OLIGONUCLEOTIDE DERIVATIVES DIRECTED AGAINST HUMAN BCL-XL AND HUMAN BCL-2 MRNA

Abstract: The present invention relates to antisense oligonucleotide derivatives directed against human bcl-xL mRNA and being capable of modulating the biosynthesis of human bcl-xL protein. Furthermore, the present invention relates to antisense oligonucleotide derivatives directed against both human bcl-xL nRNA and human bcl-2 mRNA, and being capable of modulating the biosynthesis of both human bcl-xl protein and human bcl-2 protein. The present invention further relates to a pharmaceutical composition comprising such oligonucleotide derivatives, uses thereof and methods of treatment and diagnosis utilizing such oligonucleotide derivatives.

Description: Oligonucleotide derivatives directed against human bcl-xL and human bcl-2 mRNA The present invention relates to antisense oligonucleotide derivatives directed against human bcl-xL mRNA and being capable of modulating the biosynthesis of human bcl-xL protein Furthermore, the present invention relates to antisense oligonucleotide derivatives directed against both human bcl-xL mRNA and human bcl-2 mRNA, and being capable of modulating the biosynthesis of both human bcl-xL protein and human bcl-2 protein The present invention further relates to a pharmaceutical composition comprising such oligonucleotide derivatives, uses thereof and methods of treatment and diagnosis utilizing such oligonucleotide derivatives Human bcl-xL and human bcl-2 are proteins which are closely associated with the process of programmed cell death (apoptosis) Apoptosis is an active, tightly regulated physiological process involved in development, normal cell turnover, and hormone-induced tissue athropy Lack of programmed cell death plays an important role in cancer and other hyperpro ferative diseases like re- stenosis, fibrosis, psoriasis or certain types of allergic diseases, in particular in tumor progression and, importantly, might contribute to the clinical problem of resistance to anti-neoplastic regimens, in particular standard chemotherapeutic drugs Human bcl-xL and human bcl-2 have distinct patterns of tissue expression In contrast to most normal tissues, in malignant tumors, such as a small cell lung cancer (SCLC) and non-small lung cancer (NSCLC), bcl-xL and bcl-2 are often co-expressed Oligonucleotides or, in particular, oligonucleotide derivatives directed against human bcl-xL mRNA or human bcl-2 mRNA may be used in an antisense technology strategy interfering with expression of bcl- xL and bcl-2, respectively Accordingly, there is an ongoing need for oligonucleotides or oligonucleotide derivatives showing high efficacy, preferably improved efficacy, in modulating the biosynthesis or expression of human bcl-xL and/or human bcl-2, in particular for the treatment of hyperprohferative diseases, e g those as mentioned above In the context of the present invention, surprisingly it has been identified that a specific region of the human bcl-xL mRNA encoding human bcl-xL protein, and a specific region of the human bcl-2 mRNA encoding human bcl-2 protein, are particularly accessible for hybridization with an antisense oligonucleotide derivative, in particular under physiological conditions, for example as encountered in a cellular environment Both regions share a high degree of homology In case of human bcl-xL, the said region encompasses nucleotide no 687 (5') to no 706 (3') of the human bcl-xL mRNA In case of human bcl- 2, the said region encompasses nucleotide no 2032 (5') to 2051 (3') of human bcl-2 mRNA In one aspect, the present invention relates to antisense oligonucleotide derivatives directed against these regions Surprisingly, it has been found that the oligonucleotide derivatives according to the present invention, mentioned below, show the capability to modulate human bcl-xL biosynthesis in a cell Moreover, some of the oligonucleotide derivatives of the present invention, mentioned below, in addition to be capable of modulating human bcl-xL biosynthesis, are able to modulate the biosynthesis of human bcl-2 The present oligonucleotide derivatives are therefore appropriate for the therapeutic treatment of diseases that respond to this modulation, especially inhibition, of bcl-xL or, in addition, bcl-2 biosynthesis Such an inhibition may result, as one effect, in an induction of apoptosis, resulting in inhibition of cell (hy- per)prolιferatιon Accordingly, it is an object of the present application to provide an oligonucleotide derivative which is specifically hybπdizable to a region ranging from base position no 687 (5') to no base position 706 (3') of the human bcl-xL mRNA encoding human bcl-xL protein In a preferred embodiment thereof, the oligonucleotide derivative additionally is specifically hybndizable to a region ranging from base position no 2032 (5') to base position no 2051 (3') of the human bcl-2 mRNA encoding human bcl-2 protein Within the context of the present invention, an oligonucleotide derivative is preferred which is capable of modulating the biosynthesis of the human bcl-xL protein In a further embodiment thereof, an oligonucleotide derivative is preferred which is capable of modulating the biosynthesis of both the human bcl-xL protein and the human bcl-2 protein An oligonucleotide derivative according to the present invention has a length of 8 to 25 consecutive building blocks, preferably of 15 to 20 consecutive building blocks, and most preferably of 20 consecutive building blocks The expression "oligonucleotide derivative" is familiar to the skilled person and will only be explained here for the sake of completeness Within the context of the present invention, the expression "oligonucleotide derivative" denotes, in particular, a deπvatized oligonucleotide An oligonucleotide per se is preferably an oligomer which consists of a sequence of natural nucleoside building blocks which are connected to each other by way of natural mternucleosidic bridging groups A natural nucleoside building block as such preferably consists of a sugar, in particular a β-D-erythropentofuranose, in particular β-D-πbose or β-D-2'-deoxyrιbose, together with a natural nucleic acid base which is linked to it in the β position Within the context of the present invention, nucleic acid base preferably denotes a base which, in relation to a naturally occurring nucleoside, is capable of forming Watson-Crick base pairing (for antisense oligonucleotides) Examples of natural nucleic acid bases are adenine, guanme, cyto- sine, thymine and uracil and also other bases with which the skilled person is familiar In an oligonucleotide, a natural internucleosidic bridging group, in particular a phosphodiester group, connects the individual nucleosides to each other, in each case by way of the 3'- and 5'-posιtιons In this connection, "natural preferably means that the corresponding compounds or residues occur in nature and are accessible by isolation from corresponding natural products or by means of chemical synthesis Consequently, those compounds or residues which are not regarded as being natural are termed "derivatives' or "analogues" Within the context of this invention, the term ' oligonucleotide derivative" therefore preferably denotes an oligonucleotide which is structurally modified, as compared with a corresponding natural oligonucleotide, at least one position of at least one building block (this can e g relate to the sugar or the base of a nucleoside building block, or to an internucleosidic bridging group) Thus, for example, another internucleosidic bridging group can be present in place of the naturally occurring phosphodiester bond, another linkage for example a 2'-5' linkage, can be present in place of the natural internucleosidic 3'-5' linkage of nucleoside building blocks, a base analogue which is likewise capable of hybridizing with the strand of a target nucleic acid within the sense of Watson-Crick base pairing can be present in place of a natural base, or the sugar residue can be deπvatized at different positions for example at the 2'-posιtιon or the 6'-posιtιon Moreover, an oligonucleotide derivative can also comprise, in place of at least one nucleoside building block, at least one nucleoside analogue which encompasses a non-sugar backbone to which a nucleic acid base is linked Deπvatized oligonucleotides, nucleosides, internucleosidic bridging groups and analogues have been described (cf , for example, De Mesmaeker, A et al , Curr Op Struct Biol 5 (1995), pp 343 - 355, Sanghvi, Y S et al , (Ed ), "Carbohydrate Modifications in Antisense Research", ACS Symposium Series 580 (1994), S T Crooke, "Therapeutic Applications of Oligonucleotides", R G Landes Company Publisher (1995)) Within the context of the present invention, "modulation" of the biosynthesis or expression of the human bcl-xL protein or human bcl-2 protein denotes an interference with the biosynthesis or expression, in particular a partial or complete inhibition thereof, in particular in connection with the translation or transcription process Such an inhibition, in particular due to partial or complete degradation of the target nucleic acid, due to the process for translating the target nucleic acid being completely or partially inhibited, or due to the transcription process being completely or partially inhibited, can be determined by means of known methods, for example by means of the Northern blot technique at the level of the target nucleic acid, or by means of the Western blot technique at the protein level (cf , for example, Sambrook, J , Fπtscn, E F and Maniatis, T "Molecular Cloning A Laboratory Manual", 2nd Edition, Cold Spring Harbor Laboratory Press, 1989) For further means, reference is made to the Examples as mentioned below In this regard, the term "hybridization" in