound according to the invention. Compounds having such protecting groups can be prepared, for example, using the corres¬ ponding chlorosilanes, such as dimethylchlorosilane, as silylating agents.

An N-lower alkylpyrrolidinylidene group is preferably N-methylpyrrolidin-2-ylidene.

Preferred amino-protecting groups are lower alkoxycarbonyl, phenyl-lower alkoxycarbonyl, fluorenyl-lower alkoxycarbonyl, 2-lower alkanoyl-lower alk-1 -en-2-yl and lower alkoxycar¬ bonyl-lower alk-1-en-2-yl, with most preference being given to isobutyryl, benzoyl, phenoxy- acetyl, 4-tert-butylphenoxyacetyl, N,N-dimethylformamidinyl and/or N-methylpyrrolidin-2-yli- dene.

Primary amino (for example in the definition of R 2, Rb3 and Rb5) contains preferably from 1 to 12, and especially from 1 to 6, carbon atoms, and secondary amino (for example in the definition of Rb2, Rb3 and Rb5) contains preferably from 2 to 12, and especially from 2 to 6, carbon atoms.

Some examples of alkyl, alkoxy, alkylthio, hydroxyalkyi and aminoalkyi containing preferably from 1 to 6 carbon atoms are methyl, ethyl and the isomers of propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl and dodecyl, and corresponding alkoxy, alkylthio, hydroxyalkyi and aminoalkyi radicals. Alkyl, alkoxy, alkylthio, hydroxyalkyi and aminoalkyi contain especially from 1 to 4 carbon atoms. Preferred alkyl, alkoxy, alkylthio, hydroxyalkyi and aminoalkyi radicals are methyl, ethyl, n- and iso-propyl, n-, iso- and tert-butyl, methoxy, ethoxy, methylthio and ethylthio, aminomethyl, aminoethyl, hydroxymethyl and hydroxyethyl.

The primary amino and the secondary amino may be, for example, radicals of the formula F.aiR__.N, wherein Ra is hydrogen or, independently, has the definition of Ra2, and Ra2 is d- C20-, preferably Cι-C12- and especially d-C6-alkyl, Cι-C2o-, preferably Cι-C12- and especially d-C6-aminoalkyl, Cι-C2o-, preferably Cι-C12- and especially Ci-Cβ-hydroxyalkyI; carboxyalkyi or carbalkoxyalkyl, the carbalkoxy group containing from 2 to 8 carbon atoms and the alkyl group from 1 to 6, preferably from 1 to 4, carbon atoms; C2-do-, preferably C2-Cι2- and especially C2-C6-alkenyl; phenyl, mono- or di-(d-C4alkyl or d-C alkoxy)phenyl, benzyl, mono- or di-(d-C4alkyl or d-C alkoxy) benzyl; or 1 ,2-, 1 ,3- or 1 ,4-imidazolyl-d-C6alkyl, or Ra1 and Ra2 together are tetra- or penta-methylene, 3-oxa-1 ,5-pentylene, -CH2-NRa3-CH2- CH2- or -CH2CH2-NRa3-CH2CH2-, wherein Ra3 is hydrogen or d-C4alkyl. The amino group in aminoalkyi may be substituted by one or two d-C4alkyl or d-C4hydroxyalkyl groups. The hydroxy group in hydroxyalkyi may be etherified by d-C4alkyl.

Examples of alkyl are given hereinbefore. Examples of aminoalkyi are aminomethyl, amino¬ ethyl, 1 -aminoprop-2-yl or -3-yl, 1 -amino-but-2-yl or -3-yl or -4-yl, N-methyl- or N,N-dimethyl- or N-ethyl- or N,N-diethyl- or N-2-hydroxyethyl- or N,N-di-2-hydroxyethyl-aminomethyl or - aminoethyl or -aminopropyl or -aminobutyl. Examples of hydroxyalkyi are hydroxymethyl, 1- hydroxy-eth-2-yl, 1 -hydroxy- prop-2- or -3-yl and 1-hydroxy-but-2-yl, -3-yl or -4-yl. Examples of carboxyalkyi are carboxymethyl, carboxyethyl, carboxypropyl and carboxybutyl, and ex¬ amples of carbalkoxyalkyl are those carboxyalkyi groups esterified by methyl or by ethyl. Examples of alkenyl are allyl, but-1 -en-3-yl or -4-yl, pent-3- or -4-en-1 -yl or -2-yl, hex-3- or - 4- or -5-en-1-yl or -2-yl. Examples of alkyl- and alkoxy-phenyl and alkyl- and alkoxy-benzyl are methylphenyl, dimethylphenyl, ethylphenyl, diethylphenyl, methylbenzyl, dimethylbenzyl, ethylbenzyl, diethylbenzyl, methoxyphenyl, dimethoxyphenyl, ethoxyphenyl, diethoxyphenyl, methoxy benzyl, dimethoxybenzyl, ethoxybenzyl and diethoxybenzyl. Examples of imidazol- ylalkyl in which the alkyl group preferably contains from 2 to 4 carbon atoms are 1 ,2-, 1 ,3- or 1 ,4-imidazolyl-ethyl or -n-propyl or -n-butyl. Ra3 is preferably hydrogen, methyl or ethyl.

Preferred examples of primary amino and secondary amino are methyl-, ethyl-, dimethyl-, diethyl-, allyl-, mono- or di-(1 -hydroxy-eth-2-yl)-, phenyl- and benzyl-amino, acetylamino, isobutyrylamino and/or benzoylamino.

In a preferred form, Rbι is hydrogen. In another preferred form, Rb5 is hydrogen. In a further preferred form, Rb2 and Rb3 are each independently of the other H, F, Cl, Br, OH, SH, NH2, NHOH, NHNH2, methylamino, dimethylamino, benzoylamino, isobutyrylamino, methoxy, ethoxy and methylthio. Some examples of analogues of the purine series are, in addition to purine, xanthine, hypo- xanthine, adenine, N-methyladenine, N-benzoyladenine, 2-methylthioadenine, 2-aminoade- nine, 6-hydroxypurine, 2-amino-6-chloropurine, 2-amino-6-methylthiopurine, guanine, N-iso- butyrylguanine. Especially preferred are adenine and guanine, and in a broader aspect of the invention 2-aminoadenine, or in a broader aspect of the invention the base-protected derivatives thereof.

If B in any one of formulae (Ii) to (Xi) and (Ii*) to (Xi*) is a pyrimidine radical or an analogue thereof, it is preferably a uracil, more preferably thymine or cytosine radical or an analogue thereof of formula XII, Xlla, Xllb or Xllc

Figure imgf000030_0001
wherein Rb6 is hydrogen or d-C4alkyl and R is H, OH, SH, NH2, NHNH2, NHOH, NHO-d- C12alkyl, -N=CH-N(d-C12alkyl)2, F, Cl, Br, d-C12alkyl, hydroxy-d-C12alkyl, amino-C C12. alkyl, Cι-C12alkoxy, benzyloxy or d-Cι2alkylthio, the hydroxy and amino groups being un¬ substituted or substituted by a protecting group, or is phenyl, benzyl, primary amino having from 1 to 20 carbon atoms, secondary amino having from 2 to 30 carbon atoms, C C12al- kenyl or Cι-C12alkynyl, and the NH2 group in formulaXllb is unsubstituted or substituted by d-Cβalkyl, benzoyl or by a protecting group, and the dihydro derivatives of the radicals of formulae XII, Xlla, Xllb and Xllc. R B in formula XII is preferably hydrogen, d-C6alkyl or Ci- C6hydroxyalkyl, C2-C6alkenyl or C2-C6alkynyl, F, Cl, Br, NH2, benzoylamino or mono- or di- Cι-C6alkylamino. Rb8 in formulae Xllb and Xllc is preferably hydrogen, d-C6alkyl or d-C6- alkoxy or Cι-C6hydroxyalkyl, C2-C6alkenyl or C2-C6alkynyl, F, Cl, Br, NH2, benzoylamino or mono- or di-Cι-C6alkylamino.

Rb6 is preferably hydrogen or methyl. Rb8 in formula XII is preferably H, F, Cl, Br, NH2, NHCH3, N(CH3)2, d-C4alkyl, C2-C4alkenyl or C2-C4alkyn-1-yl. Rω in formula Xllb and Xllc is preferably hydrogen, Cι-C4alkyl, especially methyl, C2-C4alkenyl, especially vinyl, or C2-C - alkyn-1-yl, especially 1-propyn-1-yl, or NH2, NHCH3 or (CH3)2N.

Some examples of pyrimidine analogues are uracil, thymine, cytosine, 5-fluorouracil, 5- chlorouracil, 5-bromouracil, dihydrouracil, 5-methylcytosine, 5-propynylthymine, 5- propynyluracil and 5-propynylcytosine, thymine, cytosine and 5-methylcytosine being most preferred.

Preferred are oligonucleotide derivatives which (in their nucleotide/nucleotide derivative sequence) comprise at least one of the following units (bivalent radicals) of the formulae given hereinafter, wherein B is a base radical as defined above, preferably of a base selec¬ ted from thymine, uracil, 5-propynyluracil, cytosine, 5-methylcytosine, 5-propynylcytosine, adenine, 2-aminoadenine and guanine; Q is H, OCH3, F, O(CH2CH2O)vCH3 wherein v is from 0 to 12, especially 1 or 3, CH2CH(CH3)OCH3 or CH2CH(OH)CH2OH, or less preferably SH; and the other moieties have the meanings given behind the respective formula:

Figure imgf000031_0001
Figure imgf000032_0001

Figure imgf000032_0002

(the phosphodiesters llh or llh* being especially preferred), especially 2'-F derivatives, 2 - deoxy derivatives (Q = H) or 2'-(2-methoxy)ethoxy (2'-O-CH2CH2OCH3) derivatives, the remaining internucleosidic bonds in the respective oligonucleotide derivative being of the phosphorothioate type; for synthesis see European Application EP 0 626 387 and International Application WO 92/20823; or

oligonucleotide derivatives which comprise at least one of the following dimeric units (bivalent radicals) of the formulae given hereinafter, wherein each B is, independently of the other, a base radical as defined above, preferably of a base selected from thymine, uracil, 5-propynyluracil, cytosine, 5-methylcytosine, 5-propynylcytosine, adenine, 2-aminoadenine and guanine; any Q is, indepently of the other, H, OCH3, F or O(CH2CH2O)vCH3 wherein v is from 0 to 12, especially 1 ; and the other moieties have the meanings given behind the respective formula:

Figure imgf000033_0001

X** Y**

Figure imgf000033_0002
CH2 N(A)

wherein A is H, methyl or phenyl, preferably H; the remaining internucleosidic bonds in the respective oligonucleotide derivative being of the phosphorothioate type; for synthesis see International Application WO 95/20597and International Application WO 92/20823;

or especially oligonucleotide derivatives, preferably oligo-2'-deoxynucleotide derivatives, where all internucleosidic bridges as of the phosphorothioate type and that have no sugar or base modification.

Preferred are also chimeric oligonucleotides which show, on the one hand, M regions of between 3 and 20 (preferably 5 to 15) modified building blocks (either with phosphodiester or with phosphorothioate backbone), especially with 2'-modification, especially 2'-(2-metho- xy)ethoxy or 2'-F modified building blocks, which are in succeeding order; and, on the other hand, U regions of 4 to 20 2'-deoxy building blocks with phosphorothioate structure that are otherwise unmodified; preferably, the M and U regions can be in one of the following arrangements: 5'-M--U~M-3'

5'-M-U-3' or

5'-U--M-3'.

These chimeric oligonucleotides may be an own preferred group, as may be the other mentioned oligonucleotide derivatives.

Any -SH or -OH group bound to phosphorus, e.g. in phosphorothioate or phosphodiester internucleosidic bridges, is preferably present mainly in the ionized form under e.g. physiological conditions, that is as -O('' or -S( ).

By the term "other chemotherapeutic agent" there is meant any chemotherapeutic agent that is or can be used in the treatment of tumor diseases, such as chemotherapeutics derived from the following classes:

(A) Alkylating agents, such as dacarbazine (DTIC-Dome); nitrogen mustards, such as mechlorethamine (Mustargen); ethyleneimine derivatives, e.g. triethylenethiophosphoramid (thio-tepa); procarbazine (Matulane); alkyl sulfonates such as busulfan (Myeleran); cyclo- phosphamide; 4-hydroxyperoxycyclophosphamide (4-HC); mafosfamide; ifosfamide; mel- phalan (Alkeran); chlorambucil (Leukeran); nitrosoureas such as cyclohexylnitrosourea (meCCNU; Carmustine, BCNU, BiCNU) or lomustine (CCNU, CeeNU), cis-platinum(ll)-di- aminedichloride (platinol or cisplatin); carboplatin (Paraplatin); preferably cross-linking che¬ motherapeutics, preferably bis-alkylating agents, especially nitrogen mustards, such as mechlorethamine (Mustargen); alkyl sulfonates such as busulfan (Myeleran); cyclophos- phamide; melphalan (Alkeran); chlorambucil (Leukeran); cis-platinum(ll)-diaminedichloride (platinol or cisplatin) or carboplatin (Paraplatin); or compounds that form cross-links via ionic bonds, such as ethyleneimine derivatives, e.g. triethylenethiophosphoramid (thio-tepa) (forms ionic cross-links);

(B) antitumor antibiotics, preferably selected from the group comprising bleomycine (Blenoxane); anthracyclines, such as daunomycin, dactinomycin (Cosmegen), daunorubicin (Cerubidine), doxorubicin (Adriamycin, Rubex), epirubicin, esorubicin, idarubicin (Idamycin), plicamycin (Mithracin, formerly called Mithramycin) and preferably cross-linking (bis-alkyla¬ ting) antitumor antibiotics, such as mitomycin C (Mitomycin, Mutamycin); (C) antimetabolites, for example folic acid analogues such as methotrexate (Folex, Mexate) or trimetrexate; purine nucleoside analogues such as Cladribine (Leustatin; 2-chloro-2'-de- oxy-β-D-adenosine), 6-mercaptopurine (Mercaptopurine, Purinethol, 6-MP), pentostatin (Nipent) or 6-thioguanine (6-TG, Tabloid); pyrimidine analogues such as 5-fluorouracil (Fluoruracil, 5-FU), 5-fluorodeoxyuridine (Floxuridine, FUDR), cytosine arabinoside (ara-C, cytarabine, Cytosar-U or Tarabine PFS), fludarabin phosphate (Fludara) or 5-azacytidine; hydroxyurea (Hydrea); or polyamine biosynthesis inhibitors, especially ornithine decarboxy- lase inhibitors or S-adenosylmethionine decarboxylase inhibitors, for example those men¬ tioned in EP 0 456 133, especially 4-amidino-1 -indanon-2'-amidinohydrazone;

(D) plant alkaloids, preferably vinca alkaloids, such as vinblastine (Velban), vincristine (Oncovin) or vindesine; epipodophyllotoxins, such as etoposide (VP-16, VePesid) or teniposide (VM-26, Vumon);

(E) hormonal agents and antagonists, preferably adrenocorticoids, such as prednisone (Deltasone) or dexamethasone (Decadron); progestines such as hydroxyprogesterone (Prodox), megestrol acetate (Megace) or medroxyprogesterone (Provera, Depo-Provera); androgens such as testosterone or fluoxymesterone (Halotestin); estrogens such as di- ethylstilbestrol (DES), estradiol or chlorotriansiene (Tace); synthetic analogues of LHRH, such as goserelin (Zoladex); synthetic analogues of LH-releasing hormone, such as leu- prolide (Lupron, Lupron Depot); anti-androgens such as flutamide (Eulexin); anti-estrogens such as tamoxifen; aromatase inhibitors such as aminogluthetimide (Cytadren), lentaron (Formestane, 4-hydroxy-4-androsten-3,17-dione) (see EP 0 162 510), fadrozole (5-(p-cya- nophenyl)-5,6,7,8-tetrahydroimidazo[1 ,5-a]pyridin, see EP 0 437 415 and EP 0 165 904), letrozole (4,4'-(1 H-1 ,2,4-triazol-1-yl-methylen)-bis-benzonitrile, see US 4,976,672), 4-(α-(4- cyanophenyl)-α-fiuoro-1-(1 ,2,4-triazolyl)methyl)-benzonitrile (see EP 0 490 816) or 4-(α-(4- cyanophenyl)-(2-tetrazolyl)methyl)-benzonitrile (see EP 0 408 509); adrenal cyctooxic agents, such as mitotane (Lysodren); somatostatine analogues, such as octreotide (Sando- statin); or 5α-reductase inhibitors, such as N-(1-cyano-1 -methyl-ethyl)-4-aza-3-oxo-5α-an- drost-1 -en-17β-carboxamid (see EP 0 538 192); (F) biological response modifiers, preferably lymphokines, such as aldesleukiπ (human re¬ combinant IL-2, Proleukin); or interferons, such as interferon-α (Intron-A, Roferon) or interferon "B1B2B3D4" (see EP 0 205 404);

(G) inhibitors of protein tyrosine kinases and/or serine/threonine kinases other than ODNs, such as N-{5-[4-methyl-piperazino-methyl)-benzoylamido]-2-methyl-phenyl}-4-(3-pyridyl)-2- pyrimidine (see EP 0 546 409), N-(3-chlorophenyl)-4-(2-(3-hydroxy)-propyl-amino-4-pyridyl)- 2-pyrimidinamin (see EP 0 606 046), N-beπzoyl-staurosporine (see EP 0 296 110), 4,5-bis- (anilino)-phthalimide (see EP 0 516 588), N-(5-benzoylamido-2-methyl-phenyl)-4-(3-pyridyl)- 2-pyridinamin (see EP 0 564409) or 4-(m-chloranilino)-5,6-dimethyl-7H-pyrrolo[2,3-d]pyri- midin (see EP 0 682 027);

(H) antisense oligonucleotides or oligonucleotide derivatives targeted to other targets than raf, such as those targeted to SAMDC (PCT application WO 96/05298) or protein kinase C ( = PKC) (International Application WO 93/19203 or WO 95/02069), especially a PKC- targeted oligonucleotide or oligonucleotide derivative (preferably of the types described as being preferred for the ODN of sequence SEQ-ID NO: 1) having the sequence 5'-GTT CTC GCT GGT GAG TTT CA-3' (SEQ-ID NO: 2); or

(I) miscellaneous agents or agents with other or unknown mechanism of action, preferably S-triazine derivatives, such as altrematine (Hexalen); enzymes, such as asparaginase (Elspar); methylhydrazine derivatives, such as dacarbazine and procarbazine; matrix metalloproteinase inhibitors, such as (Case 4-19421) hexamethylmelamine, pentamethyl- melamine; anthraquinones such as mitoxantrone (Novantrone); mitotic spindle poisons such as paclitaxel (Taxol); streptozocin (Zanosar); estracyt (estramustine); amsacrine; differentiating agents, such as all-trans retinoic acid (TRA); immunomodulators, such as levamisole (ergamisol); vaccines, e.g. anti-melanoma vaccines (see EP 0674 097); or antibodies with antitumor activity, such as recombinant human immunoglobulins directed at melanoma antigen (see EP 0 640 131) or antibodies for active immunotherapy of melanoma (see EP 0 428 485).

