[0001] This application is a continuation of U.S. patent application Ser. No. 09/640,101 filed Aug. 15, 2000 which is a continuation-in-part of U.S. patent application Ser. No. 09/286,904, filed Apr. 6, 1999.
[0002] This invention relates to compositions and methods for modulating expression of p38 mitogen activated protein kinase genes, a family of naturally present cellular genes involved in signal transduction, and inflammatory and apoptotic responses. This invention is also directed to methods for inhibiting inflammation or apoptosis; these methods can be used diagnostically or therapeutically. Furthermore, this invention is directed to treatment of diseases or conditions associated with expression of p38 mitogen activated protein kinase genes.
[0003] Cellular responses to external factors, such as growth factors, cytokines, and stress conditions, result in altered gene expression. These signals are transmitted from the cell surface to the nucleus by signal transduction pathways. Beginning with an external factor binding to an appropriate receptor, a cascade of signal transduction events is initiated. These responses are mediated through activation of various enzymes and the subsequent activation of specific transcription factors. These activated transcription factors then modulate the expression of specific genes.
[0004] The phosphorylation of enzymes plays a key role in the transduction of extracellular signals into the cell. Mitogen activated protein kinases (MAPKs), enzymes which effect such phosphorylations are targets for the action of growth factors, hormones, and other agents involved in cellular metabolism, proliferation and differentiation (Cobb et al.,
[0005] One of the MAPK signal transduction pathways involves the MAP kinases p38α and p38β_(also known as CSaids Binding Proteins, CSBP). These MAP kinases are responsible for the phosphorylation of ATF-2, MEFC2 and a variety of other cellular effectors that may serve as substrates for p38 MAPK proteins (Kummer et al,
[0006] Besides p38α and p38β, other p38 MAPK family members have been described, including p38γ (Li et al,
[0007] Modulation of the expression of one or more p38 MAPKs is desirable in order to interfere with inflammatory or apoptotic responses associated with disease states and to modulate the transcription of genes stimulated by ATF-2, MEFC2 and other p38 MAPK phosphorylation substrates.
[0008] Inhibitors of p38 MAPKs have been shown to have efficacy in animal models of arthritis (Badger, A. M., et al.,
[0009] Therapeutic agents designed to target p38 MAPKs include small molecule inhibitors and antisense oligonucleotides. Small molecule inhibitors based on pyridinylimidazole are described in U.S. Pat. Nos. 5,670,527; 5,658,903; 5,656,644; 5,559,137; 5,593,992; and 5,593,991. WO 98/27098 and WO 99/00357 describe additional small molecule inhibitors, one of which has entered clinical trials. Other small molecule inhibitors are also known.
[0010] Antisense therapy represents a potentially more specific therapy for targeting p38 MAPKs and, in particular, specific p38 MAPK isoforms. Nagata, Y., et al. (Blood, 1998, 6, 1859-1869) disclose an antisense phosphothioester oligonucleotide targeted to the translational start site of mouse p38b (p38β). Aoshiba, K., et al. (
[0011] There remains a long-felt need for improved compositions and methods for modulating the expression of p38 MAP kinases.
[0012] The present invention provides antisense compounds which are targeted to nucleic acids encoding a p38 MAPK and are capable of modulating p38 MAPK expression. The present invention also provides oligonucleotides targeted to nucleic acids encoding a p38 MAPK. The present invention also comprises methods of modulating the expression of a p38 MAPK, in cells and tissues, using the oligonucleotides of the invention. Methods of inhibiting p38 MAPK expression are provided; these methods are believed to be useful both therapeutically and diagnostically. These methods are also useful as tools, for example, for detecting and determining the role of p38 MAPKs in various cell functions and physiological processes and conditions and for diagnosing conditions associated with expression of p38 MAPKs.
[0013] The present invention also comprises methods for diagnosing and treating inflammatory diseases, particularly rheumatoid arthritis. These methods are believed to be useful, for example, in diagnosing p38 MAPK-associated disease progression. These methods employ the oligonucleotides of the invention. These methods are believed to be useful both therapeutically, including prophylactically, and as clinical research and diagnostic tools.
[0014] p38 MAPKs play an important role in signal transduction in response to cytokines, growth factors and other cellular stimuli. Specific responses elicited by p38 include inflammatory and apoptotic responses. Modulation of p38 may be useful in the treatment of inflammatory diseases, such as rheumatoid arthritis.