particular denotes binding by way of hydrogen bonds, known as "Watson-Cπck base-pairing", between complementary bases of an oligonucleotide derivative according to the invention, on the one hand, and of a target nucleic acid, on the other hand Guanine and cytosine are an example of complementary bases between which three hy- drogen bonds are formed Adenine and thymine, or adenine and uracil, are examples of complementary bases between which two hydrogen bonds are formed in each case "Specific hybridization" denotes that a sufficient degree of complementarity exists between the oligonucleotide derivative according to the invention and the target nucleic acid to enable specific binding between the oligonucleotide derivative and the nucleic acid to be achieved For achieving specific hybridization, there must exist a sufficiently high degree of complementarity between the oligonucleotide derivative according to the invention and the target nucleic acid An oligonucleotide derivative according to the invention hybridizes "specifically" with a target nucleic acid when the binding of the oligonucleotide to the target nucleic acid impairs the function of the latter and, furthermore, an adequate degree of complementarity is present in order to avoid non-specific binding of the oligonucleotide derivative according to the invention to a nucleic acid other than the target nucleic acιd(s) when specific binding is required, for example under physiological conditions in association with an in-vivo application, such as a therapeutic treatment Specific hybridization can be determined, for example, by means of an in-vitro hybridization assay, preferably under physiological conditions, between an oligonucleotide derivative according to the invention and a target nucleic acid Appropriate reaction conditions are known (cf , for example, Sam- brook, J , Fπtsch, E F and Maniatis, T "Molecular Cloning A Laboratory Manual", 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, Vol 2, 9 47 to 9 58) In this context, further reference is made to those methods as described in the Examples, below The term "at least a part of the said region" designates an internal part of the sequence as specified, said internal part having a length of at least 8 consecutive nucleotide building blocks The sequence of the human bcl-xL mRNA as referred to herein is accessible in the EMBL Nucleotide Sequence Database as HSBCLXL under accession no Z23115 The sequence of the human bcl-2 mRNA as referred to herein is accessible in the GenBank Data Base as HUMBCL2A under accession number M13994 (The sequence of the human bcl-2 mRNA is also available in the GeneBank Data Base under accession number NM 000633 In this case said region encompasses nucleotide no 605 (5') to no 624 (3') of the human bcl-2 mRNA) Within the context of the present application, the numbering of nucleic acids, in particular of mRNA or corresponding cDNA sequences, relates to the respective numbering of the human bcl-xL mRNA or bcl-2 mRNA as contained in said data bases under said accession numbers A corresponding cDNA sequence can be deduced from the mRNA sequence in particular by exchanging any base T of the cDNA sequence by a base U in the mRNA sequence, and vici versa Thus, the mRNA sequence ranging from nucleotide nos 687 (5') to 706 (3') of the human bcl-xL mRNA reads as follows δ'-AACGGCGGCUGGGAUACUUU-S' (SEQ ID NO 1 ) The mRNA sequence ranging from nucleotide nos 2032 (5') to 2051 (3') of the human bcl-2 mRNA reads as follows 5'-AACGGAGGCUGGGAUGCCUU-3' (SEQ ID NO 2) Hence, a preferred embodiment of the present invention is directed to an oligonucleotide derivative as mentioned above, comprising a base sequence which is complementary to at least a part of the said region of the human bcl-xL mRNA or the human bcl-2 mRNA, or wherein such base sequence contains up to 3 mispaiπng building blocks, or wherein such base sequence contains up to 3 abasic building blocks A further preferred embodiment thereof is directed to an oligonucleotide derivative as mentioned above, wherein said base sequence is selected from the group consisting of the base sequence 5'-AAGGCATCCCAGCCTCCGTT-3' (SEQ ID NO 3), the base sequence 5'-AAAGTATCCCAGCCGCCGTT-3' (SEQ ID NO 4) and the base sequence 5'-AAAGCATCCCAGCCTCCGTT-3' (SEQ ID NO 5) Even more preferred is an oligonucleotide derivative of the present invention, consisting of a base sequence selected from the group consisting of the base sequence 5'-AAGGCATCCCAGCCTCCGTT-3' (SEQ ID NO 3), the base sequence 5'-AAAGTATCCCAGCCGCCGTT-3' (SEQ ID NO 4) and the base sequence 5'-AAAGCATCCCAGCCTCCGTT-3' (SEQ ID NO 5) In the context of the present application the term "complementary" denotes complementarity between the base sequence of the oligonucleotide derivative and the target nucleic acid in the sense of Watson- Crick base pairing Such complementarity can be achieved by incorporating into the oligonucleotide derivative the respective complementary base with regard to the target nucleic acid strand or a suitable analogous base In this context, an analogous base sequence denotes a base which, although being different from the corresponding classical complementary base, upon introduction into an oligonucleotide derivative of the present invention, does not alter the oligonucleotide derivative's capability of specific hybridization with the target nucleic acid For example cytosine can be exchanged by 5-methylcytosιne, or adenine by 2-amιnoadenιne Furthermore, a given base can be replaced by an inert base like hypoxanthine, as long as the function of the oligonucleotide derivative is not impaired Such analogous or inert bases are known to the skilled person and examples for such bases are mentioned below Within the context of the present application the term "mispaiπng building block" denotes a building block of said oligonucleotide derivative which bears a nucleic acid base which is not the complementary base with respect to the respective position of the target nucleic acid strand in the sense of Wat- son-Cπck base pairing For example, where in the target nucleic acid strand at a specific position the base A is located, the complementary base of the oligonucleotide derivative would be the base T, and a mispanng building block can, for example, be the base C or the base G In the same context the term "abasic building block" denotes a building block of said oligonucleotide derivative bearing no nucleic acid base capable of hybridization with the complementary base with respect to the respective position of the target nucleic acid strand in the sense of Watson-Crick base pairing For example, an abasic building block can be a nucleoside unit solely consisting of a backbone (i e a sugar and an internucleosidic linkage) without comprising a nucleic acid base The incorporation of such a mιspa r ιng or abasic building block into an oligonucleotide derivative can be tolerated as long as the resulting oligonucleotide derivative is capable of specific hybridization with the target nucleic acid and, preferably, capable of modulating the biosynthesis of human bcl-xL protein or of both the human bcl-xL and human bcl-2 proteins Preferably, the oligonucleotide derivative according to the present invention comprises not more than up to 3 mispaiπng building blocks and/or not more that up to 3 abasic building blocks An oligonucleotide derivative according to the present invention, i e of any of the types as mentioned above, preferably comprises at least one building block of formula (I) wherein Q is H, -OCH 3 , -OCH 2 CH 2 NR 1 R 2 , wherein R, and R 2 are, independently of each other, H or -CH 3 , or, preferably, -0(CH 2 CH 2 ) n OCH 3 , wherein n is 1 , 2 or 3, preferably 1 , V and W are, independently of each other, the same or different radicals of an internucleosidic bridging group selected from the following group 5'-O-P(0)(0H)-0-3' (phosphodiester), 5'-0-P(0)(SH)-0-3' (phosphorothioate), 5'-0-P(S)(SH)-0-3' (phosphodithioate), 5'-0-P(0)(CH 3 )-0-3' (methylphosphonate), 5'-0-P(0)(NH-R 7 )-0-3' (phosphoamidate) in which R 7 is C r C 3 alkyl, 5'-0-P(0)(0R 8 )-0-3' (phosphotn- ester) in which R 8 is d-Caalkyl, 5'-0-S(0) 2 -CH 2 -3' (sulfonate), 5'-0-S(0) 2 -NH-3' (sulfamate), 5'-NH- S(0) 2 -CH 2 -3' (sulfonamide), 5'-CH 2 -S(0) 2 -CH 2 -3' (sulfone), 5'-0-S(0)-0-3' (sulfite), 5'-CH 2 -S(0)-CH 2 -3' (sulfoxide), δ'-CHz-S-CHz-S' (sulfide), 5'-0-CH 2 -0-3' (formacetal), 5'-S-CH 2 -0-3' (3'-thioformacetal), 5'-0-CH 2 -S-3' (δ'-thioformacetal), 5'-CH 2 -CH 2 -S-3' (thioether), 5'-CH 2 -NH-0-3' (hydroxylamine), 5'-CH 2 -N(CH 3 )-0-3' (methylene(methylιmιno)), 5'-CH 2 -0-N(CH 3 )-3' (methyleneoxy(methylιmιno)), 5'-0-C(0)-NH-3' (5'-N-carbamate), 5'-CH 2 -C(0)-NH-3' (amide), 5'-NH-C(0)-CH 2 -3' (amide 2), 5'-CH 2 -NH-C(0)-3' (amide 3) and 5'-C(0)-NH-CH 2 -3' (amide 4), and the tautomeric forms thereof; or one of V and W is such an internucleosidic bridging group and the other is a terminal radical selected from the group consisting of -OH and -NH 2 , preferably -OH; and B is a radical of a nucleic acid base; with the proviso that if Q is H, then at least one of V or W is an internucleosidic bridging group other than 5'-0-P(0)(OH)-0-3' (phosphodiester). Within the context of the present invention, Q is preferably -OCH 2 CH 2 NR 1 R 2 , wherein one of R, and R 2 is H and the other is -CH 3 , or each of R, and R 2 is H, or, more preferably, Q is -OCH 2 CH 2 OCH 3 . Oligonucleotide derivatives according to the present invention which comprise a building block of formula (I), wherein Q is other than H, can be prepared in accordance with the methods as described, for example, by P. Martin, Helv. Chem. Acta, 78 (1995), pp. 486 - 504, or in an analogous way. The 5' and 3' orientation of the said radicals V and W, as an internucleosidic