More preferred is any of the above-mentioned chemotherapeutic agents except for oligo¬ nucleotide derivative targeted at Protein kinase C (PKC), adriamycin (doxorubicin) and cyclophosphamide, preferably alone, or more preferably alone or in any combination. Especially preferred are the chemotherapeutic agents mentioned above under (A) as cross- linking chemotherapeutics, preferably bis-alkylating agents, especially nitrogen mustards, such as mechlorethamine (Mustargen); alkyl sulfonates such as busulfan (Myeleran); cyclo- phosphamide; melphalan (Alkeran); chlorambucil (Leukeran); cis-platinum(ll)-diaminedichlo- ride (platinol or cisplatin) or carboplatin (Paraplatin); or compounds that form cross-links via ionic bonds, such as ethyleneimine derivatives, e.g. triethylenethiophosphoramid (thio-tepa) (forms ionic cross-links); or chemotherapeutic agents mentioned under (B) as cross-linking (bis-alkylating) antitumor antibiotics, such as mitomycin C (Mitomycin, Mutamycin).

Especially preferred chemotherapeutics are also purine nucleoside analogues such as Cladribine (Leustatin; 2-chloro-2'-deoxy-β-D-adenosine), 6-mercaptopurine (Mercapto- purine, Purinethol, 6-MP), pentostatin (Nipent) or 6-thioguanine (6-TG, Tabloid); or pyri- midine analogues such as 5-fluorouracil (Fluoruracil, 5-FU), 5-fluorodeoxyuridine (Flox- uridine, FUDR), cytosine arabinoside (ara-C, cytarabine, Cytosar-U or Tarabine PFS), fludarabin phosphate (Fludara) or 5-azacytidine.

Preferably, the term "other chemotherapeutic agent" relates to a standard chemotherapeutic agent as mentioned before that is already used clinically, or in a less preferred sense aiso to a chemotherapeutic agent that is already being tested clinically.

Most preferred is one or more of the chemotherapeutics selected from the group comprising ifosfamide, cisplatin, mitomycine and 5-fluorouracil, especially one of ifosfamide, cisplatin and mitomycine.

By the term "proliferative disease that can be trated by administration of an oligonucleotide or oligonucleotide derivative targeted to raf there is preferably meant any disease that responds to such compounds; especially, by the term "where the disease responds to modulation of raf activity" there is preferably meant a proliferative disease selected from hyperproliferative conditions such as cancers, tumors, hyperplasias, fibrosis (especially pulmonary, but also other types of fibrosis, such as renal fibrosis), angiogenesis, psoriasis, atherosclerosis and smooth muscle cell proliferation in the blood vessels, such as stenosis or restenosis following angioplasty. Most preferably, the disease is one selected from cancer types which have been very difficult to treat or even practically unaffected by therapy with standard chemotherapeutics, such as small cell lung carcinoma, large cell lung carcinoma, melanoma, prostate carcinoma or further also lymphoma. Most preferably, any of the aforementioned proliferative diseases that can be treated by administration of an oligunucleotide or oligonucleotide derivative directed against human c-raf, especially by inhibition of human c-raf activity, is meant.

By the term "quantity which is jointly therapeutically effective against proliferative diseases that can be treated by an oligonucleotide or oligonucleotide derivative targeted to raf, especially c-raf, or that preferably depend on raf, especially c-raf, activity" there is prefer¬ ably meant any quantity of the components of the combinations that, in the combination, is diminishing proliferation of cells responsible for any of the mentioned proliferative diseases (e.g. diminished tumor growth) or, preferably, even causing regression, more preferably even the partial or complete disappearance, of such cells (e.g. tumor regression, preferably cure). The term "that depend on raf-activity" is preferably intended to mean any proliferative diseases that can be influenced, especially alleviated, by hybridization of a raf-specific ODN to its target, as described hereinbefore and hereinafter.

By the term "a product which comprises a) at least one oligonucleotide or oligonucleotide derivative (ODN) targeted to nucleic acids encoding raf, especially human c-raf, and b) at least one other chemotherapeutic agent where any component a) and/or b).can also be present in the form of a pharmaceutically acceptable salt, if at least one salt-forming group is present, in the presence or absence of one or more pharmaceutically acceptable carrier materials, as a combination preparation for simultaneous or chronologically staggered use within a period of time which is small enough for the active compounds both of component a) and of component b) to mutually enhance antiproliferative activity against proliferating cells, especially in a patient, for treating a proliferative disease which responds to such active compounds", there is meant especially a "kit of parts" in the sense that the effective components a) and b) of the combination can be dosed independently or by use of different fixed combinations with distinguished amounts of any components a) and b) at different time points. The parts of the kit of parts can then be administered simultaneously or chronologically staggered, that is at different time points and with equal or different time intervals for any part of the kit of parts, with the condition that the time intervals are chosen such that the effect on the proliferative disease in the combined use of the parts is larger than the effect which would be obtained by use of only any one of component a) and b), that is, stronger inhibition of proliferation or, preferably, stronger regression or even cure of the proliferative disease is found than when the same dose of only one of components a) and b) is administered alone in the same dose. That is meant by the term to mutually enhance antiproliferative activity against proliferating cells, especially in a patient"; preferably, there is meant a mutual en¬ hancing of the effect of the components a) and b), especially a synergism and/or the causing of regression of the proliferating cells, up to and including their complete destruc¬ tion, and especially a strong synergism between components a) and b).

By the term "proliferating cells", preferably abnormally proliferating cells are meant.

Preferred are combinations which show enhanced antiproliferative activity when compared with the single components alone, especially combinations that show synergism (synergistic combinations) or combinations that lead to regression of proliferative tissues and/or cure from proliferative diseases.

The term "synergism" is standing for an effect that is stronger than additive, that is, a stronger effect of the combination of any component a) with any component b) than could be reached by the factor of diminuation of proliferation obtained from mere multiplication of the factor of diminuation of proliferation for any component a) alone or any component b) alone when compared to a control without treatment when each a) and b) as such, whether alone or in combination, is administered in the same dose as in the single treatment without combination (which does not mean that the dose of a) and b) must be identical, although this may also be the case). As theoretical example for mere illustration, if a component a) alone gives a growth of tumor cells that is diminished by a factor of 2 in comparison to a control without any treatment and a component b) alone gives a diminuation of growth by a factor of 1.5, then an additive effect would be one where, by combined use of component a) and component b), a 3-fold diminuation of growth would be found (multiplication of 2 with 1.5). A synergistic effect would be present if a more than 3-fold diminuation of proliferation is found. By the term "quantity, which is jointly (therapeutically) effective for treating a proliferative disease that can be treated by administration of an oligonucleotide or oligonucleotide derivative targeted to (especially human) raf, especially c-raf (preferably that can be treated by modulation of (especially human) raf, especially c-raf, activity)", there is preferably meant a quantity of component a) and component b) which is effective in the treatment of one of the proliferative diseases mentioned above, that is, which leads to diminished proliferation or preferably even to regression of the proliferating cells (e.g. tumor regression) or even to cure from the proliferative disease. This term not only comprises combinations of any component a) and b) where a) and b) are dosed in such a manner as to be antiprolife- ratively effective already without combination, but also doses of any such component which alone would show no or only marginal effect but which in combination leads to clearly anti¬ proliferative effects, that is to diminished proliferation or preferably even to regression of the proliferating cells or even to cure from the proliferative disease. In addition, here the term "combination" is not only used to describe fixed combinations of the components, but also any combination of components a) and b) for simultaneous or chronologically staggered use within a period of time which is small enough for the active compounds both of com¬ ponent a) and of component b) to mutually enhance antiproliferative activity, e.g. in a patient.

The term "one or more pharmaceutically acceptable carrier materials" is explained below in the definition of pharmaceutical preparations.

It is to be understood that the invention relates also to any use of combinations of a com¬ ponent a) and a component b), as defined above and below, in a method of inhibiting hyperproliferation of cells comprising contacting hyperproliferating cells with a pharma¬ ceutical preparation or product in the sense of a kit of parts, especially a method of treating a proliferative disease comprising contacting a subject, cells, tissues or a body fluid of said subject, suspected of having a hyperproliferative disease. This includes especially the treat¬ ment of e.g. cells outside the body with the intent to replace hyperproliferating cells in the body of a subject with hyperproliferating cells by normal cells; for example, blood cells of the immune system may be taken from a subject, treated outside the body with a compo¬ nent a) and a component b) to select for non-hyperproliferative cells, the stem cells and the remaining blood cells of the immune system may be destroyed in the subject e.g. by irradi¬ ation or chemotherapy and then the selected normal cells may be reimplanted into the sub- ject, e.g. by injection etc. The methods to be employed in such kinds of treatment are known to the person having skill in the art.

Any of the references mentioned within this specification is incorporated by reference, especially those passages marked as preferred herein, especially International Application WO 95/32987, WO 93/19203 or WO 95/02069.

Provided that salt-forming groups are present, the ODN as well as the other chemothera¬ peutic^) may also be present in the form of salts.

Salts of oligonucleotides or oligonucleotide derivatives are especially acid addition salts, salts with bases or, when several salt-forming groups are present, optionally also mixed salts or internal salts.

Salts are especially the pharmaceutically acceptable, e.g. substantially non-toxic, salts of oligonucleotides or oligonucleotide derivatives as specified above and below.

Such salts are formed, for example, from the oligonucleotides or oligonucleotide derivatives having an acidic group, for example a carboxy, phosphodiester or phosphorothioate group, and are, for example, their salts with suitable bases, such as non-toxic metal salts derived from metals of groups la, lb, Ha and lib of the Periodic Table of Elements, especially suit¬ able alkali metal salts, for example lithium, sodium or potassium salts, or alkaline earth me¬ tal salts, for example magnesium or calcium salts, furthermore zinc or ammonium salts, also those salts that are formed with organic amines, such as unsubstituted or hydroxy-substi- tuted mono-, di- or tri-alkylamines, especially mono-, di- or tri-lower alkylamines, or with qua¬ ternary ammonium compounds, for example with N-methyl-N-ethylamine, diethylamine, tri¬ ethylamine, mono-, bis- or tris-(2-hydroxy-lower alkyl)amines, such as mono-, bis- or tris-(2- hydroxyethyl)amine, 2-hydroxy-tert-butylamine or tris(hydroxymethyl)methylamine, N,N-di- lower alkyl-N-(hydroxy-lower alkyl)-amines, such as N,N-dimethyl-N-(2-hydroxyethyl)-amine or tri-(2-hydroxyethyl)-amine, or N-methyl-D-glucamine, or quaternary ammonium salts, such as tetrabutylammonium salts. The oligonucleotides and their derivatives having a basic group, for example an amino or imino group, can form acid addition salts, for example with inorganic acids, for example a hydrohalic acid, such as hydrochloric acid, sulfuric acid or phosphoric acid, or with organic carboxylic, sulfonic, sulfo or phospho acids or N-substituted sulfamic acids, such as, for example, acetic acid, propionic acid, glycolic acid, succinic acid, maleic acid, hydroxymaleic acid, methylmaleic acid, fumaric acid, malic acid, tartaric acid, gluconic acid, glucaric acid, glucuronic acid, citric acid, benzoic acid, cinnamic acid, nrtan- delic acid, salicylic acid, 4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid, embonic acid, nicotinic acid or isonicotinic acid, also with amino acids, for example, α- amino acids, and also with methanesulfonic acid, ethanesulfonic acid, 2-hydroxyethane- sulfonic acid, ethane- 1 ,2-disulfonic acid, benzenesulfonic acid, 4-methylbenzenesulfonic acid, naphthalene-2-sulfonic acid, 2- or 3-phosphoglycerate, glucose-6-phosphate, N-cyclo- hexylsulfamic acid (with formation of the cyclamates) or with other acidic organic com¬ pounds, such as ascorbic acid. Compounds having acidic and basic groups can also form internal salts. If more than one salt-forming group is present, it is also possible that mixed salts are present. Corresponding salts can be formed from other chemotherapeutic agents provided that salt-forming groups are present therein.

For the purpose of isolation or purification, it is also possible to use pharmaceutically unacceptable salts, for example picrate or perchlorate salts.

The terms "oligonucleotides", "oligonucleotide derivatives", "compounds11 and "salts" also expressly include individual compounds or individual salts.

The antitumor activity of SEQ-ID NO: 1-ODN as single agents is tested against various hu¬ man tumors transplanted subcutaneously into nude mice. The human tumors tested are A549 lung carcinomas (ATCC No. CCL 185), T24 bladder carcinomas (ATCC No. HTB 4), MDA-MB-231 breast carcinomas (ATCC HTB 26) and Colo 205 colon carcinomas (ATCC CCL 222). The ODN is given once daily by the intravenous route of application when the tumor reaches a mean volume of approximately 100 mm3 throughout the experiments. In a standard experiment drug application is started at day 10 and continued until the end of the experiment at day 30 at doses of 6, 0.6, 0.06, 0.006 mg/kg. In all tumor types tested, the SEQ-ID NO: 1-ODN exhibits significant antitumor activity in the dose range of 0.06-6.0 mg/kg. The most sensitive tumor is A549 lung carcinoma (significant activity at 0.006 mg/kg), followed by T24 bladder and MDA-MB-231 breast carcinoma. The SEQ-ID NO: 1 - ODN is only marginally active in vivo against Colo 205 colon carcinomas transplanted into nude mice (significant activity at 6 mg/kg). The antitumor effects of the SEQ-ID NO: 1 -ODN are sequence-specific since scrambled control ODNs do not show antitumor effects. For the SEQ-ID NO: 1-ODN a series of mismatched ODNs with 1 to 5 mismatches in the central part of the SEQ-ID NO: 1-ODN gives the following results: The introduction of increasing numbers of mismatches into this sequence results in a gradual loss of antitumor activity in vivo. Introduction of one single mismatch in the center of the SEQ-ID NO: 1-ODN sequence results in a considerable loss of antitumor activity while ODNs with 3 mismatches are completely inactive. These results strongly indicate that the antitumor activities of SEQ-ID NO: 1-ODN are the result of sequence-specific inhibition of target gene expression in tumors. In A549 lung carcinomas, the SEQ-ID NO: 1-ODN downregulates c-raf mRNA levels in a sequence-specific and time-dependent manner at a dose of 6.0 mg/kg. Maximal reduction of c-raf mRNA leveis is found at day 13 after start of the ODN treatment. A control ODN with seven mismatches does not influence c-raf mRNA levels in A549 lung carcinomas which is consistent with the lack of antitumor activity of the control ODN.

The effects of combinations of a component a) (raf-targeted ODN) with a component b) (other chemotherapeutic agent) can preferably be shown in analogy to the methods shown below in the passage providing examples, preferably with the animals, tumor cell lines, conditions and combinations mentioned there.

As can be understood from the present text, the term "combination" in the following para¬ graphs which describe more specific and preferred variants of the present invention is intended to refer to

(j) combination preparations comprising at least one oligonucleotide or oligonucleotide derivative (ODN) targeted to nucleic acids encoding (especially human) raf with at least one other chemotherapeutic agent; or a method for treating a proliferative disease that can be treated by an oligonucleotide or oligonucleotide derivative targeted to raf, especially c-raf, especially where the disease responds to modulation of raf activity, where a) at least one oligonucleotide or oligonucleotide derivative (ODN) targeted to nucleic acids encoding (especially human) raf and capable of modulating (especially human) raf expression and b) at least one other chemotherapeutic agent are administered to a mammal in combination in a quantity which is jointly therapeutically effective against proliferative diseases that can be treated by an oligonucleotide or oligonucleotide derivative targeted to raf, especially c-raf, or that preferably depend on raf, especially c-raf, activity in order to treat them, or (iii) a product which comprises a) at least one oligonucleotide or oligonucleotide derivative (ODN) targeted to nucleic acids encoding (especially human) raf and b) at least one other chemotherapeutic agent in the presence or absence of one or more pharmaceutically acceptable carrier materials, as a combination preparation for simultaneous or chronologically staggered use within a period of time which is small enough for the active compounds both of component a) and of component b) to mutually enhance antiproliferative activity in the patient, for treating a pro¬ liferative disease which responds to such active compounds; or

(iv) a pharmaceutical preparation which comprises a quantity, which is jointly effective for treating a proliferative disease that can be treated by administration of an oligonucleotide or oligonucleotide derivative targeted to (especially human) raf, especially human c-raf (especially by modulation of human raf, especially c-raf, activity) of a) at least one oligonucleotide or oligonucleotide derivative (ODN) targeted to nucleic acids encoding (especially human) raf and b) at least one other chemotherapeutic agent, with one or more pharmaceutically acceptable carrier materials; or (v) the use of a combination of a) at least one oligonucleotide or oligonucleotide derivative (ODN) targeted to nucleic acids encoding (especially human) raf and b) at least one other chemotherapeutic agent, for producing pharmaceutical preparations for use as compositions against a a proliferative disease that can be treated by application of an oligonucleotide or oligonucleotide derivative targeted to raf, especially human c-raf, preferably a proliferative disease that can be treated by modulation of raf (especially human c-raf) activity.

or any combination of these subjects of the invention, as far as permissible under the respective patent laws; or the more specific and preferred variants thereof as given below;

where any component a) and/or b) can also be present in the form of a pharmaceutically acceptable salt, if at least one salt-forming group is present.

Within the following groups of more preferred embodiments of the invention, more general definitions may be replaced by more specific definitions in accordance with those given above or (especially with regard to definition of pharmaceutical compositions and methods of use) below.