[0015] The present invention employs antisense compounds, particularly oligonucleotides, for use in modulating the function of nucleic acid molecules encoding a p38 MAPK, ultimately modulating the amount of a p38 MAPK produced. This is accomplished by providing oligonucleotides which specifically hybridize with nucleic acids, preferably mRNA, encoding a p38 MAPK.
[0016] The antisense compounds may be used to modulate the function of a particular p38 MAPK isoform, e.g. for research purposes to determine the role of a particular isoform in a normal or disease process, or to treat a disease or condition that may be associated with a particular isoform. It may also be desirable to target multiple p38 MAPK isoforms. In each case, antisense compounds can be designed by taking advantage of sequence homology between the various isoforms. If an antisense compound to a particular isoform is desired, then the antisense compound is designed to a unique region in the desired isoform's gene sequence. With such a compound, it is desirable that this compound does not inhibit the expression of other isoforms. Less desirable, but acceptable, are compounds that do not “substantially” inhibit other isoforms. By “substantially”, it is intended that these compounds do not inhibit the expression of other isoforms greater than 25%; more preferred are compounds that do not inhibit other isoforms greater than 10%. If an antisense compound is desired to target multiple p38 isoforms, then regions of significant homology between the isoforms can be used.
[0017] This relationship between an antisense compound such as an oligonucleotide and its complementary nucleic acid target, to which it hybridizes, is commonly referred to as “antisense”. “Targeting” an oligonucleotide to a chosen nucleic acid target, in the context of this invention, is a multistep process. The process usually begins with identifying a nucleic acid sequence whose function is to be modulated. This may be, as examples, a cellular gene (or mRNA made from the gene) whose expression is associated with a particular disease state, or a foreign nucleic acid from an infectious agent. In the present invention, the target is a nucleic acid encoding a p38 MAPK; in other words, a p38 MAPK gene or RNA expressed from a p38 MAPK gene. p38 MAPK mRNA is presently the preferred target. The targeting process also includes determination of a site or sites within the nucleic acid sequence for the antisense interaction to occur such that modulation of gene expression will result.
[0018] In accordance with this invention, persons of ordinary skill in the art will understand that messenger RNA includes not only the information to encode a protein using the three letter genetic code, but also associated ribonucleotides which form a region known to such persons as the 5′-untranslated region, the 3′-untranslated region, the 5′ cap region and intron/exon junction ribonucleotides. Thus, oligonucleotides may be formulated in accordance with this invention which are targeted wholly or in part to these associated ribonucleotides as well as to the informational ribonucleotides. The oligonucleotide may therefore be specifically hybridizable with a transcription initiation site region, a translation initiation codon region, a 5′ cap region, an intron/exon junction, coding sequences, a translation termination codon region or sequences in the 5′- or 3′-untranslated region. Since, as is known in the art, the translation initiation codon is typically 5′-AUG (in transcribed mRNA molecules; 5′-ATG in the corresponding DNA molecule), the translation initiation codon is also referred to as the “AUG codon,” the “start codon” or the “AUG start codon.” A minority of genes have a translation initiation codon having the RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUG have been shown to function in vivo. Thus, the terms “translation initiation codon” and “start codon” can encompass many codon sequences, even though the initiator amino acid in each instance is typically methionine (in eukaryotes) or formylmethionine (prokaryotes). It is also known in the art that eukaryotic and prokaryotic genes may have two or more alternative start codons, any one of which may be preferentially utilized for translation initiation in a particular cell type or tissue, or under a particular set of conditions. In the context of the invention, “start codon” and “translation initiation codon” refer to the codon or codons that are used in vivo to initiate translation of an mRNA molecule transcribed from a gene encoding p38, regardless of the sequence(s) of such codons. It is also known in the art that a translation termination codon (or “stop codon”) of a gene may have one of three sequences, i.e., 5′-UAA, 5′-UAG and 5′-UGA (the corresponding DNA sequences are 5′-TAA, 5′-TAG and 5′-TGA, respectively). The terms “start codon region” and “translation initiation codon region” refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from a translation initiation codon. This region is a preferred target region. Similarly, the terms “stop codon region” and “translation termination codon region” refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from a translation termination codon. This region is a preferred target region. The open reading frame (ORF) or “coding region,” which is known in the art to refer to the region between the translation initiation codon and the translation termination codon, is also a region which may be targeted effectively. Other preferred target regions include the 5′ untranslated region (5′UTR), known in the art to refer to the portion of an mRNA in the 5′ direction from the translation initiation codon, and thus including nucleotides between the 5′ cap site and the translation initiation codon of an mRNA or corresponding nucleotides on the gene) and the 3′ untranslated region (3′UTR), known in the art to refer to the portion of an mRNA in the 3′ direction from the translation termination codon, and thus including nucleotides between the translation termination codon and 3′ end of an mRNA or corresponding nucleotides on the gene). mRNA splice sites may also be preferred target regions, and are particularly useful in situations where aberrant splicing is implicated in disease, or where an overproduction of a particular mRNA splice product is implicated in disease. Aberrant fusion junctions due to rearrangements or deletions may also be preferred targets.