bridging bond, in the above-mentioned formula (I), may be clarified as follows: When V for example is a radical 5'-CH 2 -C(0)-NH-3' (amide), the corresponding nucleoside building block of the above-defined formula (I) has the following structure (1.1): / CH 2 When W for example is a radical 5'-CH 2 -C(0)-NH-3" (amide), the corresponding nucleoside building block of the above-defined formula (I) has the following structure (I.2): (1.2) Some of the said radicals of internucleosidic bridging groups can exist in different tautomeric forms, depending, inter alia, on the solvent and on the degree of lonization of lonizable groups Thus, for example, the bridging group in a phosphorothioate [0-(P-SH)(=0)-0] can be tautomerized to [0-(P-OH)(=S)-0], with the more stable form depending, inter alia, on the solvent and the ionization state Within the context of the present invention, the term "oligonucleotide derivative" also encompasses those tautomeric forms which are familiar to the skilled person In general, within the context of the present application reference to a particular formula includes all definitions and preferences as stated for that formula Particularly preferably, V and W, as the radical of an internucleosidic bridging group, are selected, independently, from the following group 5'-0-P(0)(OH)-0-3' (phosphodiester), 5'-0-P(0)(SH)-0-3' (phosphorothioate) and 5'-CH 2 -C(0)-NH-3' (amide). In particular, one of the radicals V or W, as the radical of an internucleosidic bridging group, is 5'-0-P(0)(OH)-0-3' (phosphodiester) and the other radical is 5'-0-P(0)(SH)-0-3' (phosphorothioate) V and W are also preferably, as the radical of an internucleosidic bridging group, in each case 5'-0-P(0)(OH)-0-3' (phosphodiester) or, mostly preferred, in each case 5'-0-P(0)(SH)-0-3' (phosphorothioate). Preferred is an oligonucleotide derivative according to the present invention, comprising only building blocks of formula (I), wherein V and W each are phosphorothioate and Q is H Within the context of the present invention, a nucleic acid base, in particular a nucleic acid base B of formula (I), is understood as being, in particular, natural nucleic acid bases and known analogues (cf , for example, Accounts of Chem. Res. 28 (1955), pp. 366-374; Sanghvi, Y.S. in: Antisense Research and Applications, Crooke, ST. and Lebleu, B. (Ed.), CRC Press, Boca Raton (1993), pp. 273-288) As is familiar to the skilled person, nucleic acid bases B can exist in tautomeric forms depending on the ambient conditions. According to the invention, such tautomeric forms are also encompassed by the oligonucleotide derivatives according to the invention, including the preferred embodiments. The invention preferably relates to an oligonucleotide derivative according to the invention, including the said preferences, in which said nucleic acid base, in particular said nucleic acid base B of formula (I), is a radical of the formula (V1 ), (V2), (V3), (V4) or (V5) in which R b1 is -NH 2 , -SH or -OH; R b2 is H, -NH 2 or -OH; and R b3 is H, Br, I, -CN, -C≡C-CH 3 , -C(0)NH 2 or -CH 3 ; R M is -NH 2 or -OH; and R b5 is H, F, Br, I, -CN, -C≡C-CH 3 , -C(0)NH 2 or -CH 3 . In a preferred embodiment thereof, said nucleic acid base, in particular said nucleic acid base B of formula (I), is a radical of the formula (V1 ) or (V5) in which R bl is -NH 2 , -SH or -OH; R b2 is H, -NH 2 or -OH; R b4 is -NH 2 or -OH; and R b5 is H, F, Br, I, -CN, -C≡C-CH 3 , -C(0)NH 2 or -CH 3 . Particularly preferably, said nucleic acid base, in particular said nucleic acid base B of formula (I), is selected from the group of the following radicals: xanthine, hypoxanthine, adenine, 2-aminoadenine, guanine, 6-thioguanine, uracil, thymine, cytosine, 5-methylcytosine, 5-propynyluracil, 5-fluorouracil and 5-propynylcytosine. Preferred are oligonucleotide derivatives which (in their nucleotide/nucleotide derivative sequence) comprise at least one building block of formula (I), wherein B and Q are as defined herein, including the respective preferences and embodiments, and V and W are selected from the group consisting of the following radicals: 5'-0-P(0)(OH)-0-3' (phosphodiester), 5'-0-P(0)(SH)-0-3' (phosphorothioate) and 5'-CH 2 -C(0)-NH-3' (amide), In case at least one of V or W is 5'-CH 2 -C(0)-NH-3' (amide), the oligonucleotide derivative comprises preferably the following dimeric unit (bivalent radicals) of the formula (II) wherein Q a and Q b , independently of each other, are H, -OCH 3 or -OCH 2 CH 2 OCH 3 , or preferably Q a is -OCH 3 and Q b is H, or more preferably, Q a and Q b are each H; and B, V and W are as defined herein, inclusive of the respective preferences and embodiments. Mostly preferred is an oligonucleotide derivative according to the present invention, consisting of a base sequence selected from the group consisting of the base sequence 5'-AAGGCATCCCAGCCTCCGTT-3' (SEQ ID NO: 3) and the base sequence 5'-AAAGTATCCCAGCCGCCGTT-3' (SEQ ID NO: 4), wherein each V and each W as radicals of an internucleosidic bridging group of the building blocks according to formula (I) are of the 5'-0-P(0)(SH)- 0-3' (phosphorothioate) type and wherein each Q according to formula (I) is -H. Preference is furthermore given to an oligonucleotide derivative according to the present invention which has a "chimeric" structure. Within the context of the present invention, a "chimeric structure", also termed a "chimera", is to be understood as meaning an oligonucleotide derivative which contains 2 or more chemically different regions which are in each case synthesized from one type of nucleic acid building block. Such chimeric oligonucleotide derivatives typically comprise at least one region of modified nucleic acid building blocks which confer one or more advantageous property/properties (for example increased resistance to nucleases, increased binding affinity or diminished occurrence of sequence-independent side-effects) on the oligonucleotide derivative, the so-called "wing", also designated the M region in that which follows, and a region which enables RNAse H-mediated cleavage of the target nucleic acid to take place, i.e. the so-called "RNAse H window", also designated the U region in that which follows. The affinity of an oligonucleotide or an oligonucleotide derivative is customarily determined by measuring the T m value of the oligonucleotide (derivative)Aarget nucleic acid hybrid. The T m value is the temperature at which the oligonucleotide, or its derivative, and the target nucleic acid dissociate from a previously formed hybrid The dissociation is determined spectrophotometπcally The higher the T m value, the higher is the affinity of the oligonucleotide, or the derivative, for the target nucleic acid Methods for determining the T m value belong to the state of the art (cf , for example, Fπtsch and Maniatis, "Molecular Cloning -A Laboratory Manual", 2nd Edition, Cold Spring Harbor Laboratory Press, 1989) Within the context of the present invention, increased resistance to nucleases denotes decreased or slowed-down degradation of the oligonucleotide derivatives according to the invention by exonucleases or endonucleases which are present in a cell The resistance to nucleases or the degradation of an oligonucleotide or a derivative can be monitored by gel electrophoresis, for example RNAse H is a cellular endonuclease which cleaves the RNA strand of an RNA DNA duplex Activation of the enzyme therefore leads to cleavage of the target RNA and consequently increases the efficacy of the antisense mechanism Cleavage of the target RNA can customarily be demonstrated by gel electrophoresis Since, in a chimera, different advantageous properties are present in one and the same molecule, oligonucleotide derivatives according to the invention possess a pronounced antisense effect with regard to inhibiting the expression of a protein or RNA In one embodiment, a chimeric oligonucleotide derivative according to the present invention comprises at least one M region, which consists of at least one nucleic acid building block of the formula (I) as mentioned above, and at least one U region, which enables RNAse H-mediated cleavage of the target nucleic acid to take place The U region consists, in particular, of customary 2'-deoxyrιbonucleιc acid building blocks which are linked to each other by way of phosphodiester bonds, or preferably phosphorothioate bonds, as the internucleosidic group The M region of a chimeric oligonucleotide derivative according to the present invention consists, in particular, of nucleic acid building blocks of the formula (I) in which Q is as defined, preferably -OCH 2 CH 2 OCH 3 , in which W and V, as the radical of an internucleosidic bridging group, are a phosphodiester bond, a phosphorothioate bond or an amide bond, with a phosphodiester bond being preferred Chimeric oligonucleotide derivatives according to the invention of the above-mentioned type, which preferably consist of a total of 8 to 25, in particular of 8 to 20, and preferably of 20 consecutive building blocks, preferably of the nucleoside type, and preferably comprise one or more, preferably one, U rehaving, preferably 4 to 13, for example 10 or 13, nucleoside building blocks of formula III wherein B is as defined above, including the respective preferences and specific embodiments, and wherein V and W in each case are a phosphodiester group or phosphorothioate group, preferably a phosphorothioate group, as the radical of an internucleosidic bridging group, and further comprise one or more, preferably one or two, M regιon(s) comprising the remaining nucleoside building blocks, which are of formula (I), in which V and W are, as the radical of an internucleosidic bridging group, in particular, in each case, a phosphodiester, phosphorothioate or amide