Preferred is a combination (preferably synergistic and/or causing regression up to and including complete cure) of a) at least one oligonucleotide derivative (ODN) targeted to nucleic acids encoding human raf and preferably c-raf; the oligonucleotide derivative preferably being one that corresponds to an oligonucleotide derivative as published in PCT application WO 95/32987, especially in the examples thereof, more preferably an oligonucleotide derivative that corresponds to the following sequence:

5-TCCCGCCTGTGACATGCATT-3' SEQ.-ID NO: 1,

(or also a version thereof which is terminally shortened on one or both ends, e.g. with 15 to 19 building blocks, most especially the derivative as such)

which is preferably present as an oligonucleotide derivative which (in its nucleotide/nucleo- tide derivative sequence) comprises at least one of the following units of the formulae given hereinafter, wherein B is a base radical as defined above, preferably of a base selected from thymine, uracil, 5-propynyluracil, cytosine, 5-methylcytosine, 5-propynylcytosine, adenine, 2-aminoadenine and guanine; Q is H, OCH3, F, O(CH2CH2O)vCH3 wherein v is from 0 to 12, especially 1 or 3, CH2CH(CH3)OCH3 or CH2CH(OH)CH2OH, or less preferably SH; and the other moieties have the meanings given behind the respective formula:

Figure imgf000045_0001
Figure imgf000046_0001

Figure imgf000046_0002

(the phosphodiβsters llh or llh* being especially preferred), especially 2'-deoxy derivatives (Q = H) or 2'-(2-methoxy)ethoxy (2'-O-CH2CH2OCH3) derivatives, the remaining internu¬ cleosidic bonds in the respective oligonucleotide derivative being of the phosphorothioate type; for synthesis see European Application EP 0 626 387 and International Application WO 92/20823; or

as oligonucleotide derivative which comprises at least one of the following dimeric units (bivalent radicals) of the formulae given hereinafter, wherein each B is, independently of the other, a base radical as defined above, preferably of a base selected from thymine, uracil, 5-propynyiuracil, cytosine, 5-methylcytosine, 5-propynylcytosine, adenine, 2-aminoadenine and guanine; any Q is, indepently of the other, H, OCH3, F or O(CH2CH2O)vCH3 wherein v is from 0 to 12, especially 1 ; and the other moieties have the meanings given behind the respective formula:

Figure imgf000047_0001

(Vie)

** Y**

Figure imgf000047_0002
CH2 N(A)

wherein A is H, methyl or phenyl, preferably H; the remaining internucleosidic bonds in the respective oligonucleotide derivative being of the phosphorothioate type; for synthesis see International Application WO 95/20597and International Application WO 92/20823; or

(alternatively or in addition) as oligonucleotide derivative which is present in the form of a chimeric oligonucleotide which shows, on the one hand, M regions of between 3 and 20 (preferably 5 to 15) modified building blocks (either with phosphodiester or with phospho¬ rothioate backbone), especially 2'-(2-methoxy)ethoxy (more preferred) or 2'-F modified building blocks, which are in succeeding order; and, on the other hand, U regions of 4 to 20 2'-deoxy building blocks with phosphorothioate structure that are otherwise unmodified; preferably, the M and U regions being in one of the following arrangements:

5'-M-U~M-3'

5'-M-U-3' or

5'-U-M-3'; or especially oligonucleotide derivatives, preferably oligo-2'-deoxynucleotide derivatives, where all internucleosidic bridges as of the phosphorothioate type and that have no sugar or base modification; and

b) at least one other chemotherapeutic agent, preferably selected from

(A) alkylating agents, such as dacarbazine (DTIC-Dome); nitrogen mustards, such as mechlorethamine (Mustargen); ethyleneimine derivatives, e.g. triethylenethiophosphoramid (thio-tepa); procarbazine (Matulane); alkyl sulfonates such as busulfan (Myeleran); cyclo- phosphamide; 4-hydroxyperoxycyclophosphamide (4-HC); mafosfamide; ifosfamide; mel- phalan (Alkeran); chlorambucil (Leukeran); nitrosoureas such as cyclohexyl nitrosourea (meCCNU; Carmustine, BCNU, BiCNU) or lomustine (CCNU, CeeNU), cis-platinum(ll)-di- aminedichloride (platinol or cisplatin); carboplatin (Paraplatin); preferably cross-linking che¬ motherapeutics, preferably bis-alkylating agents, especially nitrogen mustards, such as mechlorethamine (Mustargen); alkyl sulfonates such as busulfan (Myeleran); cyclophos- phamide; melphalan (Alkeran); chlorambucil (Leukeran); cis-platinum(ll)-diaminedichloride (platinol or cisplatin) or carboplatin (Paraplatin); or compounds that form cross-links via ionic bonds, such as ethyleneimine derivatives, e.g. triethylenethiophosphoramid (thio-tepa) (forms ionic cross-links);

(B) antitumor antibiotics, preferably selected from the group comprising bleomycine (Blenoxane); anthracyclines, such as daunomycin, dactinomycin (Cosmegen), daunorubicin (Cerubidine), doxorubicin (Adriamycin, Rubex), epirubicin, esorubicin, idarubicin (Idamycin), plicamycin (Mithracin, formerly called Mithramycin) and preferably cross-linking (bis-alkyla¬ ting) antitumor antibiotics, such as mitomycin C (Mitomycin, Mutamycin);

(C) antimetabolites, for example folic acid analogues such as methotrexate (Folex, Mexate) or trimetrexate; purine nucleoside analogues such as Cladribine (Leustatin; 2-chloro-2'-de- oxy-β-D-adenosine), 6-mercaptopurine (Mercaptopurine, Purinethol, 6-MP), pentostatin (Nipent) or 6-thioguanine (6-TG, Tabloid); pyrimidine analogues such as 5-fluorouracil (Fluoruracil, 5-FU), 5-fluorodeoxyuridine (Floxuridine, FUDR), cytosine arabinoside (ara-C, cytarabine, Cytosar-U or Tarabine PFS), fludarabin phosphate (Fludara) or 5-azacytidine; hydroxyurea (Hydrea); or polyamine biosynthesis inhibitors, especially ornithine decarboxy- lase inhibitors or S-adenosylmethionine decarboxylase inhibitors, for example those men¬ tioned in EP 0 456 133, especially 4-amidino-1-indanon-2'-amidinohydrazone; (D) plant alkaloids, preferably vinca alkaloids, such as vinblastine (Velban), vincristine (Oncovin) or vindesine; epipodophyllotoxins, such as etoposide (VP-16, VePesid) or teniposide (VM-26, Vumon);

(E) hormonal agents and antagonists, preferably adrenocorticoids, such as prednisone (Deltasone) or dexamethasone (Decadron); progestines such as hydroxyprogesterone (Prodox), megestrol acetate (Megace) or medroxyprogesterone (Provera, Depo-Provera); androgens such as testosterone or fluoxymesterone (Halotestin); estrogens such as di- ethylstilbestrol (DES), estradiol or chlorotriansiene (Tace); synthetic analogues of LHRH, such as goserelin (Zoladex); synthetic analogues of LH-releasing hormone, such as leu- prolide (Lupron, Lupron Depot); anti-androgens such as flutamide (Eulexin); anti-estrogens such as tamoxifen; aromatase inhibitors such as aminogluthetimide (Cytadren), ientaron (Formestane, 4-hydroxy-4-androsten-3,17-dione) (see EP 0 162 510), fadrozole (5-(p-cya- nophenyl)-5,6,7,8-tetrahydroimidazo[1 ,5-a]pyridin, see EP 0 437 415 and EP 0 165 904), letrozole (4,4,-(1 H-1 ,2,4-triazol-1-yl-methylen)-bis-benzonitrile, see US 4,976,672), 4-(α-(4- cyanophenyl)-α-fluoro-1-(1 ,2,4-triazolyl)methyl)-benzon"ιtrile (see EP 0 490 816) or 4-(α-(4- cyanophenyl)-(2-tetrazolyl)methyl)-benzonith!e (see EP 0 408 509); adrenal cyctooxic agents, such as mitotane (Lysodren); somatostatine analogues, such as octreotide (Sando- statin); or 5α-reductase inhibitors, such as N-(1-cyano-1-methyl-ethyl)-4-aza-3-oxo-5α-an- drost-1-en-17β-carboxamid (see EP 0 538 192);

(F) biological response modifiers, preferably lymphokines, such as aldesleukiπ (human re¬ combinant IL-2, Proleukin); or interferons, such as interferon-α (Intron-A, Roferon) or interferon "B^BaD ' (see EP 0 205 404);

(G) inhibitors of protein tyrosine kinases and/or serine/threonine kinases other than ODNs, such as N-{5-[4-methyl-piperazino-methyl)-benzoylamido]-2-methyl-phenyl}-4-(3-pyridyl)-2- pyrimidine (see EP 0 546 409), N-(3-chlorophenyl)-4-(2-(3-hydroxy)-propyl-amino-4-pyridyl)- 2-pyrimidinamin (see EP 0 606 046), N-benzoyl-staurosporine (see EP 0 296 110), 4,5-bis- (aniiino)-phthalimide (see EP 0 516 588), N-(5-benzoylamido-2-methyl-pheπyl)-4-(3-pyridyl)- 2-pyridinamin (see EP 0 564 409) or 4-(m-chloranilino)-5,6-dimethyl-7H-pyrrolo[2,3-d]pyri- midin (see EP 0 682 027);

(H) antisense oligonucleotides or oligonucleotide derivatives targeted to other targets than raf, especially targeted to SAMDC (PCT application WO 96/05298) or (less preferably) protein kinase C (International Application WO 93/19203 or WO 95/02069); and (I) miscellaneous agents or agents with other or unknown mechanism of action, preferably S-triazine derivatives, such as altrematine (Hexalen); enzymes, such as asparaginase (Elspar); methylhydrazine derivatives, such as dacarbazine and procarbazine; matrix metalloproteinase inhibitors, such as (Case 4-19421) hexamethylmelamine, pentamethyl- melamine; anthraquinones such as mitoxantrone (Novantrone); mitotic spindle poisons such as paclitaxel (Taxol); streptozocin (Zanosar); estracyt (estramustine); amsacrine; differentiating agents, such as all-trans retinoic acid (TRA); immunomodulators, such as levamisole (ergamisol); vaccines, e.g. anti-melanoma vaccines (see EP 0 674 097); or antibodies with antitumor activity, such as recombinant human immunoglobulins directed at melanoma antigen (see EP 0 640 131) or antibodies for active immunotherapy of melanoma (see EP 0 428 485),

more preferably selected from the chemotherapeutics mentioned above under (A) as cross-linking chemotheraDeutics, most preferably bis-alkylating agents, especially nitrogen mustards, such as mechlorethamine (Mustargen); alkyl sulfonates such as busulfan (Myeleran); cyclophosphamide; melphalan (Alkeran); chlorambucil (Leukeran); cis-platinum(ll)- diaminedichloride (platinol or cisplatin) or carboplatin (Paraplatin); compounds that form cross-links via ionic bonds, such as ethyleneimine derivatives, e.g. triethylenethiophos¬ phoramid (thio-tepa) (forms ionic cross-links); mentioned under (B) as cross-linking (bis- alkylating) antitumor antibiotics, such as mitomycin C (Mitomycin, Mutamycin); and purine nucleoside analogues such as Cladribine (Leustatin; 2-chloro-2'-deoxy-β-D- adenosine), 6-mercaptopurine (Mercaptopurine, Purinethol, 6-MP), pentostatin (Nipent) or 6-thioguanine (6-TG, Tabloid); or pyrimidine analogues such as 5-fiuorouracil (Fluoruracil, 5- FU), 5-fluorodeoxyuridine (Floxuridine, FUDR), cytosine arabinoside (ara-C, cytarabine, Cytosar-U or Tarabine PFS), fludarabin phosphate (Fludara) or 5-azacytidine;

where any component a) and/or b) can also be present in the form of a pharmaceutically acceptable salt, if at least one salt-forming group is present.

Preferred is especially a combination of a) at least one oligonucleotide derivative (ODN) targeted to nucleic acids encoding human c-raf and that corresponds to the following sequence: 5'- TCC CGC CTG TGA CAT GCA TT -3' SEQ.-ID NO: 1 ,

(or also a version thereof which is terminally shortened on one or both ends, e.g. with 15 to 19 building blocks, most especially the derivative as such)

which is preferably present as an oligonucleotide (especially oligo-2'-deoxynucleotide) deri¬ vative which (in its nucleotide/nucleotide derivative sequence) comprises at least one of the following units of the formulae given hereinafter, wherein B is a base radical as defined above, preferably of a base selected from thymine, uracil, 5-propynyluracil, cytosine, 5- methylcytosine, 5-propynylcytosine, adenine, 2-aminoadenine and guanine; Q is H, OCH3, F, O(CH2CH2O)vCH3 wherein v is from 0 to 12, especially 1 or 3, CH2CH(CH3)OCH3 or CH2CH(OH)CH2OH; and the other moieties have the meanings given behind the respective formula:

Figure imgf000051_0001

/ O

X— P I -=Y

Figure imgf000051_0002

Radical of formula type

(Hg), phosphorothioate X = SH Y = O or S (iig*)

(llh) phosphodiester X = OH Y = O

(llh*)

(the phosphodiesters llh or llh* being especially preferred), especially 2'-deoxy derivatives (Q = H) or 2'-(2-methoxy)ethoxy (2'-O-CH2CH2OCH3) derivatives, the remaining internu¬ cleosidic bonds in the respective oligonucleotide derivative being of the phosphorothioate type; for synthesis see European Application EP 0 626 387 and International Application WO 92/20823; or

as oligonucleotide derivative which comprises at least one of the following dimeric units (bivalent radicals) of the formulae given hereinafter, wherein each B is, independently of the other, a base radical as defined above, preferably of a base selected from thymine, uracil, 5-propynyluracil, cytosine, 5-methylcytosine, 5-propynylcytosine, adenine, 2-aminoadenine and guanine; any Q is, indepently of the other, H, OCH3, F or O(CH2CH2O)vCH3 wherein v is from 0 to 12, especially 1 ; and the other moieties have the meanings given behind the respective formula:

Figure imgf000052_0001

Radical of formula type X** Y**

(Vie), (Vie*) amide CH2 N(A) wherein A is H, methyl or phenyl, preferably H; the remaining internucleosidic bonds in the respective oligonucleotide derivative being of the phosphorothioate type; for synthesis see International Application WO 95/20597and International Application WO 92/20823; or

(alternatively or in addition) as oligonucleotide derivative which is present in the form of a chimeric oligonucleotide which shows, on the one hand, M regions of between 3 and 20 (preferably 5 to 15) modified building blocks (either with phosphodiester or with phospho¬ rothioate backbone), especially 2'-(2-methoxy)ethoxy (more preferred) or 2'-F modified building blocks, which are in succeeding order; and, on the other hand, U regions of 4 to 20 2'-deoxy building blocks with phosphorothioate structure that are otherwise unmodified; preferably, the M and U regions being in one of the following arrangements:

5'-M~U"M-3'

5'-M-U-3' or

5'-U--M-3';

or especially oligonucleotide derivatives, preferably oligo-2'-deoxynucleotide derivatives, where all internucleosidic bridges as of the phosphorothioate type and that have no sugar or base modification; and

and b) at least one other chemotherapeutic agent selected from cross-linking chemotherapeutic agents, most preferably bis-alkylating agents, especially ni¬ trogen mustards, such as mechlorethamine (Mustargen); alkyl sulfonates such as busulfan (Myeleran); cyclophosphamide; melphalan (Alkeran); chlorambucil (Leukeran); cis-plati- num(ll)-diaminedichloride (platinol or cisplatin) or carboplatin (Paraplatin); from compounds that form cross-links via ionic bonds, such as ethyleneimine derivatives, e.g. triethylene¬ thiophosphoramid (thio-tepa) (forms ionic cross-links); from cross-linking (bis-alkylating) antitumor antibiotics, such as mitomycin C (Mitomycin, Mutamycin), and from purine nucleo- side analogues such as Cladribine (Leustatin; 2-chloro-2'-deoxy-β-D-adenosine), 6-mer- captopurine (Mercaptopurine, Purinethol, 6-MP), pentostatin (Nipent) or 6-thioguanine (6- TG, Tabloid); or pyrimidine analogues such as 5-fluorouracil (Fluoruracil, 5-FU), 5-fluoro- deoxyuridine (Floxuridine, FUDR), cytosine arabinoside (ara-C, cytarabine, Cytosar-U or Tarabine PFS), fludarabin phosphate (Fludara) or 5-azacytidine; most preferably selected from chemotherapeutic agents selected from the group comprising ifosfamide, cisplatin, mitomycine and 5-fluorouracil, especially one of ifosfamide, cisplatin and mitomycine;

where any component a) and/or b) can also be present in the form of a pharmaceutically acceptable salt, if at least one salt-forming group is present.

Still more preferred is a combination where in any of the embodiments of the invention defined above component b) is selected from the mentioned other chemotherapeutic agents except for adriamycin (doxorubicin), cyclophosphamide or an oligonucleotide or oligonucleotide derivative targeted at (especially human) PKC.

Even more preferred is a combination of a) at least one oligonucleotide derivative (ODN) targeted to nucleic acids encoding human c-raf and that corresponds to the following sequence:

5*- TCC CGC CTG TGA CAT GCA TT -3' SEQ.-ID NO: 1 ,

(or also a version thereof which is terminally shortened on one or both ends, e.g. with 15 to 19 building blocks, most especially the derivative as such)

which is preferably present as an oligonucleotide derivative which (in its nucleotide/nucleo¬ tide derivative sequence) comprises at least one of the following units of the formulae given hereinafter, wherein B is a base radical as defined above, preferably of a base selected from thymine, uracil, 5-propynyluracil, cytosine, 5-methylcytosine, 5-propynyicytosine, adenine, 2-aminoadenine and guanine; Q is H, OCH3, F, O(CH2CH2O)vCH3 wherein v is from 0 to 12, especially 1 or 3, CH2CH(CH3)OCH3 or CH2CH(OH)CH2OH, or less preferably SH; and the other moieties have the meanings given behind the respective formula:

Figure imgf000055_0001

/

X — P=Y

Figure imgf000055_0002

Radical of formula type

(Hg), phosphorothioate X = SH Y = O or S

(Hg*)

(llh) phosphodiester X = OH Y = O

(llh*)

(the phosphodiesters llh or llh* being especially preferred), especially 2'-deoxy derivatives (Q = H) or 2'-(2-methoxy)ethoxy (2'-O-CH2CH2OCH3) derivatives, the remaining internu¬ cleosidic bonds in the respective oligonucleotide derivative being of the phosphorothioate type; for synthesis see European Application EP 0 626 387 and International Application WO 92/20823; or

as oligonucleotide derivative which comprises at least one of the following dimeric units (bivalent radicals) of the formulae given hereinafter, wherein each B is, independently of the other, a base radical as defined above, preferably of a base selected from thymine, uracil, 5-propyπyluracil, cytosine, 5-methylcytosine, 5-propynylcytosine, adenine, 2-aminoadenine and guanine; any Q is, indepently of the other, H, F, OCH3 or O(CH2CH2O)vCH3 wherein v is from 0 to 12, especially 1 ; and the other moieties have the meanings given behind the respective formula:

Figure imgf000056_0001

Radical of formula type X** Y**

(Vie), (Vie*) amide CH2 N(A)

wherein A is H, methyl or phenyl, preferably H; the remaining internucleosidic bonds in the respective oligonucleotide derivative being of the phosphorothioate type; for synthesis see International Application WO 95/20597and International Application WO 92/20823; or

(alternatively or in addition) as oligonucleotide derivative which is present in the form of a chimeric oligonucleotide which shows, on the one hand, M regions of between 3 and 20 (preferably 5 to 15) modified building blocks (either with phosphodiester or with phospho¬ rothioate backbone), especially 2'-(2-methoxy)ethoxy (more preferred) or 2'-F modified building blocks, which are in succeeding order; and, on the other hand, U regions of 4 to 20 2'-deoxy building blocks with phosphorothioate structure that are otherwise unmodified; preferably, the M and U regions being in one of the following arrangements:

5'-M--U"M-3' 5'-M-U-3' or 5'-U--M-3';

or especially oligonucleotide derivatives, preferably oligo-2'-deoxynucleotide derivatives, where all internucleosidic bridges as of the phosphorothioate type and that have no sugar or base modification; and

b) at least one other chemotherapeutic agent selected from the group comprising ifosfamide, cisplatin, mitomycine and 5-fluorouracil, especially one of ifosfamide, cisplatin and mitomycine;

where any component a) and/or b) can also be present in the form of a pharmaceutically acceptable salt, if at least one salt-forming group is present.