[0019] Once the target site or sites have been identified, oligonucleotides are chosen which are sufficiently complementary to the target, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired modulation.
[0020] “Hybridization”, in the context of this invention, means hydrogen bonding, also known as Watson-Crick base pairing, between complementary bases, usually on opposite nucleic acid strands or two regions of a nucleic acid strand. Guanine and cytosine are examples of complementary bases which are known to form three hydrogen bonds between them. Adenine and thymine are examples of complementary bases which form two hydrogen bonds between them.
[0021] “Specifically hybridizable” and “complementary” are terms which are used to indicate a sufficient degree of complementarity such that stable and specific binding occurs between the DNA or RNA target and the oligonucleotide.
[0022] It is understood that an oligonucleotide need not be 100% complementary to its target nucleic acid sequence to be specifically hybridizable. An oligonucleotide is specifically hybridizable when binding of the oligonucleotide to the target interferes with the normal function of the target molecule to cause a loss of utility, and there is a sufficient degree of complementarity to avoid non-specific binding of the oligonucleotide to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment and, in the case of in vitro assays, under conditions in which the assays are conducted.
[0023] Hybridization of antisense oligonucleotides with mRNA interferes with one or more of the normal functions of mRNA. The functions of mRNA to be interfered with include all vital functions such as, for example, translocation of the RNA to the site of protein translation, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and catalytic activity which may be engaged in by the RNA.
[0024] The overall effect of interference with mRNA function is modulation of p38 MAPK expression. In the context of this invention “modulation” means either inhibition or stimulation; i.e., either a decrease or increase in expression. This modulation can be measured in ways which are routine in the art, for example by Northern blot assay of mRNA expression as taught in the examples of the instant application or by Western blot or ELISA assay of protein expression, or by an immunoprecipitation assay of protein expression, as taught in the examples of the instant application. Effects on cell proliferation or tumor cell growth can also be measured, as taught in the examples of the instant application.
[0025] The oligonucleotides of this invention can be used in diagnostics, therapeutics, prophylaxis, and as research reagents and in kits. Since the oligonucleotides of this invention hybridize to nucleic acids encoding a p38 MAPK, sandwich, calorimetric and other assays can easily be constructed to exploit this fact. Furthermore, since the oligonucleotides of this invention hybridize specifically to nucleic acids encoding particular isoforms of p38 MAPK, such assays can be devised for screening of cells and tissues for particular p38 MAPK isoforms. Such assays can be utilized for diagnosis of diseases associated with various p38 MAPK isoforms. Provision of means for detecting hybridization of oligonucleotide with a p38 MAPK gene or mRNA can routinely be accomplished. Such provision may include enzyme conjugation, radiolabelling or any other suitable detection systems. Kits for detecting the presence or absence of p38 MAPK may also be prepared.
[0026] The present invention is also suitable for diagnosing abnormal inflammatory states in tissue or other samples from patients suspected of having an inflammatory disease such as rheumatoid arthritis. The ability of the oligonucleotides of the present invention to inhibit inflammation may be employed to diagnose such states. A number of assays may be formulated employing the present invention, which assays will commonly comprise contacting a tissue sample with an oligonucleotide of the invention under conditions selected to permit detection and, usually, quantitation of such inhibition. In the context of this invention, to “contact” tissues or cells with an oligonucleotide or oligonucleotides means to add the oligonucleotide(s), usually in a liquid carrier, to a cell suspension or tissue sample, either in vitro or ex vivo, or to administer the oligonucleotide(s) to cells or tissues within an animal. Similarly, the present invention can be used to distinguish p38 MAPK-associated diseases, from diseases having other etiologies, in order that an efficacious treatment regime can be designed.