group, preferably phosphodiester or phosphorothioate, in particular phosphodiester, and in which Q and B are as defined above, including the respective preferences and specific embodiments, Q being in particular - 0-CH 2 CH 2 OCH 3 The M and U regions in chimeric oligonucleotide derivatives according to the invention are preferably present in one of the following arrangements 5'-M-U--M-3' 5'-M-U-3' or 5'-U-M-3'. Additional oligonucleotide derivatives according to the invention are conjugated with other units, for example a micelle-forming group, 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 dendrimer, a phospholipid and other lipophilic groups. Conjugating in this way confers advantageous properties with regard to the pharmacokinetic characteristics on the oligonucleotide derivative according to the invention. In particular, conjugating in this way achieves increased cellular uptake In a very particularly preferred embodiment, an oligonucleotide derivative according to the present invention consists exclusively of nucleoside building blocks of the formula (I) which are connected to each other by way of phosphodiester bonds as the internucleosidic bridging groups V and/or W. In another very particularly preferred embodiment, an oligonucleotide derivative according to the invention exclusively comprises nucleoside building blocks of the formula III which are connected to each other by way of phosphorothioate bonds as the internucleosidic bridging groups V and/or W. Accordingly, the present invention furthermore relates to an oligonucleotide derivative as mentioned herein, consisting of a base sequence selected from the group consisting of the base sequence 5'-AAGGCATCCCAGCCTCCGTT-3' (SEQ ID NO 3), the base sequence 5'-AAAGTATCCCAGCCGCCGTT-3' (SEQ ID NO. 4), and the base sequence 5'-AAAGCATCCCAGCCTCCGTT-3' (SEQ ID NO: 5), wherein each V and each W as radicals of an internucleosidic bridging group of all building blocks according to formula (I) are of the 5'-0-P(0)(SH)- 0-3' (phosphorothioate) type, and wherein each Q according to formula (I) of the nucleotides being underlmed is -OCH 2 CH 2 OCH 3 and wherein each Q according to formula (I) of the remaining nucleo- tides is -H Provided that salt-forming groups are present, the term "oligonucleotide derivative also encompasses salts, in particular acid addition salts, salts with bases or, if several salt-forming groups are present possibly also mixed salts or internal salts Salts of oligonucleotide derivatives according to the invention are, in particular pharmaceutically tolerated salts, i e essentially non-toxic salts Such salts are formed, for example, from the oligonucleotide derivatives according to the invention which possess an acidic group, for example a carboxyl group, a phosphodiester group or a phosphorothioate group, and are, for example, salts with suitable bases These salts include, for example, non- toxic metal salts which are derived from metals of groups la, lb, lla and lib of the Periodic System of the elements, in particular suitable alkali metal salts, for example lithium, sodium or potassium salts, or alkaline earth metal salts, for example magnesium or calcium salts They furthermore include zinc and ammonium salts and also salts which are formed with suitable organic amines, such as unsubstituted or hydroxyl-substituted mono-, di- or tri-alkylammes, in particular mono-, di- or tπ-alkylamines, or with quaternary ammonium compounds, for example with N-methyl-N-ethylamine, diethylamine, tnethyla- mine, mono-, bis- or tπs-(2-hydroxy-lower alkyl)amιnes, such as mono-, bis- or trιs-(2-hydroxy- ethyl)amιne, 2-hydroxy-tert-butylamιne or trιs(hydroxymethyl)methylamιne, N,N-dι-lower alkyl-N-(hy- droxy-lower alkyl)amιnes, such as N,N-dιmethyl-N-(2-hydroxyethyl)amιne or trι-(2-hydroxyethyl)amιne or N-methyl-D-glucamine, or quaternary ammonium compounds such as tetrabutylammonium salts Lithium salts, sodium salts, magnesium salts, zinc salts or potassium salts are preferred, with sodium salts being particularly preferred Oligonucleotide derivatives according to the invention which possess a basic group, for example an ammo group or imino group, can form acid addition salts, for example with inorganic acids, for example with a hydrohalic acid, such as hydrochloric acid, sulfuπc acid or phosphoric acid, or with organic car- boxylic acids, sulfonic acids, sulfo acids or phospho acids or N-substituted sulfamic acid, for example acetic acid, propionic acid, glyco c acid, succinic acid, maleic acid, hydroxymaleic acid, methylmaleic acid, fumaπc acid, malic acid, tartaπc acid, gluconic acid, glucaπc acid, glucuronic acid, citric acid, ben- zoic acid, cinnamic acid, mandelic acid, salicylic acid, 4-amιnosalιcylιc acid, 2-phenoxybenzoιc acid, 2-acetoxybenzoιc acid, embonic acid, nicotonic acid or isonicotonic acid, and, in addition, with ammo acids, for example with α-amino acids, and also with methaπesulfonic acid, ethanesulfonic acid 2-hydroxymethanesulfonιc acid, ethane-1 ,2-dιsulfonιc acid, benzenedisulfonic acid, 4-methylbenzene- sulfonic acid, naphthalene-2-sulfonιc acid, 2- or 3-phosphoglycerate, glucose-6-phosphate or N-cyclo- hexylsulfamic acid (with formation of the cyclamates) or with other acidic organic compounds, such as ascorbic acid Oligonucleotide derivatives according to the invention which possess both acidic and basic groups can also form internal salts Oligonucleotide conjugates according to the invention which possess more than one group which is suitable for salt formation can also form mixed salts Pharmaceutically unsuitable salts, for example picrates or perchlorates, can also be used for isolation and purification It is only the pharmaceutically tolerated salts, which are non-toxic when used correctly, which are employed for therapeutic purposes and which are therefore preferred In a further embodiment the present invention relates to a process for preparing an oligonucleotide derivative according to the present invention, said process comprising incorporating at least one building block of formula (I) as mentioned above into the oligonucleotide derivative during oligonucleotide synthesis The compounds of the formula (I), wherein V and W each are a terminal group as defined above, are employed as nucleoside building blocks in the synthesis of the oligonucleotide derivatives according to the invention The oligonucleotide derivatives according to the invention can be prepared, in a manner known per se, in accordance with a variety of methods, in DNA synthesis equipment which can be automated and which can be obtained commercially in conjunction with method protocols For example, in the case of a phosphodiester group as the internucleosidic bridging group, the phosphotπester method, the phosphite triester method or the H-phosphonate method, which are familiar to the skilled person, can be used (cf , for example, Eckstein, F , "Oligonucleotides and Analogues, A Practical Approach", IRL Press (1991 )) In the case of the phosphite triester method, the approach can, for example, be to react, for example, a nucleoside building block of the formula (I), in which V a and W a are in each case -OH, with a protecting group reagent, for example 4,4'-dιmethoxytrιphenylmethyl chloride, to give a nucleoside of the formula (lb) in which V a is a protected hydroxyl group and Q and B are defined as above for the compound of the formula I including the said preferences, reactive groups thereof being protected by a suitable protective group where necessary, and to bind the compound of the formula lb with the aid of a linker, for example succinic anhydride, to a solid support material, for example to "Controlled Pore Glass" (CPG), which contains long-chain alkylamino groups In a separate procedure, the hydroxyl group of another nucleoside building block of the formula (lb) is denvatized, for example using R x O-P[N(ι-propyl) 2 )] 2 to give a phosphoramidite of the formula (lc) in which R x is a customary protecting group, for example β-cyanoethyl, wherein B and Q are defined above Protecting groups for the radical V a as protected hydroxyl group in compounds of the formulas (lb) and (lc) preferably are of the tπtyl-type, in particular tπtyl (Tr), 4-monomethoxytπtyI (MMTr), preferably 4,4'-dιmethoxytπtyl (DMTr) and, likewise preferably, 4,4',4"-trιs-tert-butyltπtyI (TTTr) After the protecting group on the radical V a , for example the DMTr- or the TTTr group, of the support bound material has been eliminated, this material is coupled, with elimination of -N(ι-C 3 H 7 ) 2 to the compound of the formula (lc), any free hydroxyl groups which may be present are blocked ("capping") and the phosphite which has been formed is then oxidized, thereby leading, for example, to the phosphate or phosphorothioate After the dimer has been deprotected, the reaction cycle is repeated with a compound of the formula (lc) until an oligomer having the desired number of monomer units has been synthesized, and the product is then detached from the support material In this way, an oligonucleotide derivative according to the invention is obtained which is synthesized entirely from nucleoside building blocks of the formula (I), above, having phosphodiester groups or phosphorothioate groups, depending on the oxidation conditions, as the internucleosidic bridging group Depending on the use of appropnate nucleoside building blocks in the individual reaction cycles, oligonucleotides according to the invention of any arbitrary sequence can be prepared in an analogous manner, in particular those oligonucleotides according to the invention which, in addition to one or more nucleoside building block(s) of the formula (I), contain other nucleoside building blocks, in particular those comprising structures of formulas (II) and/or (III) Oligonucleotide derivatives according to the present invention which do not contain, or which do not exclusively contain, phosphodiester groups or phosphorothioate groups as the internucleosidic bridging groups can be prepared in a manner known per se (cf , for example, the above-mentioned publications of De Mesmaeker, A , or Crooke, S T ) The oligonucleotide derivatives according to the invention possess a number of advantageous properties These include, in particular, a high binding affinity for a target nucleic acid and a high resistance to nucleases Furthermore, they are capable of a sequence-specific effect, are taken up satisfactorily by a cell and have adequate bioavailability These properties make the oligonucleotides according to the invention particularly suitable for pharmaceutical applications, in particular for modulating the biosynthesis or expression of the human bcl-xL protein or both of the human bcl-xL and bcl-2 proteins Consequently, in a further aspect the present application relates to an oligonucleotide derivative according to the present invention for use in medicine An oligonucleotide derivative according to the invention can be used, in particular, as an antisense oligonucleotide The expression "antisense" is known to the skilled person and, in the context of the present invention, characterizes, in particular, the relationship between an oligonucleotide derivative according to the invention and the sequence, which is complementary to it, of a target nucleic acid (i e human bcl-xl mRNA or bcl-2 mRNA), namely that the oligonucleotide derivative and the complementary sequence are able to hybridize to each other The identification of a suitable antisense oligonucleotide is a multi-step process First of all, a target nucleic acid is identified which underlies the protein whose expression characterizes a pathological state in humans, and is to be modulated In the present case, the target nucleic acid is, in particular, the RNA which is transcribed from the gene which encodes the protein of interest, such as pre-mRNA or, preferably, the (mature) mRNA Within the target nucleic acid, a sequence or sequences is/are identified which interact, in particular by means of hybridization, with the oligonucleotide derivative according to the invention such that expression of the protein of interest is modulated An oligonucleotide derivative according to the invention must possess a complementarity to the target nucleic acid which is adequate, due to sufficiently powerful and sufficiently specific hybridization, to achieve the desired effect Consequently, the invention also provides the use of an oligonucleotide derivative according to the invention, including the said preferences, as an antisense oligonucleotide According to the invention, oligonucleotide derivatives are preferred which are capable of modulating the expression (biosynthesis) of the human bcl-xL protein. Even more preferred are oligonucleotide derivatives which are capable of modulating the expression (biosynthesis) of both the human bcl-xL protein and the human bcl-2 protein. The invention furthermore relates to a pharmaceutical composition which comprises an oligonucleotide derivative according to the invention, or a pharmaceutically tolerated salt thereof, in a pharmaceutically effective quantity, if desired together with a pharmaceutically tolerated excipient and/or auxiliary substance. Such a pharmaceutical composition preferably is suitable for administration to humans suffering from a disease that responds to the modulation of human bcl-xL expression or that responds to the modulation of human bcl-xl and human bcl-2 expression. Pharmaceutical compositions according to the invention (and also oligonucleotide derivatives according to the invention) can be used, for example, for the therapeutic or prophylactic treatment of hyper- plastic or neoplastic states, for example of cancer, in particular solid tumors, or of restenosis, fribrosis or psoriasis, preferably in a cancer selected from the group consisting of colorectal cancer, gastric cancer, prostate cancer, neuroblastoma, melanoma, thyroid cancer, renal cancer, breast cancer and, preferably, lung cancer, the letter being, for example, in particular NSCLC (non-small cell lung cancer) or, most preferably, SCLC (small cell lung cancer). In particular, pharmaceutical compositions according to the invention (and also oligonucleotide derivatives according to the invention) are capable of killing cancer cells, preferably by induction of apoptosis, and/or of reverting multidrug resistance of tumors. Pharmaceutical compositions which are preferred in accordance with the invention comprise preferred oligonucleotide derivatives as described above. The pharmaceutical compositions according to the invention are preferably present in the form of preparations which can be administered parenterally or of infusion solutions. Aqueous solutions of the active substance in water-soluble form, for example in the form of one of the above-mentioned water- soluble salts, in the presence or absence of salts, such as NaCI, and/or pharmaceutically tolerated excipient materials, such as sugar alcohols, for example mannitol, are suitable, in particular, for par- enteral administration, for example for intravenous or intraperitoneal administration. Aqueous suspensions for injection which comprise viscosity-increasing substances, such as sodium carboxymethyl cellulose, sorbitol and/or dextran, are also suitable for parenteral administration. These preparations or solutions are preferably isotonic aqueous solutions or suspensions. The active substance can be present, for example, in the form of a lyophilisate. if necessary together with a pharmaceutically tolerated excipient material, which lyophilisate is brought into solution, before its use for parenteral administration, by adding a suitable solvent. These solutions which are suitable for parenteral administration can also be employed as infusion solutions The pharmaceutical compositions according to the invention can be sterilized and/or comprise auxiliary substances, for example preservatives, stabilizers, wetting agents and/or emulsifying agents, solubilizmg agents, salts for regulating the osmotic pressure and/or buffers The pharmaceutical preparations, which, if desired, can comprise additional pharmacologically (or pharmaceutically) active compounds, for example antibiotics, are prepared in a manner known per se, for example by means of conventional solubilizmg or iyophilizing methods, and comprise from about 0 0001 % by weight to about 95 % by weight, preferably from about 0 1 % by weight, to about 90 % by weight, in particular from about 0 5 % by weight to about 30 % by weight, for example from 1 % by weight to 5 % by weight, of active compound(s) Dosage forms in the form of individual doses comprise, for example, from about 0 001 % by weight to about 20 % by weight, of active compound(s) dosage forms which are not in the form of individual doses comprise, for example, from about 0 001 % by weight to about 10 % by weight of active compound(s) Dose units preferably comprise from about 0 0005 mg to about 0 5 mg, preferably from about 0 005 mg to about 40 mg of active compound(s) depending on the nature of the mammalian subject, including man, to be treated, on the disease to be treated and on the condition of the patient, in particular its/his/her body weight, its/his/her age and its/his/her individual state of health, and also on individual pharmacokmetics contributing factors and the route of administration In order to improve activity, the pharmaceutical compositions according to the invention can comprise cationic lipids Pharmaceutical compositions according to the invention are also preferred which additionally comprise a customary cytostatic agent Such combination preparations are preferably employed for treating hy- perplastic or neoplastic states such as cancer The present invention furthermore relates to an oligonucleotide derivative according to the invention, including the above-mentioned preferences, or a pharmaceutically tolerated salt thereof, for use in the prophylactic or therapeutic treatment of humans, in particular of a pathological state, which is characterized by the expression or biosynthesis of human bcl-xL protein or of both human bcl-xL and human bcl-2 proteins The present invention furthermore relates to the use of an oligonucleotide derivative according to the invention, including the above-mentioned preferences, for preparing a pharmaceutical composition for the prophylactic or therapeutic treatment of a pathological state in humans, which is characterized by the expression or biosynthesis of human bcl-xL protein of both human bcl-xL and human bcl-2 proteins Over and above this, the present invention relates to a method for the prophylactic or therapeutic treatment of a pathological state in a mammalian subject, including man, which state is characterized by the expression or biosynthesis of human Bcl-xL protein or of both human bcl-xL and human bcl-2 proteins, which method comprises administering a pharmaceutical composition according to the inven¬ Moreover, the invention relates to a method for modulating the expression of human Bcl-xl protein or of both human bcl-xL and human bcl-2 proteins in a cell, which comprises bringing the cell, or a tissue or body fluid which contains this cell, into contact with an oligonucleotide denvat ve according to the invention, including the above-mentioned preferences or with a pharmaceutical composition according to the invention Such a process for modulating the expression or biosynthesis of a protein in a cell can be advantageously applied both in vitro and in vivo The oligonucleotide derivatives according to the invention, including