Much more preferred is a combination of a) one of the oligonucleotide derivatives mentioned in any previous definition and b) at least one, preferably one or two, of the chemotherapeutic agents mentioned in any of the preceding definitions.

Still more preference is given to a combination according to any of the preceding embodiments of the invention where the disease to be treated is selected from small cell lung carcinoma, large cell lung carcinoma, melanoma and prostate carcinoma.

Very high preference is given to a combination wherein

a) an oligonucleotide derivative that corresponds to the following sequence:

5'- TCC CGC CTG TGA CAT GCA TT -3' SEQ.-ID NO: 1 ,

(or also a version thereof which is terminally shortened on one or both ends, e.g. with 15 to 19 building blocks, most especially the derivative as such)

which is present in the form of a phosphorothioate oligo-2'-deoxynucleotide analogue, and b) any one of the following other chemotherapeutic agents for the treament of the respective proliferative disease:

- cisplatin for human prostate carcinomas;

- mitomycin for small lung cell carcinomas;

- cisplatin for small cell lung cancers: or

- mitomycin for large cell lung carcinomas

are in combination; where any component a) and b) can also be present in the form of a pharmaceutically acceptable salt, if at least one salt-forming group is present.

Very high preference is also given to a combination wherein

a) an oligonucleotide derivative that corresponds to the following sequence:

5'- TCC CGC CTG TGA CAT GCA TT -3' SEQ.-ID NO: 1 ,

(or also a version thereof which is terminally shortened on one or both ends, e.g. with 15 to 19 building blocks, most especially the derivative as such)

which is present in the form of a phosphorothioate oiigo-2'-deoxynucleotide analogue, and

b) any one of the following other chemotherapeutic agents for the treament of the respective proliferative disease:

- fluorouracil for colon cancer; or

- mitomycin for melanoma

are in combination; where any component a) and b) can also be present in the form of a pharmaceutically acceptable salt, if at least one salt-forming group is present.

Highest preference is given to the combinations provided in the examples, more especially the synergistic combinations given there, most specific the combinations where component b) is not selected from adriamycin, an oligonucleotide or oligonucleotide derivative targeted at protein kinase C and cyclophosphamide alone. Explicitely preferred is a synergistic combination and/or a combination leading to regression of proliferating cells up to and including complete cure, especially a synergistic combination, according to any of the embodiments of the invention defined above or given in the examples.

Explicitely preferred among all combinations is one wherein component a) is an oligo¬ nucleotide derivative which is present in the form of a chimeric oligonucleotide which shows, on the one hand, M regions of between 3 and 20 (preferably 5 to 15) modified building blocks (either with phosphodiester or with phosphorothioate backbone), especially 2'-(2- methoxy) ethoxy modified building blocks, which are in succeeding order; and, on the other hand, U regions of 4 to 20 2'-deoxy building blocks with phosphorothioate structure that are otherwise unmodified; preferably, the M and U regions being in one of the following arrangements:

5'-M«U-M-3'

5'-M-U-3' or

5'-U-M-3'.

The oligonucleotides and their derivatives in accordance with this invention may be conve¬ niently and routinely made in analogy to or through methods and using starting materials well-known in the art (for reviews, see, inter alia, Milligan et al., J. Med. Chem. 36(14), 1923- 37 (1993) and Uhlmann et al., Chemical Rev. 90(4), 543-84 (1990); see also International Application WO 92/20823 published 11. Nov. 1992), for example by the well-known techni¬ que of solid phase synthesis. Equipment for such synthesis is sold by several vendors (e.g., Applied Biosystems Inc., Foster City, California, USA). Any other means for such synthesis may also be employed; the actual synthesis of the oligonucleotides is well within the talents of the routineer. It is also well known to use similar techniques to prepare other oligonucle¬ otide derivatives such as the phosphorothioates and nucleoside modified derivatives.

Most preferably, phosphorothioate analogues of the invention can be made by methods known in the art, preferably by reacting a starting material which contains a 5' terminal fragment of the formula XIII,

Figure imgf000060_0001

(or a tautomer thereof) wherein D is a hydroxy protecting group, B and B' independently re¬ present bases as defined above for B in any one of formulae I to X, Q and Q' independently represent H, OH, SH, SCH3, F, N3, CN, OCN, OCH3, O(CH2)NH2 or O(CH2)zCH3 where z is from 1 to about 10, O(CH2CH2O)vCH3 wherein v is from 0 to 12, especially 1 or 3, CH2CH(CH3)OCH3 or CH2CH(OH)CH2OH, or in a broader sense another' substituent having similar properties, for example selected from Cl, Br, CF3, ONO2, NO2, NH2 and O-, S- or NH- lower alkyl, most especially Q being hydroxy or preferably -O-CH2CH2OCH3 or (most prefer¬ ably) hydrogen; and the other moieties have the meanings given behind the respective for¬ mula; G is hydrogen, lower alkoxy or 2-cyanoethoxy and E is a hydroxy protecting group, a carrier or a 3' free or carrier-bonded mono- or oligonucleotide analogue wherein in place of any phosphodiester group {O-[P(=O)(-OH)]-O} a phosphorothioate analogue {O-[P(=S)(-OH)]-O}/{O-[P(-SH)(=O)] or a group of the formula {O-[(P=O)-H]-O}/{O-(P-OH)-O} is present, with a sulfurylating reagent with simultaneous oxidation of any trivalent phos¬ phorus being present, where necessary further functional groups being in protected form, and, where necessary, removing any protecting groups and/or carriers, and, if desired,

separating any resulting mixtures of isomers into the individual isomers, and/or transforming a resulting free phosphorothioate oligonucleotide into a salt, and/or transforming a resulting salt into the free form or into a different salt.

In detail, the synthesis is preferably carried out as follows: Any functional groups being present can be in unprotected or protected form, the protecting groups being selected from those mentioned above for OH or amino/imino groups (SH pro¬ tecting groups can be selected from those given above for hydroxy groups). A characteristic of these protecting groups is that they are not present in the final products. Protecting groups can be removed by standard methods known in the art, such as those mentioned in the references given above.

D is preferably the dimethoxytrityl group; this group can be removed preferably by acid hy¬ drolysis, for example with mild acids such as formic acid, acetic acid, dichloroacetic acid or furthermore trifluoroacetic acid, in water or organic solvents such as halogenated solvents, for example dichloromethane, cyclic ethers, for example tetrahydrofurane, or lower-alkylcya- nides, for example acetonitrile, or mixtures thereof.

It is most preferred that the terminal hydroxy protecting group D in the resulting phosphoro¬ thioate oligonucleotide is removed in a step separate from and after the removal of further protecting groups, such as acetyl, benzoyl, phenoxyacetyl, 4-tert-butylphenoxyacetyl, N,N- dimethylformamidinyl, N-methylpyrrolidin-2-yliden, succinyl, 2-cyanoethyl and similar protec¬ ting groups that can be removed in a first deprotection step by basic hydrolysis, preferably in the presence of a nitrogen base, such as ethanolamine in an alcohol, such as ethanol, or preferably in the presence of ammonium hydroxide in an aqueous solvent, such as water, at temperatures ranging preferably from about 10 to about 80 °C, and then purify the resulting OH-protected oligonucleotide derivative by chromatography on lipophilic adsorbents, such as reverse phase HPLC material, and then, in the purified oligonucleotide derivative, finally removing the OH protecting group, preferably the dimethoxy trityl group, by acid hydrolysis as described above.

B and B' preferably each represent a radical derived from one of the bases adenine, gua¬ nine, thymine, cytosine or 5-methylcytosine.

G is preferably 2-cyanoethoxy, the 3' free or carrier-bonded mono- or oligonucleotide ana¬ logue then being one wherein, in place of any phosphodiester group {O-[P(=O)(-OH)]-O}, a phosphorothioate analogue {O-[P(=S)(-OH)]-O}/{O-[P(-SH)(=O)] is present. If G is OH, the 3' free or carrier-bonded mono- or oligonucleotide analogue can be one wherein either in place of any phosphodiester group {O-[P(=O)(-OH)]-O} a phosphorothioate group {O-[P(=S)(-OH)]-O}/{O-[P(-SH)(=O) - two tautomeric forms] is present or one ore more groups of the formula {O-[(P=O)-H]-O}/{O-(P-OH)-O} (two tautomeric forms) are present in¬ stead of one or more, maximally all, phosphorothioate bonds. In the latter case, in the pro¬ cess according to the invention all phosphorus atoms in the respective oligonucleotide can be thiolated simultaneously.

E is preferably a 3' free or carrier-bonded mono- or oligonucleotide analogue wherein in place of any phosphodiester group {O-[P(=O)(-OH)]-O} a phosphorothioate analogue {O- [P(=S)(-OH)]-O}/{O-[P(-SH)(=O)] or a group of the formula {O-[(P=O)-H]-O}/{O-(P-OH)-O} (two tautomeric forms) is present.

Where possible, any starting material can also be present in the form of a salt.

A sulfurylating reagent which is capable of reaction with simultaneous oxidation of any tri- valent phosphorus being present, is, for example, selected from the group comprising S8 in an organic solvent in the presence of a nitrogen base, such as S8 in pyridine/triethylamine or Sθ in 2,6-lutidine; sulfur in CS2 in the presence of a nitrogen base, such as triethylamine or pyridine; 3H-1 ,2-benzodithiol-3-one-1 ,1 -dioxide in acetonitrile; and most preferably diiso- propoxy-thiophosphoric acid disulfide of the formula XIV

Figure imgf000062_0002
Figure imgf000062_0001

in an organic solvent, such as acetonitrile, in the presence of a tertiary nitrogen base, such as pyridine. The preferred temperatures are in the range from 10 to 80 °C, most preferably around room temperature. The starting material with a fragment according to formula XIII (which makes the respective oligonucleotide complete with respect to the number of nucleotide units) can be synthesized according to methods known in the art, preferably by a combination of standard cyanoethyl phosphoramidite chemistry and simultaneous sulfurylation plus oxidation plus a further step according to standard cyanoethyl amidite chemistry to yield the corresponding triester star¬ ting materials (G being lower alkyl or 2-cyanoethyl in formula XIII) or (especially if several phosphodiester groups are present in the starting material of formula XIII due to an appro¬ priate group E corresponding to an oligonucleotide analogue wherein in place of any phos¬ phodiester group a group of the formula {O-[(P=O)-H]-O}/{O-(P-OH)-O} is present) H-phos- phonate chemistry (see Uhlmann et al., Chemical Reviews 90(4), 543-84 (1990) for review and further references).

To synthesize oligonucleotide derivatives with one or more building blocks of any one of formulae I and I* to X and X* wherein Q is O(CH2CH2O)vCH3 wherein v has the meanings defined above, and the other moieties are as defined, the respective starting material (either a building block or a complete oligonucleotide or its derivative) wherein at least one Q is hydroxy can, for example, be reacted in an inert solvent with a compound of formula XV,

X-(CH2-CH2-O)v-CH3 (XV)

wherein X is a leaving group and v is as defined above, functional groups being present in the starting materials being protected by protecting groups as defined above which can be removed at appropriate stages.

A leaving group X may, for example, be halogen, such as Cl, Br or I, arylsulfonyl, such as 4- toluolsulfonyl, or lower alkane sulfonyl, such as mesyl-sulfonyl.

The reaction is carried out preferably in the presence of a strong base, such as an alkali metal hydride, for example NaH, in an inert solvent, such as an ether, for example a cyclic ether, such as tetrahydrofurane, at temperatures ranging from 30 °C to the boiling point of the reaction mixture, preferably under reflux conditions.

For the synthesis of oligonucleotide derivatives which (in their nucleotide/nucleotide deriva¬ tive sequence) comprise at least one of the units (bivalent radicals) of the formulae Hg and llh or Hg* and llh* given hereinbefore, wherein B is a base radical as defined above, prefer¬ ably of a base selected from thymine, uracil, 5-propynyluracil, cytosine, 5-methylcytosine, 5- propynylcytosine, adenine, 2-aminoadenine or guanine; Q is H, O(CH2CH2O)vCH3 wherein v is from 0 to 12, especially 1 or 3, CH2CH(CH3)OCH3 or CH2CH(OH)CH2OH; and the other moieties have the meanings given behind the respective formula, synthesis is possible ac¬ cording to or in analogy to known procedures (see European Application EP 0 626 387 and International Application WO 92/20823); for oligonucleotide derivatives which comprise at least one of the units (bivalent radicals) of the formulae Vie and Vie* given hereinbefore, wherein B is a base radical as defined above, preferably of a base selected from thymine, uracil, 5-propynyluracil, cytosine, 5-methylcytosine, 5-propynylcytosine, adenine, 2-ami- noadenine or guanine; Q is OCH3 or O(CH2CH2O)vCH3 wherein v is from 0 to 12, especially 1 ; and the other moieties have the meanings given behind the respective formula, synthesis is also possible in accordance with or in analogy to known procedures (see International Application WO 95/20597and International Application WO 92/20823).

The separating of any resulting mixtures of isomers into the individual isomers can be done according to methods well-known in the art, for example using chromatographic procedures in order to separate diastereomers. Separation at the stage of a starting material or small intermediates is preferred due to possible difficulties in the separation of larger oligonucleo- tides/oligonucleotide derivatives. Separation of isomers is not mandatory to achieve the desired biological activity.

The transformation of resulting free compounds into their salts can be achieved according to standard methods, salts of cationic groups for example being available by treatment with appropriate acids or anion exchangers, and salts of anionic groups being available by treat¬ ment with an appropriate base or cation exchanger; or preferably by dialysis of the respecti¬ ve compound against a solution of the desired cation.

Salts can be transformed into the free compounds according to standard procedures, metal or ammonium salts for example by treatment with an appropriate acid or an acidic ion ex¬ changer, acid addition salts by treatment with an appropriate base or basic ion exchanger.

The transfer of salts into different salts is possible in analogy to the transformation of a free compound into a salt, as mentioned above. The above-mentioned reactions can be carried out under reaction conditions that are known per se, in the absence or customarily in the presence of solvents or diluents, preferably those solvents and diluents that are inert towards the reagents used and are solvents there¬ fore, in the absence or presence of catalysts, condensation agents or neutralising agents, depending on the nature of the reaction and/or the reactants at reduced, normal or elevated temperature, e.g. in a temperature range of from approximately -80°C to approximately 250°C, preferably from approximately -20°C to approximately 150°C, for example from room temperature to the reflux temperature, under atmospheric pressure or in a closed vessel, if desired under pressure, for example at the pressure produced in the reaction mixture under the reaction conditions in a closed tube, and/or in an inert atmosphere, e.g. under an argon or nitrogen atmosphere. The reaction conditions specifically mentioned in PCT application WO 95/32987 are preferred.

Solvents and diluents are, for example, water, alcohols, for example lower alkanols, such as methanol, ethanol or propanoi, diols, such as ethylene glycol, triols, such as glycerol, or aryl alcohols, such as phenol, acid amides, for example carboxylic acid amides, such as di¬ methylformamide, dimethylacetamide or 1 ,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone (DMPU), or amides of inorganic acids, such as hexamethylphosphoric acid triamide, ethers, for example cyclic ethers, such as tetrahydrofuran or dioxane, or acyclic ethers, such as di¬ ethyl ether or ethylene glycol dimethyl ether, halogenated hydrocarbons, such as halo-lower alkanes, for example methylene chloride or chloroform, ketones, such as acetone, nitriles, such as acetonitrile, acid anhydrides, such as acetic anhydride, esters, such as ethyl ace¬ tate, bisalkane sulfines, such as dimethyl sulfoxide, nitrogen heterocycles, such as pyridine, hydrocarbons, for example lower alkanes, such as heptane, or aromatic compounds, such as benzene or toluene, or mixtures of those solvents, it being possible to select the particular solvents that are suitable for each of the above-mentioned reactions.

In view of the close relationship between the oligonucleotides or oligonucleotide derivatives that have the properties according to the invention and the precursors thereof in free form and in the form of salts and/or tautomβrs, hereinbefore and hereinafter any reference to the free compounds and starting materials or the salts and/or tautomers thereof should be un¬ derstood as including the corresponding salts or free compounds and/or tautomers, respec¬ tively, as appropriate and expedient, provided that the compounds contain one or more salt- forming groups, e.g. basic groups, such as amino or imino groups, and/or acidic groups, such as carboxy, phosphoric acid radicals or sulfo (SO3H), and/or tautomerisable groups. In connection with starting materials, intermediates or final products, any reference made hereinbefore and hereinafter to a substituent, a compound, a tautomer or a salt, or to sub- stituents, compounds, tautomers or salts, is to be understood, irrespective of whether the singular or the plural is used, as meaning "one or more" as appropriate and expedient. Star¬ ting materials may also be used in protected form, where necessary, appropriate and expe¬ dient, it being possible for the protecting groups to be removed at suitable times. Protecting groups, their introduction and their removal are especially as defined above.

The compounds, including their salts, may also be obtained in the form of hydrates, or their crystals may include, for example, the solvent used for crystallisation.

In the process of the present invention the starting materials used are preferably those that result in the compounds described at the beginning as being preferred.

The description relates also to those forms of the process in which a compound obtainable as intermediate at any stage of the process is used as starting material and the remaining process steps are carried out, or in which a starting material is formed under the reaction conditions or is used in the form of a derivative, for example a salt, thereof.

The sequence and reaction conditions of all the described reactions are preferably to be so selected as considered appropriate and expedient to the person skilled in the art.

Pharmaceutical Compositions and Processes:

The pharmaceutical compositions that can find use in a combination according to the inven¬ tion are comprising either one or more of the components a) (an oligonucleotide or an oli¬ gonucleotide derivative) and b) (other chemotherapeutic agent) with the properties accor¬ ding to the invention as active ingredient, which can be used alone (e.g. as fixed combi¬ nation) or as kit of parts. Especially preferred are compositions for enteral, especially oral, or parenteral administration. The compositions comprise the active ingredient or combina¬ tions thereof as such or, preferably, together with a pharmaceutically acceptable carrier. The dose of any active ingredient depends on the disease to be treated, and on the species, age, weight and individual condition, as well as the method of administration.

Preferred is a pharmaceutical composition or combination that is suitable for administration to a warm-blooded animal, especially man, suffering from a proliferative disease selected from hyperproliferative conditions such as cancers, tumors, hyperplasias, fibrosis, angiogenesis, psoriasis, atherosclerosis and smooth muscle cell proliferation in the blood vessels, such as stenosis or restenosis following angioplasty. Most preferably, the disease is one selected from cancer types which have been very difficult to treat or even practically unaffected by therapy with standard chemotherapeutics, such as small cell lung carcinoma, large cell lung carcinoma, melanoma, prostate carcinoma or further also iymphoma. Most preferably, any of the aforementioned proliferative diseases that can be treated by inhibition of human c-raf activity is meant.