[0027] The oligonucleotides of this invention may also be used for research purposes. Thus, the specific hybridization exhibited by the oligonucleotides may be used for assays, purifications, cellular product preparations and in other methodologies which may be appreciated by persons of ordinary skill in the art.
[0028] In the context of this invention, the term “oligonucleotide” refers to an oligomer or polymer of ribonucleic acid or deoxyribonucleic acid. This term includes oligonucleotides composed of naturally-occurring nucleobases, sugars and covalent intersugar (backbone) linkages as well as oligonucleotides having non-naturally-occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced binding to target and increased stability in the presence of nucleases.
[0029] The antisense compounds in accordance with this invention preferably comprise from about 5 to about 50 nucleobases. Particularly preferred are antisense oligonucleotides comprising from about 8 to about 30 nucleobases (i.e. from about 8 to about 30 linked nucleosides). Preferred embodiments comprise at least an 8-nucleobase portion of a sequence of an antisense compound which inhibits the expression of a p38 mitogen activated kinase. As is known in the art, a nucleoside is a base-sugar combination. The base portion of the nucleoside is normally a heterocyclic base. The two most common classes of such heterocyclic bases are the purines and the pyrimidines. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to either the 2=, 3= or 5=hydroxyl moiety of the sugar. In forming oligonucleotides, the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. In turn the respective ends of this linear polymeric structure can be further joined to form a circular structure, however, open linear structures are generally preferred. Within the oligonucleotide structure, the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3=to 5=phosphodiester linkage.
[0030] Specific examples of some preferred modified oligonucleotides envisioned for this invention include those containing phosphorothioates, phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages. Most preferred are oligonucleotides with phosphorothioates (usually abbreviated in the art as P═S) and those with CH
[0031] The oligonucleotides of the invention may additionally or alternatively include nucleobase modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include adenine (A), guanine (G), thymine (T), cytosine (C) and uracil (U). Modified nucleobases include nucleobases found only infrequently or transiently in natural nucleic acids, e.g., hypoxanthine, 6-methyladenine and 5-methylcytosine, as well as synthetic nucleobases, e.g., 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N
[0032] Another preferred additional or alternative modification of the oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more lipophilic moieties which enhance the cellular uptake of the oligonucleotide. Such lipophilic moieties may be linked to an oligonucleotide at several different positions on the oligonucleotide. Some preferred positions include the 3′ position of the sugar of the 3′ terminal nucleotide, the 5′ position of the sugar of the 5′ terminal nucleotide, and the 2′ position of the sugar of any nucleotide. The N
[0033] The present invention also includes oligonucleotides which are chimeric oligonucleotides. “Chimeric” oligonucleotides or “chimeras,” in the context of this invention, are oligonucleotides which contain two or more chemically distinct regions, each made up of at least one nucleotide. These oligonucleotides typically contain at least one region wherein the oligonucleotide is modified so as to confer upon the oligonucleotide increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the oligonucleotide may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of antisense inhibition of gene expression. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art. This RNAse H-mediated cleavage of the RNA target is distinct from the use of ribozymes to cleave nucleic acids. Ribozymes are not comprehended by the present invention.
[0034] Examples of chimeric oligonucleotides include but are not limited to “gapmers,” in which three distinct regions are present, normally with a central region flanked by two regions which are chemically equivalent to each other but distinct from the gap. A preferred example of a gapmer is an oligonucleotide in which a central portion (the “gap”) of the oligonucleotide serves as a substrate for RNase H and is preferably composed of 2′-deoxynucleotides, while the flanking portions (the 5′ and 3′ “wings”) are modified to have greater affinity for the target RNA molecule but are unable to support nuclease activity (e.g., 2′-fluoro- or 2′-O-methoxyethyl-substituted). Other chimeras include “wingmers,” also known in the art as “hemimers,” that is, oligonucleotides with two distinct regions. In a preferred example of a wingmer, the 5′ portion of the oligonucleotide serves as a substrate for RNase H and is preferably composed of 2′-deoxynucleotides, whereas the 3′ portion is modified in such a fashion so as to have greater affinity for the target RNA molecule but is unable to support nuclease activity (e.g., 2′-fluoro- or 2′-O-methoxyethyl-substituted), or vice-versa. In one embodiment, the oligonucleotides of the present invention contain a 2′-O-methoxyethyl (2′-O—CH
[0035] The oligonucleotides used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including Applied Biosystems. 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 well known to use similar techniques to prepare oligonucleotides such as the phosphorothioates and 2′-alkoxy or 2′-alkoxyalkoxy derivatives, including 2′-O-methoxyethyl oligonucleotides [Martin, P.,
[0036] The antisense compounds of the present invention include bioequivalent compounds, including pharmaceutically acceptable salts and prodrugs. This is intended to encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound which, upon administration to an animal including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to pharmaceutically acceptable salts of the nucleic acids of the invention and prodrugs of such nucleic acids.