the above-mentioned preferences, are also suitable for use as diagnostic agents and can be employed, for example, in a manner known per se, as gene probes for detecting genetically determined diseases or viral infections by means of selective interaction at the level of single-stranded or double-stranded target nucleic acids In particular, a diagnostic application is possible in vivo as well as in vitro, due to the increased stability towards nucleases The diagnosis can take place, for example, on isolated tissue samples, blood plasma, blood serum or other body fluids, and, in the case of m-vivo diagnosis, on tissues, cells or body fluids in the patient to be investigated as well In a preferred embodiment thereof, such diagnosis takes place under physiological conditions Another aspect of the present invention consequently relates to an oligonucleotide derivative according to the invention, including the above-mentioned preferences, for use in a diagnostic method As mentioned above, the oligonucleotide derivatives according to the invention are suitable both for m-vivo and for in-vitro diagnostic methods It is to note that the entire content of the references, patents and publications cited in this application is hereby incorporated by reference The following examples clarify the invention but do not restrict it Examples are in particular directed to preferred embodiments of the present invention Examples Example 1 : Cell Culture The SW2 SCLC cell line is obtained from the Dana Farber Cancer Institute Boston, MA and The NSCLC cell line NCI-H125 cell line is obtained from the American Type Tissue Culture Collection (ATCC, Rockville, MD) Lung cancer cell lines are cultured in RPMI-1640 medium (Gibco Life Technologies Ltd, Paisley, UK) supplemented with 2 mM L-glutamine, 10% fetal calf serum (FCS Hy-Clone II Fetal Clone II), 50 lU/mL penicillin and 50 μg/mL streptomycin at 37°C in a humidified atmosphere with 5% CO2 SW2 cells are grown as floating aggregates, H125 cells are grown as monolayers Example 2. Synthesis of oligonucleotide derivatives The oligonucleotides of the present invention can be prepared according to methods known in the art For 1 μmole scale syntheses, a Perseptive Expedite MOSS synthesizer is used The syntheses are performed on Polystyrene Primer support (Pharmacia) loaded with the 3'-end residue Solutions at 0 05M in Acetonitrile of β -cyanoethyl deoxynbonucleosides phosphoramidites (Amersham Pharmacia Biotech) or β -cyanoethyl 2'-methoxyethylrιbonucleosιdes phosphoramidites (P Martin, Helvetica Chimica Acta, 78 (1995), 486 - 504) are used In the coupling step, phosphoramidites are activated by Benzimidazolium Tπflate (0 2M in Acetonitrile, R Noyoπ, J Org Chem 61 , 1996, 7996 - 7997) Sulfuπ- zation can be performed by a 0 5 M solution of phenyl acetyl disulfide or PADS in acetonitπle/picoline (3 1 ) as described (van Boom et al , Tet Letters 1989, 30(48), pp 6757-6760) Capping and washing steps are carried out by standard reagents and solvents The synthetic cycle is depicted in table 1 Table 1 : Synthetic cycle for 1 μmole Synthesis at larger scales (synthesis scale of 150 μmole or more) are on the Oligopilot II (Amersham Pharmacia Biotech) DNA synthesizer performed according the cycle described in table 2 with the same solutions except for the phosphoramidites which are 0.2 M in Acetonitrile. Table 2: CV is column volume Crude product of approximately 70 % purity is further purified by reverse phase chromatography using C18 stationary phases. The purified material is lyophilized, redissolved in water, quantified by UV absorption at 260 nm and checked using standard procedures by capillary gel electrophoresis for purity and by MALDI-TOF MS for identification. For larger scale oligonucleotides, the purified material is re- dissolved in 100 nriM NaCI and further desalted by ultrafiltration. The oligonucleotides are finally lyophilized and stored as dry materials. The following oligonucleotide derivatives (OD) are synthesized, all being 20-mer phosphorothioates purified by high pressure liquid chromatography (HPLC). The sequences are OD1 : 5'-AAGGCATCCCAGCCTCCGTT-3' (SEQ ID NO: 3) OD2: 5'-AAAGTATCCCAGCCGCCGTT-3' (SEQ ID NO: 4) OD3: 5'-AAGGCATCCCAGCCTCCGTT-3' (SEQ ID NO: 3) OD4: 5'-AAAGTATCCCAGCCGCCGTT-3' (SEQ ID NO: 4) OD5: 5'-AAAGCATCCCAGCCTCCGTT-3' (SEQ ID NO: 5) The underlined nucleotides of OD3, OD4 and OD5 denote building blocks bearing a 2'-methoxyethoxy substituent. These oligonucleotides OD1 , OD2, OD3, OD4 and OD5 are the most preferred embodiment of the present invention The following oligonucleotide derivative C01 for use as scrambled control oligonucleotide derivative to OD2 is likewise synthesised C01 5'-CCATGCTATCGCCTACACGG-3' (SEQ ID NO 6) The following oligonucleotide derivative C02 targets the translation initiation site of the bcl-2 mRNA and is used as a bcl-2 specific control C02 5'-CCAGCGTGCGCCATCCTTCC-3' (SEQ ID NO 7) The following oligonucleotide derivatives C03 and C04 are synthesized in the same manner C03 and C04 are used as scrambled controls to OD3 and OD4, respectively C03 5'-CACGTCACGCGCGCACTATT-3' (SEQ ID NO 8) C04 5'-CATATCACGCGCGCACTATG-3' (SEQ ID NO 9) The underlined nucleotides of C3 and C4 designate building blocks bearing a 2'-methoxyethoxy sub- stituent Example 3. Delivery of oligonucleotide derivatives to tumor cells Antisense oligonucleotide derivatives OD1 and OD2 and the C01 control oligonucleotide derivative are delivered to cells in the form of complexes with the cationic lipid N-[1-(2,3-dιoleoyloxy)propyl]-N,N,N- trimethylammonium methylsulfate (DOTAP) (Boehnnger Mannheim, Germany) under serum free conditions Equal volumes of ODNs (6 μM) and DOTAP (0 2 mM) in HEPES-buffered saline are mixed and allowed to complex for 10 mm at room temperature Different dilutions of the stock solutions are prepared in RPMI-1640 depending on the experiment, and the mixtures are further diluted into 9 volumes of cell suspension to achieve a final density of 2 x 10^ cells/ml in the cultures After 4 h of incubation serum is added to the cultures to a final concentration of 10% Control cultures ware treated with equivalent concentrations of DOTAP or medium alone (untreated) Cells are incubated at 37°C under standard cell culture conditions for different time periods depending on the experiment Example 4: Northern blot analysis To examine the effect of the antisense oligonucleotide derivatives to downregulate bcl-2 and bcl-xL expression on the transcπptional level, Northern blot analyses of SW2 cells is performed after treat- ment with OD1 or OD2, respectively, at a concentration of 150 nM In this study the SW2 cell line is used for the molecular analyses of tne antisense effects because it expresses nearly equivalent amounts of both death antagonists bcl-2 and bcl-xL The antisense oligonucleotide derivative C02 that targets the translation initiation site of the bcl-2 mRNA is used as a bcl-2-specιfιc control Total RNA is prepared from 5 x 10 6 SW2 cells by use of the RNeasy Mini Kit (Qiagen GmbH, Hilden Germany) and QIAshredder (Quiagen GmbH) for homogenization For each sample, 5 μg total RNA are electrophoresed on a denaturing 1 % formaldehyde-agarose gel, transferred to a NYTRAN NY 13 N membrane (Schleicher & Schuell, Dassel, Germany) according to standard procedures (Anony- mous1994), and cross-linked using a Stratalinker (Stratagene, La Jolla, USA) Equal loading and blotting of the RNA is confirmed and documented by staining with 0,03% methylenblue in 0,3M sodium acetate, pH 5,2 Membranes are pre-hybπdized for 1 h at 68°C in QuickHyb Solution (Stratagene) Membranes are hybridized for 2 hours at 68°C with an EcoRI restriction fragment composed of nucleotides 1410 to 2340 of the bcl-2 complementary DNA (cDNA), or an EcoRI / Smal restriction fragment composed or nucleotides 77 to 373 of the bcl-xL cDNA which have been radiolabelled with 32 by use of random hexanucleotide primers (Pπme-a-Gene Labelling System, Promega Corp, Madison, USA) Hybridization with a 1 3 kb EcoRI / H/πdlll restriction fragment of the 3 93 kb full-length chicken-actin cloned into plasmid pUC9 as described (Miyashita et al , 1994), or a 900 bp Apa\ restriction fragment of the 1 2 kb human GAPDH cloned into plasmid pBlueskπpt KS (+) is used as probe for reference Blots are exposed to a storage phosphor screen scanned by use of a phosphoπmager (Molecular Dynamics, Sunnyvale, CA) Relative messenger RNA levels are quantified by use of the ImageQuant software (Molecular Dynamics) As a result, OD1 efficiently reduces the level of the bcl-2 mRNA and, despite the 3-base mismatches compared with the natural base sequence of bcl-xL mRNA, also effectively reduces the level of the bcl- xL mRNA This finding clearly indicates that OD1 can tolerate mismatches, in particular those which comprise an A to G, a T to C, and a G to T substitution at positions 3, 5, and 15 of OD1 , respectively, said oligonucleotide derivative still being effective in modulating the biosynthesis both of bcl-xL and bcl- 2 On the other hand, OD2 only downregulates bcl-xL expression and does not reduce the level of the bcl-2 mRNA species As expected for the control oligonucleotide derivative C02, it selectively down- regulates the expression of bcl-2 Example 5 Western blot analysis using OD1 and OD2 To confirm the findings of the Northern blot analyses Western blot analyses of bcl-2 and bcl-xL protein levels are performed after treatment of SW2 cells with OD1 or OD2 at a concentration of 150 nM For each treatment, 14 ml of SW2 cells/oligonucleotide mixture are plated in a 25 cm^ flask and incubated for 48 h under cell culture conditions Soluble protein extracts are prepared as described (M Hanada et al , Cancer Res 53 (1993), 4978 - 4986, A Ziegler et al , J Natl Cancer Inst 