The pharmaceutical compositions comprise from approximately 0.0001 % to approximately 95% active ingredient, dosage forms that are in single dose form preferably comprising from approximately 0.001 % to approximately 20 % active ingredient, and dosage forms that are not in single dose form preferably comprising from approximately 0.001 % to approximately 10 % active ingredient. Unit dose forms, such as dragees, tablets, ampoules or capsules, comprise from approximately 0.0005mg to approximately 0.5 g of the active ingredient, preferably from 0.005 mg to approximately 40 mg.

The pharmaceutical compositions are prepared in a manner known per se, for example by means of conventional mixing, granulating, confectioning, dissolving or lyophilising pro¬ cesses. For example pharmaceutical compositions for oral administration can be obtained by combining the active ingredient with one or more solid or liquid carriers, where necessary granulating a resulting mixture and processing the mixture or the granules, if desired or appropriate with the addition of further excipients, to form tablets or dragee cores or solutions, respectively.

Suitable carriers are especially fillers, such as sugars, e.g. lactose, saccharose, mannitol or sorbitol, cellulose preparations and/or calcium phosphates, e.g. tricalcium phosphate or calcium hydrogen phosphate, and binders, such as starches, e.g. corn, wheat, rice or potato starch, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylceliu- lose and/or polyvinyipyrrolidone, and/or, if desired, disintegrators, such as the above- mentioned starches, and also carboxymethyl starch, crosslinked polyvinyipyrrolidone or alginic acid or a salt thereof, such as sodium alginate. Additional excipients are especially flow conditioners and lubricants, e.g. silicic acid, talc, stearic acid or salts thereof, such as magnesium or calcium stearate, and/or polyethylene glycol, or derivatives thereof.

Dragee cores may be provided with suitable, optionally enteric, coatings, there being used, inter alia, concentrated sugar solutions which may comprise gum arabic, talc, polyvinyi¬ pyrrolidone, polyethylene glycol and/or titanium dioxide, or coating solutions in suitable organic solvents or solvent mixtures, or, for the preparation of enteric coatings, solutions of suitable cellulose preparations, such as acetylcellulose phthalate or hydroxypropylmethyl- cellulose phthalate. Dyes or pigments may be added to the tablets or dragee coatings, e.g. for identification purposes or to indicate different doses of active ingredient.

Orally administrable pharmaceutical compositions are also dry-filled capsules consisting of gelatin, and also soft sealed capsules consisting of gelatin and a plasticiser, such as gly¬ cerol or sorbitol. The dry-filled capsules may contain the active ingredient in the form of granules, for example in admixture with fillers, such as corn starch, binders and/or glidants, such as talcum or magnesium stearate, and, where appropriate, stabilisers. In soft capsules, the active ingredient is preferably dissolved or suspended in suitable liquid excipients, e.g. fatty oils, *Lauroglycol (Gattefosse S.A., Saint Priest, France), "Gelucire (Gattefosse S.A., Saint Priest, France) or sesame oil, paraffin oil or liquid polyethylene glycols, such as PEG 300 or 400 (Fluka, Switzerland), or polypropylene glykols, to each of which stabilisers or detergents may also be added, on in water comprising further soluble carriers as mentioned above, such as methylcellulose or mannitol.

Other oral forms of administration are, for example, solutions or syrups prepared in custo¬ mary manner that comprise the active ingredient e.g. in suspended form and in a concen¬ tration of approximately from 0.001 % to 20 %, preferably approximately 0.001% to about 2%, or in a similar concentration that provides a suitable single dose when administered, for example, in measures of 0.5 to 10 ml. Also suitable, for example, are powdered or liquid concentrates for preparing shakes, e.g. in milk. Such concentrates can also be packed in single-dose quantities. Transdermal Delivery Systems are possible, especially with neutral active ingredients. Suitable formulations comprise, for example, about 0.0001% to about 2% by weight of active ingredient. In a preferred aspect, there are provided formulations which comprise about 2 % to 99.9999 % (or the balance to 100 %) of a short chain aliphatic alcohol. Suitable alcohols include ethanol, isopropanol, propylene glycol and glycerol. In a more preferred aspect, these formulations may additionally comprise a flux enhancer. Suitable flux enhancers include, for example, decylmethylsulfox.de, dimethylsufoxide as well as cyclic ketones, lactones, anhydrides and esters. Some of these flux enhancers also in¬ crease retention of the active ingredient and thus act to increase the concentration of it in the skin itself. For formulations for direct (local) treatment, such as topical application to the skin, it is preferred to use a flux enhancer which not only maximizes transdermal flux, but increases retention of the active ingredient in the skin. Certain cyclic ketone and lactone enhancers have been reported to increase local retention as well and, thus, comprise a preferred class of enhancers for topical administration of the active ingredient. In formu¬ lations for systemic treatment, it is preferable to use a flux enhancer which maximizes flux with a minimal local retention of the active ingredient.

Suitable rectally administrable pharmaceutical compositions are e.g. suppositories that consist of a combination of the active ingredient with a suppository base. Suitable suppos¬ itory bases are e.g. natural or synthetic triglycerides, paraffin hydrocarbons, polyethylene glycols or higher alkanols.

For parenteral administration there are suitable, especially, aqueous solutions of an active ingredient in water-soluble form, e.g. in the form of a water-soluble salt, in the presence or absence of salts, such as sodium chloride, and/or sugar alcohols, such as mannitol, or aqueous injection suspensions that comprise viscosity-increasing substances, e.g. sodium carboxymethylcellulose, sorbitol and/or dextran, and, where appropriate, stabilisers. The active ingredient, where appropriate together with excipients, may also be in the form of a lyophilisate and may be made into a solution prior to parenteral administration by the addi¬ tion of suitable solvents.

Solutions as used e.g. for parenteral administration may also be used as infusion solutions. Preferred formulations comprising any component b) (other chemotherapeutic agent) are those that are customary for the respective clinical use of any (especially standard) chemotherapeutic agent which are known in the art.

Preferred formulations for component a) are those mentioned in the examples.

The invention relates also to a method of treating the above-mentioned pathological con¬ ditions. For this purpose, in the combinations as hereinbefore described any active ingre¬ dient, or a pharmaceutically acceptable salt thereof, may be administered prophylactically or therapeutically, preferably in an amount that is effective against the mentioned diseases, to a warm-blooded animal, e.g. man, requiring such treatment, preferably in the form of a pharmaceutical composition. The dose of any active ingredient depends on the species of the warm-blooded animal to be treated, its body weight, its age and individual status, individual pharmacokinetic circumstances, the disease to be treated and the application route. Preferably, for a body weight of approximately 70 kg a daily dose of from 0.001 mg to 1000 mg, e.g. from approximately 0.01 mg to approximately 100 mg, preferably from approximately 0.05 mg to approximately 50 mg, of any active ingredient is administered.

The following examples illustrate the present invention without being intended to limit the scope thereof:

Examples:

(A) Chemotherapeutic drugs:

Adriamycin is purchased from Farmitalia Carlo Erba (Italy). Estracyt is purchased from Kabi Pharmacia (Uppsala, Sweden). Cisplatin is from Bristol Meyers Squibb (USA). 5- Fluorouracil is from Hoffmann La Roche (Switzerland). Mitomycin is from Kyowa Hakko (Japan). Ifosfamide is purchased from Asta (Germany). Tamoxifen is from Farmos (Finland). The drugs are dissolved in saline and applied either p.o. (tamoxifen) or i.v. (adriamycin, ifosfamide, estracyt, cis-platin, 5-fluorouracil, mitomycin) according to the schedules indicated in the tables in the examples . ln combination studies the chemotherapeutic agents are applied as follows (if not indicated otherwise in the tables):

For i.v. application, the concentrations of the solutions are chosen such that the amount of active ingredient mentioned below is applied in 10 ml/kg of the indicated solution. For p.o. application, the concentrations of the solutions are chosen such that the amount of active ingredient mentioned below is applied in 25 ml/kg of the indicated solution.

- Tamoxifen: 20 mg/kg p.o., day 6-8, 13-15, 21 -23 and 28-30 in 0.5 % methylcellulose.

Formula of tamoxifen:

Figure imgf000071_0001

- Adriamycin: 9 mg/kg i.v., once weekly.

Formula of adriamycine:

Figure imgf000071_0002

Ifosfamide: 150 mg/kg i.v., once weekly.

Formula of ifosfamide:

Figure imgf000071_0003

- Estracyt: 50 mg/kg i.v., daily in 5 % mannitol. Formula of estracyt

Figure imgf000072_0001

- Cisplatin: 10 or 11 mg/kg i.v., once weekly in pharmacopoe (10 or 11 mg of USP cisplatin RS in 10 ml 0.9 % NaCI/1% D-mannitol).

Formula of cisplatin:

Figure imgf000072_0002

- 5-Fluorouracil: 75 mg/kg i.v., once weekly.

Formula of 5-fluorouracil:

Figure imgf000072_0003

Mitomycin: 3.5 mg/kg i.v., once weekly.

Formula of mitomycin:

Figure imgf000072_0004

(B) ODNs:

Phosphorothioate oligodeoxynucleotides (ODNs) are synthesized on a 0.5 mmole scale using a Milligen model 8800 DNA synthesizer (Bedford, MA) using modified phosphor¬ amidite chemistries with β-cyanoethoxyphosphoramidites. Crude product of approximately 70 % purity is further purified by orthogonal columns chromatography using a Millipore HC18-HA column followed by anion exchange chromatography using a Millipore Q-15 strong anion exchanger. The purified material is precipitated from ethanol, redissolved and further desalted by ultrafiltration. The samples are depyrogenated by ultrafiltration with endotoxin levels reduced to below detectable levels using a standard endotoxin assay. 5'-TCC CGC CTG TGA CAT GCA TT-3'; SEQ-ID NO: 1 is a 20-mer phosphorothioate ODN targeting the 3'-untranslated region of c-raf mRNA which is used in the following examples, where it is named SEQ-ID NO: 1-ODN.

(C) Antitumor activity in vivo:

Female or male Balb/c nude mice are obtained from Bomholtgaard, Kopenhagen, Denmark or Ciba animal breeding facility, Sisseln, Switzerland. For the testing of in vivo antitumor activity, SEQ-ID NO: 1-ODN is dissolved in sterile saline (0.6 mg/ml) and is tested in a dose- range of 0.006 to 6 mg/kg once daily by the intravenous route of application. If not indicated otherwise, data with standard error of the mean are given for 6 mice per time point, respec¬ tively. Placebo treated controls receive carrier as indicated in examples. The following hu¬ man tumor cells are used for the experiments:

Estrogen receptor-positive breast cancer: MCF-7. Estrogen receptor-negative breast cancer: MDA-MB-231. Colon cancers: Colo 205, HCT 116, WiDr. Small cell lung carcinomas: NCI-H69, NCI-H209. Large cell lung carcinoma: NCI-H460. Squamous cell carcinoma: NCI-H520. Melanomas: SK-mel 3, SK-mel 1. Ovarian carcinomas: NIH:Ovcar3. Prostate carcinomas: PC3, DU145.

All the cell lines used are obtained from the American Type Culture Collection (ATCC) and are cultured in the suggested media and additives (ATCC culture conditions):

(i) MCF-7: ATCC HTB 22. These cells were taken from the pleural effusion of a 69-year old female Caucasian (see H.D. Soule et al., J. Natl. Cancer Inst. 5J_, 1409-16 (1973)). Medium for propagation: Medium: Eagle's MEM with non-essential amino acids, sodium pyruvate, 20 ug insulin/ml, 10 % fetal calf serum. (ii) MDA-MB-231 : ATCC HTB 26. This line was isolated from the pleural effusion of a 51- year-old female Caucasian (see J. Natl. Cancer Inst. (Bethesda) 53, 661-74 (1974)). Medium for propagation: Leibovitz's L-15 medium, 85%; fetal bovine serum, 15%.

(iii) Colo 205: ATCC CCL 222. This cell line was isolated from ascitic fluid of a 70-year-old Caucasian male with carcinoma of the colon (see Cancer Res. 38, 1345-55 (1978)). Medium for propagation: RPMI 1640, 90%; heat-inactivated fetal bovine serum, 10%.

(iv) HCT 1 16: ATCC CCL 247. The cells belong to one of three strains of malignant cells isolated in 1979 from a male patient with colon carcinoma (see Cancer Res. 4 _, 1751 -56 (1981)). Medium for propagation: McCoy's 5a, 10 % fetal calf serum.

(v) WiDr: ATCC CCL 218. The cell line was initiated through explant culture of tissue from a primary adenocarcinoma of the rectosigmoid colon from a 78-year-old female (see In Vitro 15, 401-8 (1979)). Medium for propagation: Eagle's Minimal Essential Medium with non- essential amino acids and Earle's BSS, 90%; fetal bovine serum, 10%.

(vi) NCI-H69: ATCC HTB 119. The cell line was derived from the pleural fluid of a 55-year- old Caucasian male with small cell carcinoma of the lung (see Cancer Res. 40, 3502-7 (1980)). Medium for propagation: RPMI 1640, 90%; fetal bovine serum, 10%.

(vii) NCI-H209: ATCC HTB 172. This cell line was derived from the bone marrow of a Caucasian male with small cell cancer of the lung (see Cancer Res. 45, 2913-23 (1985)). Medium for propagation: Iscove's modified Dulbecco's medium, 90%; fetal bovine serum, 10%.

(viii) NCI-H460: ATCC HTB 177 . This cell line was designed from the plueral fluid of a male with large lung cell carcinoma in 1982 (see A.F Gazdar et al., Science 246, 491 -494 (1989)) Medium for propagation: RPMI 1640, 10 % fetal calf serum.

(ix) NCI-H520: ATCC HTB 182. This cell line was established in serum-free medium in 1982 from a lung mass taken from a male with squameous cell carcinoma of the lung (see Cancer Res. 46, 798-806 (1986)). Medium for propagation: RPMI 1640, 90 %; fetal bovine serum, 10%. (x) SK-mel 3: ATCC HTB 69. The cell line was isolated from tumor cells released by trypsinization of lymph node metastases (see "Human Tumor Cells In Vitro", pp. 1 15-159, J. Fogh (ed.), Plenum Press, New York (1975)). Medium for propagation: McCoy's 5a medium, 90%; fetal bovine serum, 10%.

(xi) SK-mel 1 : ATCC HTB 67. The cell line was isolated using cells obtained from the thoracic duct of a patient with widespread and rapidly progressing malignant melanoma (J. Natl. Cancer Inst. (Bethesda) 41, 827-31 (1968)). Medium for propagation: Eagle's Minimal Essential Medium with non-essential amino acids, sodium pyruvate (1 mM) and Earle's BSS, 90%; fetal bovine serum, 10%.

(xii) NIH:Ovcar3: ATCC HTB 161 . This cell line was established from the malignant ascites of a patient with progressive adenocarcinoma of the ovary (see Cancer Res. 43, 5379-89 (1983)). Medium for propagation: RPMI with 10 μg/ml insulin, 80%; fetal bovine serum, 20%.

(xiii) PC3: ATCC CRL 1435. The cell line was initiated from a grade IV prostatic adenocarcinoma from a 62-year-old male Caucasian (see Invest. Urol. 17, 16-23 (1979) and Cancer Res. 40, 524-34 (1980)). Medium for propagation: Ham's F12K medium, 93 %; fetal bovine serum, 7%.

(xiv) DU145: ATCC HTB 81. This cell line was isolated by K.R. Stone and associates fro a lesion in the brain of a patient with widespread metastatic carcinoma of the prostate and a 3-year history of lymphocytic leukemia (see D.D. Mickey et al. Cancer Res. 37, 4049-4058, (1977)). Medium for propagation: Eagle's MEM, 10 % fetal calf serum.

Animals are kept under sterile conditions with free access to food and water. For all in vivo experiments, tumors are serially passaged by a minimum of three consecutive transplanta¬ tions prior to start of treatment. Tumor fragments (approx. 25 mg) are implanted s.c. into the left flank of the animals with a 13-gauge trocar needle under Forene (Abbott, Switzerland) anesthesia. Treatments are started when the tumors reach a mean tumor volume of ap¬ proximately 100 mm3, Tumor growth is monitored twice weekly and 24 hours after the last treatment by measuring perpendicular diameters. Tumor volumes are calculated as descri- bed (Evans, BD, Mith, IE, Shorthouse, AJ, Millar, JJ. A comparison of the response of hu¬ man cell carcinoma to vindesine and vincristine. Brit. J. Cancer 45, 466-468 (1982)). T/C % data are percent values of Tumor versus Control.

Treatment with the respective compounds or combinations was made according to the following time schedule in the Examples mentioned hereinafter:

Example Time schedule for administration:

Ex. 1 placebo and SEQ-ID NO: 1-ODN: once daily for 32 consecutive days starting with day 6 after tumor transplantation; adriamycin: once weekly on days 6 and 13; ifosfamide: once weekly for 4 consecutive weeks (on days 6, 13, 20 and 27); tamoxifen: three times weekly on days 6-8, 13-15, 21 -23 and 28-30, respectively.

Ex. 2 placebo: once daily for 21 consecutive days, starting with day 7 after tumor transplantation; SEQ-ID NO: 1 -ODN: once daily for 29 consecutive days, starting with day 7 after tumor transplantation; adriamycin: once weekly on day 7, 14 and 28, respectively.

Ex. 3 placebo: twice daily for 21 days, starting with day 12 after tumor transplantation; SEQ-ID NO: 1 -ODN and estracyt: once daily for 21 consecutive days, starting with day 12 after tumor transplantation; cisplatin: once a week on days 12 and 19 after tumor transplantation.

Ex. 4 placebo, estracyt and SEQ-ID NO: 1 -ODN: once daily for 14 consecutive days, starting from day 9 after transplantation; cisplatin: once weekly on days 9 and 16 after transplantation.

Ex. 5 placebo and SEQ-ID NO: 1 -ODN: once daily for 17 consecutive days, starting on day 12 after transplantation; 5-fluorouracil and adriamycin: once weekly on days 12, 19 and 26 after transplantation. Ex. 6 placebo and SEQ-ID NO: 1 -ODN: once daily for 15 consecutive days, starting on day 4 after transplantation; adriamycin, cisplatin and 5-fluoro uracil: once weekly on days 4 and 1 1 after tumor transplantation.

Ex. 7 placebo and SEQ-ID NO: 1 -ODN: once daily for 25 consecutive days, starting on day 10 after transplantation; 5-fluorouracil: once weekly for 4 consecutive weeks on day 10, 17, 24 and 31 ; adriamycin: once weekly for 3 consecutive weeks (day 10, 17 and 24)

Ex. 8 placebo and SEQ-ID NO: 1-ODN: once daily for 22 consecutive days, starting on day 13 after transplantation; mitomycin and ifosfamide: once weekly on days 13, 20 and 27; cisplatin: once weekly on days 13 and 20

Ex. 9 placebo: once weekly on day 4 and 11 after transplantation; SEQ-ID NO: 1- ODN: once daily for 14 consecutive days, starting on day 4 after transplan tation; mitomycin: once weekly on days 4, 11 and 18 after tumor transplan tation; ifosfamide: once weekly on days 4, 1 1 and 18 after transplantation; cisplatin: once weekly on days 4 and 11 after transplantation.