[0037] Pharmaceutically acceptable “salts” are physiologically and pharmaceutically acceptable salts of the nucleic acids of the invention: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto [see, for example, Berge et al., “Pharmaceutical Salts,”
[0038] For oligonucleotides, examples of pharmaceutically acceptable salts include but are not limited to (a) salts formed with cations such as sodium, potassium, ammonium, magnesium, calcium, polyamines such as spermine and spermidine, etc.; (b) acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; (c) salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d) salts formed from elemental anions such as chlorine, bromine, and iodine.
[0039] The oligonucleotides of the invention may additionally or alternatively be prepared to be delivered in a “prodrug” form. The term “prodrug” indicates a therapeutic agent that is prepared in an inactive form that is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes or other chemicals and/or conditions. In particular, prodrug versions of the oligonucleotides of the invention are prepared as SATE [(S-acetyl-2-thioethyl)phosphate] derivatives according to the methods disclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993.
[0040] For therapeutic or prophylactic treatment, oligonucleotides are administered in accordance with this invention. Oligonucleotide compounds of the invention may be formulated in a pharmaceutical composition, which may include pharmaceutically acceptable carriers, thickeners, diluents, buffers, preservatives, surface active agents, neutral or cationic lipids, lipid complexes, liposomes, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients and the like in addition to the oligonucleotide. Such compositions and formulations are comprehended by the present invention.
[0041] Pharmaceutical compositions comprising the oligonucleotides of the present invention may include penetration enhancers in order to enhance the alimentary delivery of the oligonucleotides. Penetration enhancers may be classified as belonging to one of five broad categories, i.e., fatty acids, bile salts, chelating agents, surfactants and non-surfactants (Lee et al.,
[0042] The compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions may contain additional compatible pharmaceutically-active materials such as, e.g., antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the composition of present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the invention.
[0043] Regardless of the method by which the oligonucleotides of the invention are introduced into a patient, colloidal dispersion systems may be used as delivery vehicles to enhance the in vivo stability of the oligonucleotides and/or to target the oligonucleotides to a particular organ, tissue or cell type. Colloidal dispersion systems include, but are not limited to, macromolecule complexes, nanocapsules, microspheres, beads and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, liposomes and lipid:oligonucleotide complexes of uncharacterized structure. A preferred colloidal dispersion system is a plurality of liposomes. Liposomes are microscopic spheres having an aqueous core surrounded by one or more outer layers made up of lipids arranged in a bilayer configuration [see, generally, Chonn et al.,
[0044] The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic, vaginal, rectal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular injection, pulmonary administration, e.g., by inhalation or insufflation, or intracranial, e.g., intrathecal or intraventricular, administration. Oligonucleotides with at least one 2′-O-methoxyethyl modification are believed to be particularly useful for oral administration.
[0045] Formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful.
[0046] Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets or tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable.