89 (1997), 1027 - 1036), and protein concentrations are determined using the BCA protein assay reagent (Pierce, Rockford, IL) One hundred μg of soluble protein per sample are separated on a 12% polyacrylamide SDS gel at 50 V for about 16 h Electrotransfer to a polyvmylidene fluoride membrane (Immobilon P, Millipore, Bedford, USA) is performed in a semi-dry blotting chamber (Schleicher and Schuell) at 1 mA/cm^ for 1 h The blots are blocked in Tris-buffered saline containing 5% bovine serum albumin and 5% non-fat milk, and then incubated overnight at 4 °C with 200 ng/mL mouse anti-human bcl-2 monoclonal antibody (Dako Diagnostics AG, Glostrup, Denmark) or 250 ng/ml anti bcl-xL rabbit polydonal serum (Transduction Laboratories, Lexington, USA) To detect the primary antibody, blots are incubated with a rabbit anti-mouse immunoglobulin peroxidase conjugate, or a goat anti-rabbit immuno- globulm peroxidase conjugate (Sigma Chemical Co , St Louis, MO) for 2 h at room temperature Visualization of the immunocomplex is achieved by enhanced chemiluminescence using the ECL™ kit (Amersham), followed by exposure to x-ray film (FUJI RX) Relative protein levels are quantified after scanning of the films using a flat bed scanner (Hewlett Packard ScanJet Ilex) and the ImageQuant software (Molecular Dynamics) In agreement with the findings obtained on the mRNA level, the Western blot results indicate that OD1 also efficiently downregulates the expression of both the bcl-2 and the bcl-xL protein In contrast, OD2 efficiently downregulates only the bcl-xL protein Example 6: Western Blot analysis using the 2'-methoxyethoxy oligonucleotide derivatives OD3, OD4 and OD5 The effect of the 2'-methoxyethoxy modified oligonucleotide derivatives OD3, OD4 and OD5 on bcl-2 and bcl-xL gene expression is tested as described for the unmodified oligonucleotide derivatives OD1 and OD2 (above), using SW2 SCLC cells as target cells expressing equal levels of the bcl-2 and bcl-xL proteins SW2 cells are suspended in RPMI1640 cell culture medium and the oligonucleotide derivatives OD3, OD4 and OD 5, respectively, are added at a final concentration of 600 nM in the presence of the cationic lipid DOTAP (N-[1-(2,3-dιoleoyloxy)propyl]-N,N,N-tπmethylammonιum methylsulfate) The cells are allowed to incubate with the oligonucleotide derivatives for 24 - 48 hours and are then subjected to Western Blot analysis as described above As determined by Western blot analysis 48 h after transfection the antisense oligonucleotide derivative OD3 reduces the level of the bcl-2 protein to 40 1 % and of the bcl-xL protein to 32.6% of the respective untreated controls OD5 reduces the level of the bcl-2 protein to 48 3% and of the bcl-xL protein to 29.1 %. For comparison OD4 reduces only the level of bcl-xL to 53% The scrambled control sequence C03 does not reveal a repressive effect on the expression of the two anti-apoptotic proteins bcl-2 and bcl-xL These data demonstrate the bispecificity of the oligonucleotide derivatives OD3 and OD5 on bcl-2 and bcl-xL expression, and quantitatively correlate with the numbers of base-mismatches on the two distinct mRNAs Apparently, the 3- and 2- base-mismatches of OD3 and OD5, respectively, on the bcl-xL mRNA can be well compensated by the 2'-methoxyethoxy modifications In contrast, the 3 base mismatches of OD4 on the bcl-2 mRNA cannot be compensated by this modification In a similar experiment on H125 NSCLC, specifically OD4 reduces the level of bcl-xL expression by more than 90%, but does not alter bcl-2 expression The control oligonucleotide derivative C04 does not show an effect on bcl-xL protein expression Example 7: Caspase-3 activation assay following treatment with the 2'-methoxyethoxy modified oligonucleotide derivatives OD3, OD4 and OD5 Caspase activity in cells is analyzed by use of a colorimetπc test system SW2 cells are treated with the 2'-methoxyethoxy modified oligonucleotide derivatives 0D3, OD4 and OD5, respectivley, as described above The cells are lysed in buffer by freeze/thawing essentially as described (Olie et al , Curr Biol 8 (1998), pp 955 - 958) and lysates are centifuged at 13000 rpm at 4°C for 15 mm Cytosolic protein is mixed with 50 μM of the caspase-3 specific substrate DEVC-pNA (Asp-Glu-Val-Asp-p-nitroaniiides) (Bachem, Dubendorf, Switzerland) and incubated at 37°C Subsequently, substrate cleavage is monitored at 405nm using a SPECTRAmax 340 microplate reader and analyzed using SOFTmax PRO software (Molecular Devices) As determined in such a caspase activation assay, 24h after transfection with the antisense oligonucleotide derivatives OD3, OD4 or OD5, respectively, there is no evidence of caspase activation Following 48 h transfection, however, there is more than a 2-fold increase in caspase activation when cells have been treated with the bispecific oligonucleotide derivatives OD3 or OD5, respectively No effect on caspase activation is observed with the bcl-xL specific antisense oligonucleotide derivative OD4 nor with the scrambled sequence control C03 This result suggests that in SW2 cells repression of bcl-2 expression, modulated by OD3 and OD5, respectively, rather than repression of bcl-xL expression induces apoptosis and that these SCLC cells are dependent on the bcl-2 protein for survival In a similar experiment, performed with OD4 on H125 NSCLC, OD4 induces apoptosis The control oligonucleotide C04 does not show an effect on the tumor cells This indicates that OD4 efficiently and specifically tackles bcl-xL expression Example 8. Determination of the viability of lung cancer cell lines after treatment with OD1 or OD2 The SW2 SCLC and the H125 NSCLC cell lines both co-express bcl-2 and bcl-xL, although bcl-2 expression in H125 cells is rather low This, however, does not necessarily mean that both death antagonists are equally important survival factors for the different lung cancer cell types To compare the effects of downregulatmg bcl-2 and/or bcl-xL, SW2 and H125 cells are treated with the antisense oligonucleotide derivatives OD1 or OD2, and for control purposes, with C01 or C02, and the cytotoxic effects are measured in coloπmetric cell viability assays For SW2 cells a test system which is based on the cleavage of the tetrazo um salt WST-1 (Boehrmger Mannheim) by mitochondπal dehydrogenases in viable cells (Cory et al , 1991 ) is used For each treatment with oligonucleotide derivatives, 100 μL of cells/oligonucleotide derivative mixture are plated in triplicates in 96-well plates Cells are incubated for 96 h under cell culture conditions, and then 10 μL of WST-1 reagent are added per well and allowed to react for 3 h at 37°C Absorbance at 450 nm is measured by use of an enzyme-linked immunosorbent assay reader (2550 EIA reader, Bio Rad Laboratories Ltd , Hercules, CA) The viability of H125 cells is determined by use of a standard MTT For each treatment, 100 μL of cells/oligonucleotide derivative mixture are plated in triplicates in 96-well plates Cells are incubated for 96 h under cell culture conditions before 10 μL of MTT reagent are added per well and allowed to react for 2 h Cells are lysed for 3 hours under 37°C with 100 μl lysis buffer, containing 20% SDS in 50% dimethyl-formamide and 50% H 2 0, pH 4,7 Absorbance at 570 nm is measured by use of an enzyme- linked immunosorbent assay reader (2550 EIA reader, Bio Rad Laboratories Ltd , Hercules, CA) Data are expressed as percent of the absorbance of untreated control cells As a result, the viability of SW2 cells is strongly reduced by treatment with the bi-specific oligonucleotide derivative OD1 and the bcl-2-specιfιc control oligonucleotide derivative C02 In contrast, treatment with the bcl-xL-specific oligonucleotide derivative OD2 is not effective when compared with the SC1 control oligonucleotide derivative This indicates that bcl-2 but not bcl-xL is the critical survival factor for this lung cancer cell line In contrast to the SW2 cell line, the H125 cell line is highly susceptible to treatment with oligonucleotide derivative OD2 and also to treatment with oligonucleotide derivative OD1 Since the control oligonucleotide derivative C02 is less effective in reducing the viability of these cells, it can be concluded that bcl-xL is the more critical survival factor for this NSCLC cell line and that bcl-2 only plays a minor protective role Example 9. Detection of apoptotic cells based on light scattering properties and electron microscopy To demonstrate that death of SW2 cells after treatment with oligonucleotide derivative OD1 is apoptotic by nature, flow cytometπc analysis of cell morphology based on their light scattering properties according to the method of Cotter et al , 1992 Approximately 0 2 x 10 6 of ODN-treated cells are fixed in 2% paraformaldehyde, permeabi zed in 0 05% Tπton-X100 and measured by use of a FACSCalibur cytofluorometer (Becton Dickinson, Mountain View, CA) Cells showing changes in light scattering are also subjected to electron microscopy Briefly, for electron microscopic examination SW2 cells are fixed in 2,5% glutaraldehyde, 0,8% paraformaldehyde in 0.05M Na-Cacodylate buffer For transmission electron microscopy, cells are dehydrated in ethanol and pro- pylene oxide After dehydrating, cells are embeded in Epon 812, cut into 60nm slices, contrasted with Reynolds Solution (uranyl acetate and lead nitrate), and visualized by use of a Philips CM 420 electron microscope For scanning electron microscopy, cells are dried in aceton, stained with gold and visualized by use of a Philips SEM 505 Treatment of cells with 150 nM OD1 for 3 days results in extensive plasma membrane blebbing which is considered as a hallmark of apoptosis The FSC/SSC analysis shows that this membrane blebbing also dramatically increases the side-light scattering intensities of the cells.