Ex. 10 placebo and SEQ-ID NO: 1-ODN: once daily for 11 consecutive days, starting on day 8 after transplantation; adriamycin, mitomycin and ifosfamide: once weekly on days 8 and 15 after transplantation

Ex. 1 1 placebo and SEQ-ID NO: 1 -ODN: once daily for 15 consecutive days, starting on day 14 after transplantation; adriamycin and cisplatin: once weekly on days 14 and 21 after transplantation.

Ex. 12 placebo: once weekly on day 1 1 , 18 and 25 after transplantation; adriamycin and mitomycin: once weekly on day 1 1 , 18 and 25 after tumor transplantation; SEQ-ID NO: 1 -ODN: once daily starting on day 1 1 after transplantation.

Ex. 13 placebo: twice daily for 35 consecutive days, starting on day 6 after transplantation; adriamycin: once weekly on days 6, 13 and 20 after transplantation; mitomycin: once weekly on days 6, 13, 20 and 27 after transplantation

(D) Results:

Example 1 : Effect of SEQ-ID NO: 1 -ODN in combination with tamoxifen. adriamvcine or ifosfamide on the growth of s.c. transplanted human breast carcinoma MCF-7 in female Balb/c nude mice:

Treatment Tumor volume in mm (mean ± sem) on T/C%

Figure imgf000078_0001

Placebo treated controls 116 199 262 455 718 928 1375 100

(NaCl 0.9 %, 10 ml/kg i.v.) ±15 ±38 ±49 ±55 ±120 ±102 ±126

SEQ-ID: NO-1 -ODN alone 1 10 155 206 271 376 503 683 46

(NaCl 0.9 %, 10 ml/kg i.v.) ±21 ±45 ±38 ±36 ±63 ±85 ±110

Adriamycine alone 93 125 171 209 269 334 441 28

(NaCl 0.9%, 9 mg/kg i.v.) ±36 ±33 ±33 ±43 ±62 ±69 ±55

SEQ-ID: NO-1 -ODN + 81 86 91 91 1 12 128 147 5 adriamycine (6 mg/kg i.v. ±25 ±22 ±26 ±26 ±30 ±41 ±48 and 9 mg/kg i.v.)

Ifosfamide alone (NaCl 83 115 134 203 310 378 542 36

0.9%, 150 mg/kg i.v.) ±26 ±27 ±31 ±76 ±168 ±170 ±269

SEQ-ID: NO-1 -ODN + 94 1 10 132 161 195 258 345 20 ifosfamide (6 mg/kg i.v. ±22 ±21 ±27 ±45 ±55 ±107 124 and 150 mg/kg i.v.) Tamoxifen alone (methyl- 80 107 162 250 375 512 687 48 cell. 0.5%, 20 mg/kg p.o.) ±18 ±19 ±24 ±20 ±43 ±61 ±67

SEQ-ID: NO-1 -ODN + 116 118 133 159 213 261 327 U_ tamoxifen (6 mg/kg i.v. + ±38 ±35 ±28 ±33 ±52 ±67 ±84

20 mg/kg p.o. in NaCl 0.9 % and 0.5% meth.-cellul.)

Example 2: Effect of SEQ-ID: NO-1-ODN in combination with adriamycin on the growth of s.c. transplanted human breast carcinoma MDA-MB 231 (estrogen independent) in female Balb/c nude mice:

Treatment Tumor volume in mm3 (mean ± sem) on T C

% day day day day day day day 7 1 1 15 18 22 25 ' 28

Placebo treated controls 77 126 301 442 780 1076 1419 100 (NaCl 0.9 %, 10 ml/kg i.v.) ±10 ±16 ±29 ±47 ±90 ±121 ±119

SEQ-ID: NO-1 -ODN alone 77 105 142 196 261 339 447 28

(NaCl 0.9 %, 6 mg/kg i.v.) ±10 ±10 ±23 ±38 ±56 ±72 ±92

Adriamycine alone 81 102 160 278 439 708 936 64

(NaCl 0.9 %, 9 mg/kg i.v.) ±10 ±19 ±28 ±57 ±51 ±88 ±68

SEQ-ID: NO-1 -ODN + 70 77 85 107 130 154 181 8 adriamycine (6 mg/kg i.v. ±7 ±10 ±14 ±25 ±32 ±32 ±41 + 9 mg/kg i.v., NaCl 0.9%)

SEQ-ID: NO-1 -ODN alone 70 101 128 163 197 259 348 21 (NaCI 0.9%, 0.6mg/kg i.v.) ±7 ±21 ±34 ±58 ±78 ±84 ±106

SEQ-ID: NO-1 -ODN alone 67 97 134 187 270 391 570 37

(NaCl 0.9 %, 0.06 mg/kg ±6 ±13 ±24 ±42 ±59 ±58 70 i.v.)

SEQ-ID: NO-1 -ODN + ad- 70 99 123 175 235 361 422 26 riamycin (0.06 mg/kg i.v. + ±7 ±26 ±35 ±40 ±73 ±103 ±138 9.0 mg/kg i.v., NaCl 0.9%)

Example 3: Effect of SEQ ID: NO-1-ODN in combination with estracyt or cisplatin against the s.c. transplanted human prostate carcinoma PC3 in male Balb/c nude mice

Treatment Tumor volume in mm3 (mean ± sem) on T/C%

Figure imgf000080_0001

Placebo treated controls 113 281 769 1110 1525 100

(water, 25 ml/kg p.o.) ±0 ±16 ±78 ±96 ±160

SEQ-ID: NO-1 -ODN alone 107 139 224 270 370 19

(NaCl 0.9%, 6 mg/kg i.v.) ±14 ±44 ±118 ±164 ±193

estracyt alone (50 mg/kg 107 293 690 945 1230 80 i.v. in 5 % mannitol) ±14 ±16 ±97 ±160 ±189

SEQ-ID: NO-1 -ODN + 113 123 183 246 291 13 estracyt (6 mg/kg and 50 ±0 ±16 ±31 ±68 ±76 mg/kg in NaCl 0.9 % and mannitol 5%)

Figure imgf000081_0001

Example 4: Effect of SEQ ID: NO-1 -ODN in combination with estracyt or cisplatin against the s.c. transplanted human prostate carcinoma DU145 in male Balb/c nude mice

Treatment Tumor volume in mm3 (mean ± sem) on T/C% day day day day day 9 13 17 20 23

Placebo treated controls 116 451 852 1323 1729 100

(NaCl 0.9%, 10 ml/kg i.v.) ±15 ±85 ±138 ±219 ±21 1

SEQ-ID: NO-1 -ODN alone 104 231 437 702 1038 58

(NaCl 0.9 %, 6 mg/kg i.v.) ±15 ±31 ±69 ±100 ±135

estracyt alone (50 mg/kg 135 390 627 916 1254 69 i.v. in 5 % mannitol) ±14 ±105 ±127 ±99 ±47

SEQ-ID: NO-1 -ODN + 113 169 282 415 644 33 estracyt (6 mg/kg and 50 ±0 ±48 ±54 ±35 ±86 mg/kg in NaCl 0.9 % and mannitol 5%) cisplatin alone (11 mg/kg 119 242 417 616 838 45 i.v. in pharmacopoe) ±10 ±52 ±49 ±83 ±65

16

Figure imgf000082_0001

Example 5: Effect of SEQ ID: NO-1-ODN in combination with 5-fluorouracil or adriamycin against the s.c. transplanted human colon carcinoma Colo 205 in female Balb/c nude mice

Treatment Tumor volume in mm3 (mean ± sem) on T/C% day day day day day 12 17 20 24 29

Placebo treated controls 1 16 253 515 1017 171 1 100

(NaCl 0.9%, 10 ml/kg i.v.) ±8 ±39 ±60 ±104 ±213

SEQ-ID: NO-1 -ODN alone 111 126 212 307 445 21

(NaCl 0.9%, 6 mg/kg i.v.) ±17 ±10 ±34 ±68 ±65

5-fluorouracil (75 mg/kg 1 13 131 142 149 157 3 i.v. in NaCl 0.9%) ±13 ±26 ±31 ±37 ±43

SEQ-ID: NO-1 -ODN + 5- 116 78 70 48 24 * fluorouracil (6 mg/kg and ±8 ±19 ±7 ±13 ±10

75 mg/kg in NaCl 0.9 %)

adriamycin alone (9 mg/kg 1 19 177 336 567 819 44 i.v. in 0.9 % NaCl) ±10 ±38 ±76 ±100 ±71 SEQ-ID: NO-1 -ODN + 119 123 142 183 226 adriamycin (6 mg/kg i.v. + ±10 ±10 ±20 ±33 ±41 9 mg/kg i.v. in NaCl 0.9%) * regression (T/C % = -79%); strong synergism

Example 6: Effect of SEQ ID: NO-1-ODN in combination with 5-fluorouracil. adriamycin or cisplatin against the s.c. transplanted human colon carcinoma HCT 116 in female Balb/c nude mice:

Treatment Tumor volume in mm3 (mean ± sem) on T/C%

Figure imgf000083_0001

Placebo treated controls 97 228 600 1066 1697 100

(NaCl 0.9%, 10 ml/kg i.v.) ±20 ±54 ±1 10 ±162 ±277

SEQ-ID: NO-1 -ODN alone 96 185 349 61 1 917 5 .

(NaCl 0.9%, 6 mg/kg i.v.) ±22 ±29 ±53 ±107 ±141

5-fluorouracil (75 mg/kg 94 175 396 672 1107 63 i.v. in NaCl 0.9%) ±20 ±34 ±99 ±147 ±237

SEQ-ID: NO-1-ODN + 5- 93 103 131 204 292 12 fluorouracil (6 mg/kg and ±1 1 ±26 ±36 ±75 ±120 75 mg/kg in NaCl 0.9 %)

adriamycin alone (9 mg/kg 95 182 326 537 857 48 i.v. in 0.9 % NaCl) ±15 ±50 ±42 ±175 ±149

SEQ-ID: NO-1 -ODN + 95 1 17 194 292 491 23 adriamycin (6 mg/kg i.v. + ±15 ±39 ±70 ±84 ±100 9 mg/kg i.v. in NaCl 0.9%)

cisplatin alone (10 mg/kg 95 209 555 1031 1544 9 . i.v. in pharmacopoe) ±15 ±43 ±134 ±141 ±265

12

Figure imgf000084_0001

Example 7: Effect of SEQ ID: NO-1 -ODN in combination with 5-fluorouracil or adriamycin against the s.c. transplanted human colon carcinoma WiDr in male Balb/c nude mice:

Treatment Tumor volume in mm3 (mean ± sem) on T/C%

Figure imgf000084_0002

Placebo treated controls 126 306 857 1139 1438 100

(NaCl 0.9%, 10 ml/kg i.v.) ±16 ±79 ±120 ±200 ±191

SEQ-ID: NO-1 -ODN alone 126 177 291 396 465 26

(NaCl 0.9%, 6 mg/kg i.v.) ±16 ±38 ±76 ±89 ±121

5-fluorouracil (75 mg/kg 1 16 201 400 514 729 47 i.v. in NaCl 0.9%) ±8 ±19 ±56 ±86 ±124

SEQ-ID: NO-1 -ODN + 5- 134 122 214 328 397 20 fluorouracil (6 mg/kg and ±25 ±33 ±33 ±60 ±68 75 mg/kg in NaCl 0.9 %)

adriamycin alone (9 mg/kg 1 19 225 550 821 1091 J i.v. in 0.9 % NaCl) ±10 ±39 ±93 ±134 ±142

SEQ-ID: NO-1 -ODN + 142 164 240 406 494 27 adriamycin (6 mg/kg i.v. + ±31 ±37 ±41 ±34 ±49 9 mg/kg i.v. in NaCl 0.9%)

Example 8: Effect of SEQ ID: NO-1-ODN in combination with mitomycin. ifosfamide or cisplatin against the s.c. transplanted human small cell lung carcinoma NCI H69 in female

Figure imgf000085_0001

(NaCl 0.9%, 10 ml/kg i.v.) ±0 ±21 ±84 ±86 ±138 ±146

SEQ-ID: NO-1 -ODN atone 113 123 156 245 413 734 29

(NaCl 0.9%, 6 mg/kg i.v.) ±0 ±10 ±29 ±47 ±96 ±228

mitomycin (3.5 mg/kg i.v. in NaCl 0.9%)

SEQ-ID: NO-1 -ODN + mitomycin (6 mg/kg and 3.5 mg/kg in NaCl 0.9 %)

SEQ-ID: NO-1 -ODN + mitomycin (6 mg/kg and

Figure imgf000085_0002
3.5 mg/kg in NaCl 0.9 %)

ifosfamide alone (150 1 13 123 140 219 267 394 1J5 mg/kg i.v. in 0.9 % NaCl) ±0 ±10 ±20 ±39 ±31 ±24

SEQ-ID: NO-1 -ODN + 113 119 127 168 191 253 ifosfamide (6 mg/kg i.v. + ±0 ±10 ±27 ±38 ±53 ±74 150 mg/kg i.v./NaCI 0.9%)

SEQ-ID: NO-1 -ODN + 1 13 119 117 160 173 253 ifosfamide (6 mg/kg i.v. + ±0 ±10 ±21 ±52 ±72 ±92 150 mg/kg i.v./NaCI 0.9%)

cisplatin alone (11 mg/kg 1 13 1 16 1 16 15 14 12 i.v. in pharmacopoe) ±0 ±8 ±8 ±2 ±0 ±4

SEQ-ID: NO-1 -ODN + 113 1 13 51 7 1 1 cisplatin (6 mg/kg i.v. ±0 ±0 ±21 ±6 ±2 ±2 and 11 mg/kg i.v. in NaCl 0.9% and pharmacopoe)

* regression (T/C %= -85%)

** strong regression; complete cure in 3 out of 6 animals (T/C% =-99%)

*** strong regression; complete cure in 6 out of 6 animals strong regression; complete cure in 5 out of 6 animals

Example 9: Effect of SEQ ID: NO-1-ODN in combination with mitomycin. ifosfamide or cisplatin against the s.c. transplanted human large cell lung carcinoma NCI H460 in female

Figure imgf000086_0001
(NaCI 0.9%, 10 ml/kg i.v.) ±12 ±83 ±186 ±182 ±232

SEQ-ID: NO-1 -ODN alone 119 343 546 776 1090 not done 46

(NaCl 0.9%, 6 mg/kg i.v.) ±10 ±69 ±123 ±161 ±164

mitomycin (3.5 mg/kg 107 85 65 65 70 54 * i.v. in NaCl 0.9%) ±14 ±14 ±0 ±0 ±7 ±14

SEQ-ID: NO-1 -ODN + 132 88 47 25 8 1 ** mitomycin (6 mg/kg and ±17 ±1 1 ±15 ±8 ±7 ±2

3.5 mg/kg in NaCl 0.9 %)

ifosfamide alone (150 1 16 206 332 568 879 not done 36 mg/kg i.v. in 0.9 % NaCl) ±8 ±61 ±167 ±198 ±210

SEQ-ID: NO-1 -ODN + 131 178 244 296 412 471 13 ifosfamide (6 mg/kg i.v. + ±26 ±61 ±122 ±144 ±222 ±214 150 mg/kg i.v./NaCI 0.9%)

cisplatin alone (11 mg/kg 1 13 152 227 415 679 1021 27 i.v. in 0.9 % NaCl) ±0 ±34 ±106 ±1 15 ±80 ±85

SEQ-ID: NO-1 -ODN + 131 157 197 254 332 676 10 cisplatin (6 mg/kg i.v. ±26 ±36 ±77 ±121 ±155 ±273 and 1 1 mg/kg i.v. in NaCl 0.9%)

* regression (T/C % = -47)

** complete cure in 4 out of 6 animals after 22 days, in 5 out of 6 animals after 29 days

T/C % on day 18 = -94%). Example 10: Effect of SEQ ID: NO-1 -ODN in combination with mitomycin. ifosfamide or adriamycin against the s.c. transplanted human squameous cell lung carcinoma NCI H520 in female Balb/c nude mice:

Treatment Tumor volume in mm3 (mean ± sem) on T/C% day day day day

8 12 16 20

Placebo treated controls 187 529 980 1739 100

(NaCl 0.9%, 10 ml/kg i.v.) ±15 ±44 ±68 ±1 15

SEQ-ID: NO-1 -ODN alone 151 225 329 557 26

(NaCl 0.9%, 6 mg/kg i.v.) ±35 ±60 ±91 ±127

mitomycin (3.5 mg/kg 197 226 238 291 6 i.v. in NaCl 0.9%) ±13 ±40 ±48 ±103

SEQ-ID: NO-1-ODN + 193 197 197 212 1 mitomycin (6 mg/kg and ±14 ±21 ±21 ±34 3.5 mg/kg in NaCl 0.9 %)

ifosfamide alone (150 232 382 790 1485 81 mg/kg i.v. in 0.9 % NaCl) ±40 ±53 ±727 ±107

SEQ-ID: NO-1 -ODN + 199 188 247 358 10 ifosfamide (6 mg/kg i.v. ÷ ±35 ±62 ±80 ±105 150 mg/kg i.v./NaCI 0.9%)

adriamycin alone (9 mg/kg 193 317 586 990 51 i.v. in 0.9 % NaCl) ±14 ±52 ±83 ±78 SEQ-ID: NO-1-ODN + 193 191 263 359 11 adriamycin (6 mg/kg i.v. + ±14 ±52 ±73 ±93 9 mg/kg i.v. in NaCl 0.9%)

Example 11 : Effect of SEQ ID: NO-1-ODN in combination with adriamycin or cisplatin against the s.c. transplanted human ovarian carcinoma NIH:Ovcar3 in female Balb/c nude mice:

Treatment Tumor volume in mm3 (mean ± sem) on T/C%

Figure imgf000089_0001

Placebo treated controls 155 363 590 1078 1709 100

(NaCl 0.9%, 10 ml/kg i.v.) ±31 ±125 ±138 ±217 ±213

SEQ-ID: NO-1 -ODN alone 138 382 577 919 1340 77

(NaCl 0.9 %, 6 mg/kg i.v.) ±50 ±141 ±203 ±192 ±231

adriamycine alone (9 62 145 256 389 543 31 mg/kg i.v. in NaCl 0.9%) ± ±54 ±1 Q2 ±g8 ±2Q5

SEQ-ID: NO-1 -ODN + 118 205 262 337 454 22 adriamycin (6 mg/kg i.v. ±35 ±37 ±89 ±127 ±186 and 9 mg/kg in NaCl 0.9%

cisplatin alone (10 mg/kg 100 105 105 97 95 * i.v. in NaCl 0.9%) ±28 ±37 ±37 ±40 ±42

SEQ-ID: NO-1 -ODN + 74 106 113 140 163 6 cisplatin (6 mg/kg i.v. ±24 ±31 ±48 ±89 ±121 and 10 mg/kg i.v. in NaCl 0.9%)

* regression (T/C % = -5%)

Example 12: Effect of SEQ ID: NO-1 -ODN in combination with mitomycin or adriamycin against the s.c. transplanted human melanoma SK-mel 1 in male Balb/c nude mice:

Treatment Tumor volume in mm3 (mean ± sem) on T/C% day day day day 1 1 18 25 32

Placebo treated controls 126 269 635 1025 100

(NaCl 0.9%, 10 ml/kg i.v.) ±16 ±36 ±118 ±130

SEQ-ID: NO-1 -ODN alone 113 86 136 184 8

(NaCl 0.9%, 6 mg/kg i.v.) ±0 ±24 ±36 ±50

mitomycin (3.5 mg/kg i.v. in NaCl 0.9%)

SEQ-ID: NO-1-ODN +

Figure imgf000090_0001
mitomycin (6 mg/kg and ±14 ±32 ±47 ±54 3.5 mg/kg in NaCl 0.9 %)

adriamycin alone (9 mg/kg 123 181 241 518 44 i.v. in 0.9 % NaCl) ±10 ±43 ±48 ±150

SEQ-ID: NO-1 -ODN + 126 101 137 172 5 adriamycin (6 mg/kg i.v. + ±16 ±42 ±47 ±58 9 mg/kg i.v. in NaCl 0.9%)

* regression (T/C % = -50) Example 13: Effect of SEQ ID: NO-1 -ODN in combination with mitomycin or adriamycin against the s.c. transplanted human melanoma SK-mel 3 in male Balb/c nude mice:

Treatment Tumor volume in mm3 (mean ± sem) on T/C%

Figure imgf000091_0001

Placebo treated controls 65 120 192 318 592 786 100

(water, 25 ml/kg p.o.) ±0 ±30 ±60 ±110 ±142 ±174

SEQ-ID: NO-1 -ODN alone 65 67 90 105 134 176 15

(NaCl 0.9%, 6 mg/kg i.v.) ±0 ±6 ±20 ±21 ±45 ±71

mitomycin (3.5 mg/kg 65 69 91 98 146 182 1 i.v. in NaCl 0.9%) ±0 ±23 ±56 ±64 ±108 ±134

SEQ-ID: NO-1 -ODN + 65 36 24 16 11 7 * mitomycin (6 mg/kg and ±0 ±5 ±10 ±1 1 ±13 ±13 3.5 mg/kg in NaCl 0.9 %)

adriamycin alone (9 mg/kg 65 98 147 21 1 299 397 46 i.v. in 0.9 % NaCl) ±0 ±22 ±49 ±69 ±95 ±123

SEQ-ID: NO-1 -ODN + 65 44 68 68 81 104 5 adriamycin (6 mg/kg i.v. + ±0 ±17 ±37 ±37 ±43 ±55

9 mg/kg i.v. in NaCl 0.9%)

* regression (T/C % = -89) and complete cure in 3 out of 6 animals

Discussion of the Examples with combination studies with the SEQ-ID NO: 1 -ODN and standard chemotherapeutic drugs:

The chemotherapeutic drugs adriamycin, estracyt, cisplatin, 5-fluorouracil, mitomycin, ifosfamide and tamoxifen are applied according to established chemotherapeutic schedules for the respective tumor types. The SEQ-ID NO: 1-ODN exerts improved antitumor effects with adriamycin, estracyt, 5-fluorouracil, ifosfamide and tamoxifen against human breast, prostate, colon, ovarian and melanoma tumors transplanted into nude mice. The combina¬ tions of SEQ-ID NO: 1 -ODN with fluorouracil in human Colo205 colon carcinomas results in strong tumor regression. The combinations of SEQ-ID NO: 1-ODN with mitomycin in SK- mel3 melanomas results in strong regression in all and complete cure in half of the animals observed. The combinations of SEQ-ID NO: 1-ODN with cisplatin in PC3 human prostate carcinomas result in an especially highly synergistic effect with complete tumor cures observed. The combination of SEQ-ID NO: 1-ODN and mitomycin in NCI-H69 lung carcinomas also results in a strong synergistic antitumor effect with complete cures. The same result is found with the combination of SEQ-ID NO: 1-ODN and cisplatin in NCI-H69 small cell lung cancers and with the combination of SEQ-ID NO: 1-ODN and mitomycin in NCI-H460 large cell carcinomas. In other lung carcinomas, positive effects with mitomycin or cisplatin and SEQ-ID NO: 1-ODN are found. Most importantly, in no case antagonistic influences between SEQ-ID NO: 1-ODN and chemotherapeutic agents or increased toxicity are observed. These results indicate that the SEQ-ID NO: 1 -ODN exerts beneficial antitumor effects both as single agent and in an improved manner in combination with chemotherapeutic drugs in the treatment of human cancer.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: CIBA-GEIGY AG

(B) STREET: Klybec str. 141

(C) CITY: Basel

(E) COUNTRY: Switzerland

(F) POSTAL CODE (ZIP): 4002

(G) TELEPHONE: +41 61 69 11 11 (H) TELEFAX: + 41 61 696 79 76 (I) TELEX: 962 991

(A) NAME: ISIS Pharmaceuticals, Inc.

(B) STREET: 2280 Faraday Avenue

(C) CITY: Carlsbad

(E) COUNTRY: United States of America

(F) POSTAL CODE (ZIP): CA 92008

(ii) TITLE OF INVENTION: Antiproliferative combinations

(iii) NUMBER OF SEQUENCES: 2

(iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

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

(D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (EPO)

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "antisense oligonucleotide"

(iv) ANTI-SENSE: YES

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

TCCCGCCTGT GACATGCATT 20

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(A) DESCRIPTION: /desc = "antisense oligonucleotide"