[0047] Compositions for parenteral administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives. In some cases it may be more effective to treat a patient with an oligonucleotide of the invention in conjunction with other traditional therapeutic modalities in order to increase the efficacy of a treatment regimen. In the context of the invention, the term “treatment regimen” is meant to encompass therapeutic, palliative and prophylactic modalities. For example, a patient may be treated with conventional chemotherapeutic agents, particularly those used for tumor and cancer treatment. Examples of such chemotherapeutic agents include but are not limited to daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosine arabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D, mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine (CA), 5-azacytidine, hydroxyurea, deoxycoformycin, 4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU), 5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine,taxol, vincristine, vinblastine, etoposide, trimetrexate, teniposide, cisplatin and diethylstilbestrol (DES). See, generally,
[0048] The formulation of therapeutic compositions and their subsequent administration is believed to be within the skill of those in the art. Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on EC
[0049] Thus, in the context of this invention, by “therapeutically effective amount” is meant the amount of the compound which is required to have a therapeutic effect on the treated mammal. This amount, which will be apparent to the skilled artisan, will depend upon the type of mammal, the age and weight of the mammal, the type of disease to be treated, perhaps even the gender of the mammal, and other factors which are routinely taken into consideration when treating a mammal with a disease. A therapeutic effect is assessed in the mammal by measuring the effect of the compound on the disease state in the animal. For example, if the disease to be treated is cancer, therapeutic effects are assessed by measuring the rate of growth or the size of the tumor, or by measuring the production of compounds such as cytokines, production of which is an indication of the progress or regression of the tumor.
[0050] The following examples illustrate the present invention and are not intended to limit the same.
[0051] Unmodified oligodeoxynucleotides are synthesized on an automated DNA synthesizer (Applied Biosystems model 380B) using standard phosphoramidite chemistry with oxidation by iodine. β-cyanoethyldiisopropyl-phosphoramidites were purchased from Applied Biosystems (Foster City, Calif.). For phosphorothioate oligonucleotides, the standard oxidation bottle was replaced by a 0.2 M solution of
[0052] 2′-methoxy oligonucleotides are synthesized using 2′-methoxy β-cyanoethyldiisopropyl-phosphoramidites (Chemgenes, Needham, Mass.) and the standard cycle for unmodified oligonucleotides, except the wait step after pulse delivery of tetrazole and base was increased to 360 seconds. Other 2′-alkoxy oligonucleotides were synthesized by a modification of this method, using appropriate 2′-modified amidites such as those available from Glen Research, Inc., Sterling, Va.
[0053] 2′-fluoro oligonucleotides are synthesized as described in Kawasaki et al.,
[0054] The synthesis of 2′-deoxy-2′-fluoroguanosine is accomplished using tetraisopropyldisiloxanyl (TPDS) protected 9-β-D-arabinofuranosylguanine as starting material, and conversion to the intermediate diisobutyryl-arabinofuranosylguanosine. Deprotection of the TPDS group is followed by protection of the hydroxyl group with THP to give diisobutyryl di-THP protected arabinofuranosylguanine. Selective O-deacylation and triflation is followed by treatment of the crude product with fluoride, then deprotection of the THP groups. Standard methodologies are used to obtain the 5′-DMT- and 5′-DMT-3′-phosphoramidites.
[0055] Synthesis of 2′-deoxy-2′-fluorouridine is accomplished by the modification of a known procedure in which 2,2′-anhydro-1-β-D-arabinofuranosyluracii is treated with 70% hydrogen fluoride-pyridine. Standard procedures are used to obtain the 5′-DMT and 5′-DMT-3′phosphoramidites.
[0056] 2′-deoxy-2′-fluorocytidine is synthesized via amination of 2′-deoxy-2′-fluorouridine, followed by selective protection to give N
[0057] 2′-(2-methoxyethyl)-modified amidites were synthesized according to Martin, P.,
[0058] Synthesis of 5-Methyl Cytosine Monomers:
[0059] 2,2′-Anhydro[1-(β-D-arabinofuranosyl)-5-methyluridine]:
[0060] 5-Methyluridine (ribosylthymine, commercially available through Yamasa, Choshi, Japan) (72.0 g, 0.279 M), diphenyl-carbonate (90.0 g, 0.420 M) and sodium bicarbonate (2.0 g, 0.024 M) were added to DMF (300 mL). The mixture was heated to reflux, with stirring, allowing the evolved carbon dioxide gas to be released in a controlled manner. After 1 hour, the slightly darkened solution was concentrated under reduced pressure. The resulting syrup was poured into diethylether (2.5 L), with stirring. The product formed a gum. The ether was decanted and the residue was dissolved in a minimum amount of methanol (ca. 400 mL). The solution was poured into fresh ether (2.5 L) to yield a stiff gum. The ether was decanted and the gum was dried in a vacuum oven (60EC at 1 mm Hg for 24 hours) to give a solid which was crushed to a light tan powder (57 g, 85% crude yield). The material was used as is for further reactions.