Claims: Claims 1. An oligonucleotide derivative which is specifically hybridizable to a region ranging from base position no. 687 (5') to no. 706 (3') of the human bcl-xL mRNA encoding human bcl-xL protein. 2. The oligonucleotide derivative according to claim 1 , which oligonucleotide derivative additionally is specifically hybridizable to a region ranging from base position 2032 (5') to 2051 (3') of the human bcl-2 mRNA encoding human bcl-2 protein. 3. The oligonucleotide derivative according to any of claims 1 or 2, comprising a base sequence which is complementary to at least a part of the said region of the human bcl-xL mRNA or the human bcl-2 mRNA, or wherein such base sequence contains up to 3 mispairing building blocks, or wherein such base sequence contains up to 3 abasic building blocks. 4. The oligonucleotide derivative according to any of claims 1 to 3 having a length of 8 to 25 consecutive building blocks. 5. The oligonucleotide derivative according to claim 4 having a length of 20 consecutive building blocks. 6. The oligonucleotide derivative according to any of claims 3 to 5, wherein said base sequence is selected from the group consisting of the base sequence 5'-AAGGCATCCCAGCCTCCGTT-3' (SEQ ID NO: 3), the base sequence 5'-AAAGTATCCCAGCCGCCGTT-3' (SEQ ID NO: 4), and the base sequence 5'-AAAGCATCCCAGCCTCCGTT-3' (SEQ ID NO: 5). 7. The oligonucleotide derivative according to any of claims 1 to 6, consisting of a base sequence selected from the group consisting of the base sequence 5'-AAGGCATCCCAGCCTCCGTT-3' (SEQ ID NO: 3), the base sequence S'-AAAGTATCCCAGCCGCCGTT-S' (SEQ ID NO: 4) and the base sequence S'-AAAGCATCCCAGCCTCCGTT-S' (SEQ ID NO: 5). 8. The oligonucleotide derivative according to any of claims 1 to 7, comprising at least one building block of formula (I) wherein Q is H, -OCH 3 , -0(CH 2 CH 2 ) n OCH 3 , or -OCH 2 CH 2 NR,R 2 , wherein R, and R 2 are, independently of each other, H or -CH 3 , and wherein n is 1 , 2 or 3; V and W are, independently of each other, the same or different radicals of an internucleosidic bridging group selected from the following group: 5'-0-P(0)(OH)-0-3' (phosphodiester), 5'-0-P(0)(SH)-0-3' (phosphorothioate), 5'-0-P(S)(SH)-0-3' (phosphodithioate), 5'-0-P(0)(CH 3 )-0-3' (methylphosphonate), 5'-0-P(0)(NH-R 7 )-0-3' (phosphoamidate) in which R 7 is CrCaalkyl, 5'-0-P(0)(0R 8 )-O-3' (phosphotri- ester) in which R 8 is C C 3 alkyl, 5'-0-S(0) 2 -CH 2 -3' (sulfonate), 5'-0-S(0) 2 -NH-3' (sulfamate), 5'-NH- S(0) 2 -CH 2 -3' (sulfonamide), 5'-CH 2 -S(0) 2 -CH 2 -3' (sulfone), 5'-0-S(0)-0-3' (sulfite), 5'-CH 2 -S(0)-CH 2 -3' (sulfoxide), 5'-CH 2 -S-CH 2 -3' (sulfide), 5'-0-CH 2 -0-3' (formacetal), 5'-S-CH 2 -0-3' (3'-thioformacetal), 5'- 0-CH 2 -S-3' (5'-thioformacetal), 5'-CH 2 -CH 2 -S-3' (thioether), 5'-CH 2 -NH-0-3' (hydroxylamine), 5'-CH 2 - N(CH 3 )-0-3' (methylene(methylimino)), 5'-CH 2 -0-N(CH 3 )-3' (methyleneoxy(methylimino)), 5'-0-C(0)- NH-3' (5'-N-carbamate), 5'-CH 2 -C(0)-NH-3' (amide), 5'-NH-C(0)-CH 2 -3' (amide 2), 5'-CH 2 -NH-C(0)-3' (amide 3) and 5'-C(0)-NH-CH 2 -3' (amide 4), and the tautomeric forms thereof; or one of V and W is such an internucleosidic bridging group and the other is a terminal radical selected from the group consisting of -OH and -NH 2 , preferably -OH; and B is a radical of a nucleic acid base; with the proviso that if Q is H, then at least one of V or W is an internucleosidic bridging group other than 5'-0-P(0)(OH)-0-3' (phosphodiester). 9. An oligonucleotide derivative according to claim 8, wherein B is a radical of the formula (V1 ), (V2), (V3), (V4) or (V5) in which R b2 is H, -NH 2 or -OH; and R b3 is H, Br, I, -CN, -C≡C-CH 3 , -C(0)NH 2 or -CH 3 ; R M is -NH 2 or -OH; and R b5 is H, F, Br, I, -CN, -C≡C-CH 3 , -C(0)NH 2 or -CH 3 . 10. An oligonucleotide derivative according to claim 9, wherein B is a radical of the formula (V1) or (V5) in which R b1 is -NH 2 , -SH or -OH; R b2 is H, -NH 2 or -OH; R M is -NH 2 or -OH; and R b5 is H, F, Br, I, -CN, -C≡C-CH 3 , -C(0)NH 2 or -CH 3 . 11. An oligonucleotide derivative according to claim 10, wherein B is selected from the group of the following radicals: xanthine, hypoxanthine, adenine, 2-aminoadenine, guanine, 6-thioguanine, uracil, thymine, cytosine, 5-methylcytosine, 5-propynyluracil, 5-fluorouracil and 5-propynylcytosine. 12. An oligonucleotide derivative according to claim 8, wherein V and W, as radicals of an internucleosidic bridging group, are, independently of each other, selected from the following group: 5'-0- P(0)(OH)-0-3' (phosphodiester), 5'-0-P(0)(SH)-0-3' (phosphorothioate) and 5'-CH 2 -C(0)-NH-3' (amide). 13. An oligonucleotide derivative according to claim 12, wherein one of the radicals V or W, as radicals of an internucleosidic bridging group, is 5'-0-P(0)(OH)-0-3' (phosphodiester) and the other radical is 5'-0-P(0)(SH)-0-3" (phosphorothioate). 14. The oligonucleotide derivate according to claim 13, wherein both V and W as radicals of an internucleosidic bridging group are 5'-0-P(0)(OH)-0-3' (phosphodiester) or are 5'-0-P(0)(SH)-0-3' (phosphorothioate). 15. The oligonucleotide derivative according to claim 8, wherein V and W, as terminal radicals, are, independently of each other, -OH or -NH 2 . 16. The oligonucleotide derivative according to claim 8, wherein Q is selected from the group consisting of 2'-0-methyl, 2'-0-methoxyethoxy, 2'-0-di(methoxyethoxy), 2'-0-tri(methoxyethoxy), 2'-0- aminoethoxy, 2'-0-monomethylaminoethoxy and 2'-0-dimethylaminoethoxy. 17. The oligonucleotide derivative according to claim 8, consisting of a base sequence selected from the group consisting of the base sequence 5'-AAGGCATCCCAGCCTCCGTT-3' (SEQ ID NO: 3) and the base sequence 5'-AAAGTATCCCAGCCGCCGTT-3' (SEQ ID NO: 4), wherein each V and each W as radicals of an internucleosidic bridging group of the building blocks according to formula (I) are of the 5'-0-P(0)(SH)-0-3' (phosphorothioate) type and wherein each Q according to formula (I) is -H. 18. The oligonucleotide derivative according to claim 8, consisting of a base sequence selected from the group consisting of the base sequence 5'-AAGGCATCCCAGCCTCCGTT-3' (SEQ ID NO: 3), the base sequence 5'-AAAGTATCCCAGCCGCCGTT-3' (SEQ ID NO: 4), and the base sequence 5'-AAAGCATCCCAGCCTCCGTT-3' (SEQ ID NO: 5), wherein each V and each W as radicals of an mternucleosidic bridging group of all building blocks according to formula (I) are of the 5'-0-P(0)(SH)- 0-3' (phosphorothioate) type, and wherein each Q according to formula (I) of the nucleotides being underlined is -OCH 2 CH 2 OCH 3 and wherein each Q according to formula (I) of the remaining nucleotides is -H 19. A process for the preparation of an oligonucleotide derivative according to any of claims 1 to 18, said process comprising incorporating at least one building block of formula (I) according to claim 8 into the oligonucleotide derivative during oligonucleotide synthesis 20. A pharmaceutical composition comprising an oligonucleotide derivative according to any of claims 1 to 18, optionally together with a pharmaceutically acceptable excipient and/or auxilliary substance, said pharmaceutical composition being suitable for administration to humans suffering from a disease that responds to the modulation of human bcl-xL expression or that responds to the modulation of human bcl-xl and human bcl-2 expression 21. An oligonucleotide derivative according to any of claims 1 to 18 for use in medicine 22. Use of an oligonucleotide derivative according to any of claims 1 to 18 in the preparation of a pharmaceutical composition for treatment of a disease status associated with the biosynthesis of human bcl-xL protein or with the biosynthesis of both the human bcl-xL protein and the human bcl-2 protein. 23. A method of treatment of a disease status accociated with the expression of human bcl-xL protein or with the expression of both the human bcl-xL protein and the human bcl-2 protein, comprising application of an oligonucleotide derivative according to any of claims 1 to 18 24. A method of modulating the biosynthesis of human bcl-xL protein in a cell, comprising application of an oligonucleotide derivative according to any of claims 1 to 18 to said cell 25. An oligonucleotide derivative according to any of claims 1 to 18 for use in a diagnostic method

Inventors: HUESKEN DIETER, LUEDKE GERD, ZANGEMEISTER-WITTKE UWE, HUESKEN, DIETER, LUEDKE, GERD, ZANGEMEISTER-WITTKE, UWE
Applicants: HUESKEN DIETER, LUEDKE GERD, UNIV ZUERICH, ZANGEMEISTER WITTKE UWE, HUESKEN, DIETER, LUEDKE, GERD, UNIVERSITAET ZUERICH, ZANGEMEISTER-WITTKE, UWE
Application year: 2000
Main classification: C12N15
IPC codes: A61P35, C12N15