(iv) ANTI-SENSE: YES

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

GTTCTCGCTG GTGAGTTTCA 20

Claims

What is claimed is
1. A method for treating a proliferative disease that can be treated by administration of an oligonucleotide or oligonucleotide derivative targeted to raf, where a) at least one oligonucleotide or oligonucleotide derivative targeted to nucleic acids encoding raf and capable of modulating raf expression and b) at least one other chemotherapeutic agent are administered to a mammal in combination in a quantity which is jointly therapeutically effective against proliferative diseases that can be treated by administration of an oligonucleotide or oligonucleotide derivative targeted to raf in order to treat them, where any component a) and/or b) can also be present in the form of a pharmaceutically acceptable salt, if at least one salt-forming group is present.
2. The method according to claim 1 wherein the combination has an enhanced effect when compared with the effect of the single components a) or b).
3. The method according to claim 1 wherein the combination of component a) and b) leads to synergism or to tumor regression, or both.
4. The method according to claim 1 wherein a) an oligonucleotide derivative that corresponds to the following sequence:
5'-TCCCGCCTGTGACATGCATT-3' SEQ.-ID NO: 1,
or also a shortened version thereof
and b) at least one other chemotherapeutic agent selected from
(A) alkylating agents selected from nitrogen mustards; ethyleneimine derivatives; procarbazine; alkyl sulfonates; cyclophosphamide; 4-hydroxyperoxycyclophosphamide; mafosfamide; ifosfamide; melphalan; chlorambucil; nitrosoureas; cis-platinum(ll)-di- aminedichloride; and carboplatin; (B) antitumor antibiotics selected from the group comprising bleomycine; anthracyclines; and cross-linking antitumor antibiotics;
(C) antimetabolites selected from the group comprising folic acid analogues; purine nucleoside analogues; pyrimidine analogues; hydroxyurea; and polyamine biosynthesis inhibitors;
(D) plant alkaloids selected from the group comprising vinca alkaloids; and epipodophyllotoxins;
(E) hormonal agents and antagonists selected from adrenocorticoids; progestines; androgens; estrogens; synthetic analogues of LHRH; synthetic analogues of LH-releasing hormone; anti-androgens; anti-estrogens; aromatase inhibitors; adrenal cyctooxic agents; somatostatine analogues; and 5α-reductase inhibitors;
(F) biological response modifiers selected from lymphokines; and interferons;
(G) inhibitors of protein tyrosine kinases and/or serine/threonine kinases other than ODNs; (H) antisense oligonucleotides or oligonucleotide derivatives targeted to other targets than raf; and
(I) miscellaneous agents or agents with other or unknown mechanism of action selected from S-triazine derivatives; enzymes; methylhydrazine derivatives; matrix metalloproteinase inhibitors; anthraquinones; mitotic spindle poisons; streptozocin; estracyt; amsacrine; differentiating agents; immunomodulators; vaccines; and antibodies with antitumor activity
are in combination; where component a) and b) show enhanced activity over the single components or synergism; and where any component a) or b) can also be present in the form of a pharmaceutically acceptable salt, if at least one salt-forming group is present.
5. The method according to claim 1 wherein a) an oligonucleotide derivative that corresponds to the following sequence:
5'- TCC CGC CTG TGA CAT GCA TT -3' SEQ.-ID NO: 1 ,
or also a version thereof with 15 to 19 building blocks which is terminally shortened on one or both ends,
which is present as an oligonucleotide derivative which comprises at least one of the following units of the formulae given hereinafter, wherein B is a base radical of a base selected from thymine, uracil, 5-propynyluracil, cytosine, 5-methylcytosιne, 5-propynyl- cytosine, adenine, 2-aminoadenine or guanine; Q is H, OCH3, F,
Figure imgf000097_0001
wherein v is from 0 to 12, CH2CH(CH3)OCH3 or CH2CH(OH)CH2OH; and the other moieties have the meanings given behind the respective formula:
Figure imgf000097_0002
Figure imgf000097_0004
Figure imgf000097_0003
Figure imgf000097_0005
the remaining internucleosidic bonds in the respective oligonucleotide derivative being of the phosphorothioate type; or
as oligonucleotide derivative which comprises at least one of the following dimenc units of the formulae given hereinafter, wherein each B is, independently of the other, a base radical selected from thymine, uracil, 5-propynyluracil, cytosine, 5-methylcytosιne, 5- propynylcytosine, adenine, 2-aminoadenine and guanine; any Q is, indepently of the other, H, OCH3, F or O(CH2CH2θ)vCH3 wherein v is from 0 to 12; and the other moieties have the meanings given behind the respective formula:
O
Figure imgf000098_0001
Figure imgf000098_0002
wherein A is H, methyl or phenyl; the remaining internucleosidic bonds in the respective oligonucleotide derivative being of the phosphorothioate type; and
b) at least one other chemotherapeutic agent selected from
(A) alkylating agents selected from dacarbazine; nitrogen mustards; triethylene¬ thiophosphoramid; procarbazine; busulfan; 4-hydroxyperoxycyclophosphamide; mafosfamide; ifosfamide; melphalan; chlorambucil; cyclohexylnitrosourea; lomustine; cis- platinum(il)-diaminedichloride; and carboplatin;
(B) antitumor antibiotics selected from bleomycine daunomycin, dactinomycin, daunorubicin epirubicin, esorubicin, idarubicin, plicamycin and mitomycin C;
(C) antimetabolites selected from methotrexate, trimetrexate, Cladribine, 6-mercaptopurine, pentostatin, 6-thioguanine, 5-fluorouracil, 5-fluorodeoxyuridine, cytosine arabinoside, fludarabin phosphate, 5-azacytidine, hydroxyurea, and 4-amidino-1-indanon-2'- amidinohydrazone;
(D) plant alkaloids selected from vinblastine, vincristine, vindesine, etoposide and teniposide;
(E) hormonal agents and antagonists selected from prednisone, dexamethasone, hydroxy- progesterone, megestrol acetate, medroxyprogesterone, testosterone, fluoxymesterone, di- ethylstilbestrol, estradiol, chlorotriansiene, goserelin, leuprolide, flutamide, tamoxifen, ami- nogluthetimide, lentaron, 5-(p-cyanophenyl)-5,6,7,8-tetrahydroimidazo[1 ,5-a]pyridin, 4,4'-
(1 H-1 ,2,4-triazol-1 -yl-methylen)-bis-benzonitrile, 4-(α-(4-cyanophenyl)-α-fluoro-1 -(1 ,2,4-tri- azolyl)methyl)-benzonitrile, 4-(α-(4-cyanophenyl)-(2-tetrazolyl)methyl)-benzonitrile, mitotane, octreotide and N-(1 -cyano-1 -methyl-ethyl)-4-aza-3-oxo-5α-androst-1 -en-17β-carboxamid;
(F) biological response modifiers selected from aldesleukin, interferon-α and interferon "B1B2B3D4";
(G) inhibitors of protein tyrosine kinases and/or serine/threonine kinases selected from N-{5- [4-methyl-piperazino-methyl)-benzoylamido]-2-methyl-phenyl}-4-(3-pyridyl)-2-pyrimidine, N- (3-chlorophenyl)-4-(2-(3-hydroxy)-propyl-amino-4-pyridyl)-2-pyrimidinamin, N-benzoyl-stau- rosporine, 4,5-bis(anilino)-phthalimide, N-(5-benzoylamido-2-methyl-phenyl)-4-(3-pyridyl)-2- pyridinamin and 4-(m-chloranilino)-5,6-dimethyl-7H-pyrrolo[2,3-d]pyrimidin;
(H) antisense oligonucleotides or oligonucleotide derivatives targeted to other targets selected from SAMDC and protein kinase C; and
(I) miscellaneous agents or agents with other or unknown mechanism of action selected from altrematine; asparaginase; dacarbazine; procarbazine; hexamethylmelamine; pentamethylmelamine; mitoxantrone; paclitaxel; streptozocin; estracyt; amsacrine; all-trans retinoic acid; levamisole; anti-melanoma vaccines, recombinant human immunoglobulins directed at melanoma antigen and antibodies for active immunotherapy of melanoma;
are in combination; and where any component a) and b) can also be present in the form of a pharmaceutically acceptable salt, if at least one salt-forming group is present.
6. The method according to claim 1 wherein the oligonucleotide sequence corresponds to the following sequence: 5'- TCC CGC CTG TGA CAT GCA TT -3' SEQ.-ID NO: 1 ,
or also a version thereof with 15 to 19 building blocks which is terminally shortened on one or both ends,
and which is present in the form of a chimeric oligonucleotide.
7. The method according to claim 1 wherein the oligonucleotide derivative corresponds to the following sequence:
5'-TCCCGCCTGTGACATGCATT-3' SEQ.-IDNO: 1,
or also a version thereof with 15 to 19 building blocks which is terminally shortened on one or both ends,
and is an oligonucleotide derivative which is present in the form of a chimeric oligonucleo¬ tide which shows, on the one hand, M regions of between 3 and 20 2'-modified building blocks either with phosphodiester or with phosphorothioate backbone, which are in succeeding order; and, on the other hand, U regions of 4 to 20 2'-deoxy building blocks with phosphorothioate structure that are otherwise unmodified.
8. The method according to claim 1 wherein a) an oligonucleotide derivative that corresponds to the following sequence:
5'- TCC CGC CTG TGA CAT GCA TT -3' SEQ.-ID NO: 1 ,
or also a version thereof with 15 to 19 building blocks which is terminally shortened on one or both ends,
which is present as an oligonucleotide derivative which comprises at least one of the following units of the formulae given hereinafter, wherein B is a base radical of a base selected from thymine, uracil, 5-propynyluracil, cytosine, 5-methylcytosine, 5-propynyl- cytosine, adenine, 2-aminoadenine or guanine; Q is H, OCH3, F, O(CH2CH2θ)vCH3 wherein v is from 0 to 12, CH2CH(CH3)OCH3 or CH2CH(OH)CH OH; and the other moieties have the meanings given behind the respective formula:
Figure imgf000101_0001
/ o
X — P I =Y
Figure imgf000101_0002
Radical of formula type
(llg), phosphorothioate X = SH Y = O or S dig*)
(llh) phosphodiester X = OH Y = O
(llh*)
the remaining internucleosidic bonds in the respective oligonucleotide derivative being of the phosphorothioate type; or
as oligonucleotide derivative which comprises at least one of the following dimeric units of the formulae given hereinafter, wherein each B is, independently of the other, a base radical selected from thymine, uracil, 5-propynyluracil, cytosine, 5-methylcytosine, 5- propynylcytosine, adenine, 2-aminoadenine and guanine; any Q is, indepently of the other, H, OCH3,F or O(CH2CH2θ)vCH3 wherein v is from 0 to 12; and the other moieties have the meanings given behind the respective formula:
Figure imgf000102_0001
O:
Figure imgf000102_0002
Radical of formula type X** Y**.
(Vie), (Vie*) amide CH2 N(A)
wherein A is H, methyl or phenyl; the remaining internucleosidic bonds in the respective oligonucleotide derivative being of the phosphorothioate type; and
b) at least one other chemotherapeutic agent selected from bis-alkylating agents selected from the group comprising mechlorethamine, busulfan, melphalan, chlorambucil, cis-platinum(ll)-diaminedichloride, carboplatin and triethylene¬ thiophosphoramid; cross-linking antitumor antibiotics selected from mitomycin C; and purine nucleoside analogues selected from Cladribine, 6-mercaptopurine, pentostatin and 6- thioguanine; and pyrimidine analogues selected from 5-fluorouracil, 5-fluorodeoxyuridine, cytosine arabinoside, fludarabin phosphate and 5-azacytidine;
are in combination; and where any component a) and b) can also be present in the form of a pharmaceutically acceptable salt, if at least one salt-forming group is present.
9. The method according to claim 1 wherein the disease to be treated is selected from cancers, tumors, hyperplasias, fibrosis, angiogenesis, psoriasis, atherosclerosis and smooth muscle cell proliferation in the blood vessels.
10. The method according to claim 1 wherein the disease to be treated is selected from small cell lung carcinoma, large cell lung carcinoma, melanoma and prostate carcinoma.
11. The method according to claim 1 wherein
a) an oligonucleotide derivative that corresponds to the following sequence:
5'- TCC CGC CTG TGA CAT GCA TT -3' SEQ.-ID NO: 1 ,
or also a version thereof with 15 to 19 building blocks which is terminally shortened on one or both ends,
which is present in the form of a phosphorothioate oligo-2'-deoxyπucleotide analogue, and
b) at least one other chemotherapeutic agent selected from ifosfamide, cisplatin, mitomycine and 5-fluorouracil
are in combination; and where any component a) and b) can also be present in the form of a pharmaceutically acceptable salt, if at least one salt-forming group is present.
12. The method according to claim 1 wherein
a) an oligonucleotide derivative that corresponds to the following sequence:
5'- TCC CGC CTG TGA CAT GCA TT -3' SEQ.-ID NO: 1 ,
which is present in the form of a phosphorothioate oligo-2'-deoxynucleotide analogue, and b) any one of the following other chemotherapeutic agents for the treament of the respective proliferative disease:
- cisplatin for human prostate carcinomas;
- mitomycin for small lung cell carcinomas;
- cisplatin for small cell lung cancers: or
- mitomycin for large cell lung carcinomas
are in combination; where any component a) and b) can also be present in the form of a pharmaceutically acceptable salt, if at least one salt-forming group is present.
13. The method according to claim 1 wherein
a) an oligonucleotide derivative that corresponds to the following sequence:
5'- TCC CGC CTG TGA CAT GCA TT -3' SEQ.-ID NO: 1 ,
which is present in the form of a phosphorothioate oligo-2'-deoxynucleotide analogue, and
b) any one of the following other chemotherapeutic agents for the treament of the respective proliferative disease:
- fluorouracil for colon cancer; or
- mitomycin for melanoma
are in combination; where any component a) and b) can also be present in the form of a pharmaceutically acceptable salt, if at least one salt-forming group is present.
14. A pharmaceutical preparation which comprises a quantity, which is jointly effective for treating a proliferative disease that can be treated by administration of an oligonucleotide or oligonucleotide derivative targeted to raf, of a) at least one oligonucleotide or oligonucleotide derivative (ODN) targeted to nucleic acids encoding raf and b) at least one other chemotherapeutic agent, where any component a) and/or b) can also be present in the form of a pharmaceutically acceptable salt, if at least one salt-forming group is present, with one or more pharmaceutically acceptable carrier materials.
15. A pharmaceutical preparation according to claim 14 wherein the combination has an enhanced effect when compared with the effect of the single components a) or b)
16. A pharmaceutical preparation according to claim 14 wherein the combination of component a) and b) leads to synergism or to tumor regression, or both
17. A pharmaceutical preparation according to claim 14 wherein a) an oligonucleotide derivative that corresponds to the following sequence:
5'- TCC CGC CTG TGA CAT GCA TT -3' SEQ.-ID NO: 1 ,
or also a shortened version thereof
and b) at least one other chemotherapeutic agent selected from
(A) alkylating agents selected from nitrogen mustards; ethyleneimine derivatives, procarbazine; alkyl sulfonates; cyclophosphamide; 4-hydroxyperoxycyclophosphamιde; mafosfamide; ifosfamide; melphalan; chlorambucil; nitrosoureas; cιs-platιnum(ll)-dι- aminedichloπde; and carboplatin,
(B) antitumor antibiotics selected from the group comprising bleomycine, anthracyclines; and cross-linking antitumor antibiotics,
(C) antimetabolites selected from the group comprising folic acid analogues; purine nucleoside analogues; pyrimidine analogues, hydroxyurea; and polyamine biosynthesis inhibitors;
(D) plant alkaloids selected from the group comprising vmca alkaloids, and epipodophyllotoxins;
(E) hormonal agents and antagonists selected from adrenocorticoids; progestines, androgens, estrogens; synthetic analogues of LHRH, synthetic analogues of LH-releasing hormone; anti-androgens; anti-estrogens; aromatase inhibitors; adrenal cyctooxic agents; somatostatine analogues; and 5α-reductase inhibitors;
(F) biological response modifiers selected from lymphokines; and interferons;
(G) inhibitors of protein tyrosine kinases and/or serine/threonine kinases other than ODNs; (H) antisense oligonucleotides or oligonucleotide derivatives targeted to other targets than raf; and
(I) miscellaneous agents or agents with other or unknown mechanism of action selected from S-triazine derivatives; enzymes; methylhydrazine derivatives; matrix metalloproteinase inhibitors; anthraquinones; mitotic spindle poisons; streptozocin; estracyt; amsacrine; differentiating agents; immunomodulators; vaccines; and antibodies with antitumor activity
are in combination; where component a) and b) show enhanced activity over the single components or synergism; and where any component a) or b) can also be present in the form of a pharmaceutically acceptable salt, if at least one salt-forming group is present.
18. A pharmaceutical preparation according to claim 14 wherein a) an oligonucleotide derivative that corresponds to the following sequence:
5'-TCCCGCCTGTGACATGCATT-3' SEQ.-ID NO: 1,
or also a version thereof with 15 to 19 building blocks which is terminally shortened on one or both ends,
which is present as an oligonucleotide derivative which comprises at least one of the following units of the formulae given hereinafter, wherein B is a base radical of a base selected from thymine, uracil, 5-propynyluracil, cytosine, 5-methylcytosine, 5-propynyl- cytosine, adenine, 2-aminoadenine or guanine; Q is H, OCH3, F, O(CH2CH2O)vCH3 wherein v is from 0 to 12, CH2CH(CH3)OCH3 or CH2CH(OH)CH2OH; and the other moieties have the meanings given behind the respective formula:
Figure imgf000107_0001
/ o
X — P=Y
\
Figure imgf000107_0002
Radical of formula type
(llg), phosphorothioate X = SH Y = O or S
(iig*)
(llh) phosphodiester X = OH Y = O
(llh*)
the remaining internucleosidic bonds in the respective oligonucleotide derivative being of the phosphorothioate type; or
as oligonucleotide derivative which comprises at least one of the following dimeric units of the formulae given hereinafter, wherein each B is, independently of the other, a base radical selected from thymine, uracil, 5-propynyluracil, cytosine, 5-methylcytosine, 5- propynylcytosine, adenine, 2-aminoadenine and guanine; any Q is, indepently of the other, H, OCH3, F or O(CH2CH2O)vCH3 wherein v is from 0 to 12; and the other moieties have the meanings given behind the respective formula: (Vie)
Figure imgf000108_0001
Radical of formula type X** Y**
(Vie), (Vie*) amide CH2 N(A)
wherein A is H, methyl or phenyl; the remaining internucleosidic bonds in the respective oligonucleotide derivative being of the phosphorothioate type; and
b) at least one other chemotherapeutic agent selected from
(A) alkylating agents selected from dacarbazine; nitrogen mustards; triethylene¬ thiophosphoramid; procarbazine; busulfan; 4-hydroxyperoxycyclophosphamide; mafosfamide; ifosfamide; melphalan; chlorambucil; cyclohexylnitrosourea; lomustine; cis- platinum(ll)-diaminedichloride; and carboplatin;
(B) antitumor antibiotics selected from bleomycine daunomycin, dactinomycin, daunorubicin epirubicin, esorubicin, idarubicin, piicamycin and mitomycin C;
(C) antimetabolites selected from methotrexate, trimetrexate, Cladribine, 6-mercaptopurine, pentostatin, 6-thioguanine, 5-fluorouracil, 5-fluorodeoxyuridine, cytosine arabinoside, fludarabin phosphate, 5-azacytidine, hydroxyurea, and 4-amidino-1 -indanon-2'- amidinohydrazone;
(D) plant alkaloids selected from vinblastine, vincristine, vindesine, etoposide and teniposide; (E) hormonal agents and antagonists selected from prednisone, dexamethasone, hydroxy- progesterone, megestrol acetate, medroxyprogesterone, testosterone, fluoxymesterone, di- ethylstilbestrol, estradiol, chlorotriansiene, goserelin, leuprolide, flutamide, tamoxifen, ami- nogluthetimide, lentaron, 5-(p-cyanophenyl)-5,6,7,8-tetrahydroimidazo[1 ,5-a]pyridin, 4,4'- (1 H-1 ,2,4-triazol-1 -yl-methylen)-bis-benzonitrile, 4-(α-(4-cyanophenyl)-α-fluoro-1-(1 ,2,4-tri- azolyl)methyl)-benzonitrile, 4-(α-(4-cyanophenyl)-(2-tetrazolyl)methyl)-benzonitrile, mitotane, octreotide and N-(1 -cyano-1 -methyl-ethyl)-4-aza-3-oxo-5α-androst-1 -en-17β-carboxamid;
(F) biological response modifiers selected from aldesleukin, interferon-α and interferon
(G) inhibitors of protein tyrosine kinases and/or serine/threonine kinases selected from N-{5- [4-methyl-piperazino-methyl)-benzoylamido]-2-methyl-phenyl}-4-(3-pyridyl)-2-pyrimidine, N- (3-chlorophenyl)-4-(2-(3-hydroxy)-propyl-amino-4-pyridyl)-2-pyrimidinamin, N-benzoyl-stau- rosporine, 4,5-bis(anilino)-phthalimide, N-(5-benzoylamido-2-methyl-phenyl)-4-(3-pyridyl)-2- pyridinamin and 4-(m-chloranilino)-5,6-dimethyl-7H-pyrrolo[2,3-d]pyrimidin;
(H) antisense oligonucleotides or oligonucleotide derivatives targeted to other targets selected from SAMDC and protein kinase C; and
(I) miscellaneous agents or agents with other or unknown mechanism of- action selected from altrematine; asparaginase; dacarbazine; procarbazine; hexamethylmelamine; pentamethylmelamine; mitoxantrone; paclitaxel; streptozocin; estracyt; amsacrine; all-trans retinoic acid; levamisole; anti-melanoma vaccines, recombinant human immunoglobulins directed at melanoma antigen and antibodies for active immunotherapy of melanoma;
are in combination; and where any component a) and b) can also be present in the form of a pharmaceutically acceptable salt, if at least one salt-forming group is present.
19. A pharmaceutical preparation according to claim 14 wherein the oligonucleotide sequence corresponds to the following seαuence:
5'- TCC CGC CTG TGA CAT GCA TT -3' SEQ.-ID NO: 1 ,
or also a version thereof with 15 to 19 building blocks which is terminally shortened on one or both ends, and is present in the form of a chimeric oligonucleotide.
20. A pharmaceutical preparation according to claim 14 wherein the oligonucleotide derivative corresponds to the following sequence:
5'- TCC CGC CTG TGA CAT GCA TT -3' SEQ.-ID NO: 1 ,
or also a version thereof with 15 to 19 building blocks which is terminally shortened on one or both ends,
and is an oligonucleotide derivative which is present in the form of a chimeric oligonucleo¬ tide which shows, on the one hand, M regions of between 3 and 20 2'-modified building blocks either with phosphodiester or with phosphorothioate backbone, which are in succeeding order; and, on the other hand, U regions of 4 to 20 2'-deoxy building blocks with phosphorothioate structure that are otherwise unmodified.
21 . A pharmaceutical preparation according to claim 14 wherein a) an oligonucleotide derivative that corresponds to the following sequence:
5'- TCC CGC CTG TGA CAT GCA TT -3' SEQ.-ID NO: 1 ,
or also a version thereof with 15 to 19 building blocks which is terminally shortened on one or both ends,
which is present as an oligonucleotide derivative which comprises at least one of the following units of the formulae given hereinafter, wherein B is a base radical of a base selected from thymine, uracil, 5-propynyluracil, cytosine, 5-methylcytosine, 5-propynyl- cytosine, adenine, 2-aminoadenine or guanine; Q is H, OCH3, F, O(CH2CH2O)vCH3 wherein v is from 0 to 12, CH2CH(CH3)OCH3 or CH2CH(OH)CH2OH; and the other moieties have the meanings given behind the respective formula:
Figure imgf000111_0001
/ o
X— -P=Y
Figure imgf000111_0002
Radical of formula type
(iig). phosphorothioate X = SH Y = O or S
(Hg*)
(llh) phosphodiester X = OH Y = O
(llh*)
the remaining internucleosidic bonds in the respective oligonucleotide derivative being of the phosphorothioate type; or
as oligonucleotide derivative which comprises at least one of the following dimeric units of the formulae given hereinafter, wherein each B is, independently of the other, a base radical selected from thymine, uracil, 5-propynyluracil, cytosine, 5-methylcytosine, 5- propynylcytosine, adenine, 2-aminoadenine and guanine; any Q is, indepently of the other, H, OCH3, F or O(CH2CH2O)vCH3 wherein v is from 0 to 12; and the other moieties have the meanings given behind the respective formula:
Figure imgf000112_0001
Radical of formula type X** Y**
(Vie), (Vie*) amide CH2 N(A)
wherein A is H, methyl or phenyl; the remaining internucleosidic bonds in the respective oligonucleotide derivative being of the phosphorothioate type; and
b) at least one other chemotherapeutic agent selected from bis-alkylating agents selected from the group comprising mechlorethamine, busulfan, melphalan, chlorambucil, cis-platinum(ll)-diaminedichloride, carboplatin and triethylene¬ thiophosphoramid; cross-linking antitumor antibiotics selected from mitomycin C; and purine nucleoside analogues selected from Cladribine, 6-mercaptopurine, pentostatin and 6- thioguanine; and pyrimidine analogues selected from 5-fluorouracil, 5-fluorodeoxyuridine, cytosine arabinoside, fludarabin phosphate and 5-azacytidine;
are in combination; and where any component a) and b) can also be present in the form of a pharmaceutically acceptable salt, if at least one salt-forming group is present.
22. A pharmaceutical preparation according to claim 14 wherein the disease to be treated is selected from cancers, tumors, hyperplasias, fibrosis, angiogenesis, psoriasis, atherosclerosis and smooth muscle cell proliferation in the blood vessels.
23. A pharmaceutical preparation according to claim 14 wherein the disease to be treated is selected from small cell lung carcinoma, large cell lung carcinoma, melanoma and prostate carcinoma.
24. A pharmaceutical preparation according to claim 14 wherein
a) an oligonucleotide derivative that corresponds to the following sequence:
5'- TCC CGC CTG TGA CAT GCA TT -3' SEQ.-ID NO: 1 ,
or also a version thereof with 15 to 19 building blocks which is terminally shortened on one or both ends,
which is present in the form of a phosphorothioate oligo-2'-deoxynucleotide analogue, and
b) at least one other chemotherapeutic agent selected from ifosfamide, cisplatin, mitomycine and 5-fluorouracil
are in combination; and where any component a) and b) can also be present in the form of a pharmaceutically acceptable salt, if at least one salt-forming group is present.
25. A Dharmaceutica! preparation according to claim 14 wherein
a) an oligonucleotide derivative that corresponds to the following sequence:
5'- TCC CGC CTG TGA CAT GCA TT -3' SEQ.-ID NO: 1 ,
which is present in the form of a phosphorothioate oligo-2'-deoxynucleotide analogue, and b) any one of the following other chemotherapeutic agents for the treament of the respective proliferative disease:
- cisplatin for human prostate carcinomas;
- mitomycin for small lung cell carcinomas;
- cisplatin for small cell lung cancers: or
- mitomycin for large cell lung carcinomas
are in combination; where any component a) and b) can also be present in the form of a pharmaceutically acceptable salt, if at least one salt-forming group is present.
26. A pharmaceutical preparation according to claim 14 wherein
a) an oligonucleotide derivative that corresponds to the following sequence:
5'- TCC CGC CTG TGA CAT GCA TT -3' SEQ.-ID NO: 1 ,
which is present in the form of a phosphorothioate oligo-2'-deoxynucleotide analogue, and
b) any one of the following other chemotherapeutic agents for the treament of the respective proliferative disease:
- fluorouracil for colon cancer; or
- mitomycin for melanoma
are in combination; where any component a) and b) can also be present in the form of a pharmaceutically acceptable salt, if at least one salt-forming group is present.
27. A method of inhibiting hyperprol iteration of cells comprising contacting hyperproliferating cells with a pharmaceutical preparation according to claim 14.
28. A method of treating a proliferative disease comprising contacting a subject, cells, tissues or a body fluid of said subject, suspected of hving a hyperproliferative diesease with a pharmaceutical composition according to claim 14.
29. A combination preparation comprising a) at least one oligonucleotide or oligonucleotide derivative targeted to nucleic acids encoding raf with b) at least one other chemotherapeutic agent; or pharmaceutically acceptable salts of any component a), b) or a) and b) if at least one salt-forming group is present.
30. A product which comprises a) at least one oligonucleotide or oligonucleotide derivative (ODN) targeted to nucleic acids encoding raf and b) at least one other chemotherapeutic agent where any component a) and/or b).can also be present in the form of a pharmaceutically acceptable salt, if at least one salt-forming group is present, in the presence or absence of one or more pharmaceutically acceptable carrier materials, as a combination preparation for simultaneous or chronologically staggered use within a period of time which is small enough for the active compounds both of component a) and of component b) to mutually enhance antiproliferative activity against proliferating cells, for treating a proliferative disease which responds to such active compounds.
31. The use of a combination of a) at least one oligonucleotide or oligonucleotide derivative (ODN) targeted to nucleic acids encoding raf and b) at least one other chemotherapeutic agent, where any component a) and/or b) can also be present in the form of a pharmaceutically acceptable salt, if at least one salt-forming group is present, for producing pharmaceutical preparations for use as compositions against a proliferative disease that can be treated by application of an oligonucleotide or oligonucleotide derivative targeted to raf.
PCT/EP1997/000875 1996-03-07 1997-02-24 Antiproliferative combinations, containing raf-targeted oligonucleotides and chemotherapeutic compounds WO1997032604A1 (en)

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EP0938328A1 (en) * 1996-08-30 1999-09-01 Biomeasure Incorporated Method of inhibiting fibrosis with a somatostatin agonist
EP1243290A3 (en) * 1998-10-19 2004-03-17 Methylgene, Inc. Modulation of gene expression by combination therapy
US6953783B1 (en) 1998-10-19 2005-10-11 Methylgene, Inc. Modulation of gene expression by combination therapy
EP1243290A2 (en) * 1998-10-19 2002-09-25 Methylgene, Inc. Modulation of gene expression by combination therapy
EP1243289A2 (en) * 1998-10-19 2002-09-25 Methylgene, Inc. Modulation of gene expression by combination therapy
EP1243289A3 (en) * 1998-10-19 2004-03-17 Methylgene, Inc. Modulation of gene expression by combination therapy
WO2000023112A1 (en) * 1998-10-19 2000-04-27 Methylgene, Inc. Modulation of gene expression by combination therapy
WO2000037141A1 (en) * 1998-12-22 2000-06-29 Warner-Lambert Company Combination chemotherapy
WO2001074346A3 (en) * 2000-04-03 2002-07-25 Hybridon Inc Sensitization of cells to cytotoxic agents using oligonucleotides directed to nucleotide excision repair or transcritpion coupled repair genes
WO2001074346A2 (en) * 2000-04-03 2001-10-11 Hybridon, Inc. Sensitization of cells to cytotoxic agents using oligonucleotides directed to nucleotide excision repair or transcritpion coupled repair genes
WO2002006293A1 (en) * 2000-07-18 2002-01-24 Leo Pharma A/S Matrix metalloproteinase inhibitors
US7902192B2 (en) 2003-05-15 2011-03-08 Arqule, Inc. Inhibitors of P38 and methods of using the same
JP2006528697A (en) * 2003-05-16 2006-12-21 イデラ ファーマシューティカルズ インコーポレイテッド The immunomer used in combination with a chemotherapeutic agent, the synergistic treatment of cancer
US7829560B2 (en) 2004-07-08 2010-11-09 Arqule, Inc. 1,4-disubstituted naphthalenes as inhibitors of P38 MAP kinase
US8114873B2 (en) 2004-07-08 2012-02-14 Arqule, Inc. 1,4-disubstituted naphthalenes as inhibitors of p38 map kinase
US8178672B2 (en) 2004-10-19 2012-05-15 Arqule, Inc. Synthesis of imidazooxazole and imidazothiazole inhibitors of p38 MAP kinase
WO2007139943A3 (en) * 2006-05-26 2008-03-06 John G Clement Therapeutic drug combinations and delivery systems comprising c-raf kinase antisense polynucleotides for treating ocular diseases and disorders
WO2007139943A2 (en) * 2006-05-26 2007-12-06 Ico Therapeutics Inc. Therapeutic drug combinations and delivery systems comprising c-raf kinase antisense polynucleotides for treating ocular diseases and disorders
US9073997B2 (en) 2007-02-02 2015-07-07 Vegenics Pty Limited Growth factor antagonists for organ transplant alloimmunity and arteriosclerosis
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