[0061] 2′-O-Methoxyethyl-5-methyluridine:
[0062] 2,2′-Anhydro-5-methyluridine (195 g, 0.81 M), tris(2-methoxyethyl)borate (231 g, 0.98 M) and 2-methoxyethanol (1.2 L) were added to a 2 L stainless steel pressure vessel and placed in a pre-heated oil bath at 160EC. After heating for 48 hours at 155-160EC, the vessel was opened and the solution evaporated to dryness and triturated with MeOH (200 mL). The residue was suspended in hot acetone (1 L). The insoluble salts were filtered, washed with acetone (150 mL) and the filtrate evaporated. The residue (280 g) was dissolved in CH
[0063] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine:
[0064] 2′-O-Methoxyethyl-5-methyluridine (160 g, 0.506 M) was co-evaporated with pyridine (250 mL) and the dried residue dissolved in pyridine (1.3 L). A first aliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) was added and the mixture stirred at room temperature for one hour. A second aliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) was added and the reaction stirred for an additional one hour. Methanol (170 mL) was then added to stop the reaction. HPLC showed the presence of approximately 70% product. The solvent was evaporated and triturated with CH
[0065] 3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine:
[0066] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (106 g, 0.167 M), DMF/pyridine (750 mL of a 3:1 mixture prepared from 562 mL of DMF and 188 mL of pyridine) and acetic anhydride (24.38 mL, 0.258 M) were combined and stirred at room temperature for 24 hours. The reaction was monitored by tlc by first quenching the tlc sample with the addition of MeOH. Upon completion of the reaction, as judged by tlc, MeOH (50 mL) was added and the mixture evaporated at 35EC. The residue was dissolved in CHCl
[0067] 3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine:
[0068] A first solution was prepared by dissolving 3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (96 g, 0.144 M) in CH
[0069] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine:
[0070] A solution of 3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine (103 g, 0.141 M) in dioxane (500 mL) and NH
[0071] N
[0072] 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (85 g, 0.134 M) was dissolved in DMF (800 mL) and benzoic anhydride (37.2 g, 0.165 M) was added with stirring. After stirring for 3 hours, tic showed the reaction to be approximately 95% complete. The solvent was evaporated and the residue azeotroped with MeOH (200 mL). The residue was dissolved in CHCl
[0073] N′-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine-3′-amidite:
[0074] N
[0075] 5-methyl-2′-deoxycytidine (5-me-C) containing oligonucleotides were synthesized according to published methods [Sanghvi et al.,
[0076] 2=-O-(dimethylaminooxyethyl) Nucleoside Amidites
[0077] 2′-(Dimethylaminooxyethoxy) nucleoside amidites [also known in the art as 2′-O-(dimethylaminooxyethyl) nucleoside amidites] are prepared as described in the following paragraphs. Adenosine, cytidine and guanosine nucleoside amidites are prepared similarly to the thymidine (5-methyluridine) except the exocyclic amines are protected with a benzoyl moiety in the case of adenosine and cytidine and with isobutyryl in the case of guanosine.
[0078] 5′-O-tert-Butyldiphenylsilyl-O
[0079] O
[0080] 5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine
[0081] In a 2 L stainless steel, unstirred pressure reactor is added borane in tetrahydrofuran (1.0 M, 2.0 eq, 622 mL). In the fume hood and with manual stirring, ethylene glycol (350 mL, excess) is added cautiously at first until the evolution of hydrogen gas subsided. 5′-O-tert-Butyldiphenylsilyl-O
[0082] 2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine
[0083] 5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine (20 g, 36.98 mmol) was mixed with triphenylphosphine (11.63 g, 44.36 mmol) and N-hydroxyphthalimide (7.24 g, 44.36 mmol). It was then dried over P
[0084] 5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine
[0085] 2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine (3.1 g, 4.5 mmol) is dissolved in dry CH
[0086] 5′-O-tert-Butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine
[0087] 5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine (1.77 g, 3.12 mmol) is dissolved in a solution of 1M pyridinium p-toluenesulfonate (PPTS) in dry MeOH (30.6 mL). Sodium cyanoborohydride (0.39 g, 6.13 mmol) is added to this solution at 10° C. under inert atmosphere. The reaction mixture is stirred for 10 minutes at 10° C. After that the reaction vessel is removed from the ice bath and stirred at room temperature for 2 hr, the reaction monitored by TLC (5% MeOH in CH
[0088] 2′-O-(dimethylaminooxyethyl)-5-methyluridine
[0089] Triethylamine trihydrofluoride (3.91 mL, 24.0 mmol) is dissolved in dry THF and triethylamine (1.67 mL, 12 mmol, dry, kept over KOH). This mixture of triethylamine-2HF is then added to 5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine (1.40 g, 2.4 mmol) and stirred at room temperature for 24 hrs. Reaction is monitored by TLC (5% MeOH in CH
[0090] 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine
[0091] 2′-O-(dimethylaminooxyethyl)-5-methyluridine (750 mg, 2.17 mmol) is dried over P
[0092] 5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-F(2-cyanoethyl)-N,N-diisopropylphosphoramiditel
[0093] 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine (1.08 g, 1.67 mmol) is co-evaporated with toluene (20 mL). To the residue N,N-diisopropylamine tetrazonide (0.29 g, 1.67 mmol) is added and dried over P
[0094] 2′-(Aminooxyethoxy) Nucleoside Amidites
[0095] 2′-(Aminooxyethoxy) nucleoside amidites [also known in the art as 2′-O-(aminooxyethyl) nucleoside amidites] are prepared as described in the following paragraphs. Adenosine, cytidine and thymidine nucleoside amidites are prepared similarly.
[0096] N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-F(2-cyanoethyl)-N,N-diisopropylphosphoramid itel
[0097] The 2′-O-aminooxyethyl guanosine analog may be obtained by selective 2′-O-alkylation of diaminopurine riboside. Multigram quantities of diaminopurine riboside may be purchased from Schering AG (Berlin) to provide 2′-O-(2-ethylacetyl) diaminopurine riboside along with aminor amount of the 3′-O-isomer. 2′-O-(2-ethylacetyl) diaminopurine riboside may be resolved and converted to 2′-O-(2-ethylacetyl)guanosine by treatment with adenosine deaminase. (McGee, D. P. C., Cook, P. D., Guinosso, C. J., WO 94/02501 A1 940203.) Standard protection procedures should afford 2′-O-(2-ethylacetyl) -5′-O-(4,4′-dimethoxytrityl)guanosine and 2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxy trityl)guanosine which may be reduced to provide 2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl) guanosine. As before the hydroxyl group may be displaced by N-hydroxyphthalimide via a Mitsunobu reaction, and the protected nucleoside may phosphitylated as usual to yield 2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphorami dite].
[0098] Oligonucleotides having methylene (methylimino) (MMI) backbones are synthesized according to U.S. Pat. No. 5,378,825, which is coassigned to the assignee of the present invention and is incorporated herein in its entirety. For ease of synthesis, various nucleoside dimers containing MMI linkages were synthesized and incorporated into oligonucleotides. Other nitrogen-containing backbones are synthesized according to WO 92/20823 which is also coassigned to the assignee of the present invention and incorporated herein in its entirety.
[0099] Oligonucleotides having amide backbones are synthesized according to De Mesmaeker et al.,
[0100] Oligonucleotides with morpholino backbones are synthesized according to U.S. Pat. No. 5,034,506 (Summerton and Weller).
[0101] Peptide-nucleic acid (PNA) oligomers are synthesized according to P. E. Nielsen et al.,
[0102] After cleavage from the controlled pore glass column (Applied Biosystems) and deblocking in concentrated ammonium hydroxide at 55EC for 18 hours, the oligonucleotides are purified by precipitation twice out of 0.5 M NaCl with 2.5 volumes ethanol. Synthesized oligonucleotides were analyzed by polyacrylamide gel electrophoresis on denaturing gels and judged to be at least 85% full length material. The relative amounts of phosphorothioate and phosphodiester linkages obtained in synthesis were periodically checked by
[0103] Antisense oligonucleotides were designed to target human p38α. Target sequence data are from the p38 MAPK cDNA sequence; Genbank accession number L35253, provided herein as SEQ ID NO: 1. Oligonucleotides was synthesized as chimeric oligonucleotides (“gapmers”) 20 nucleotides in length, composed of a central “gap” region consisting of eight 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by six-nucleotide “wings.” The wings are composed of 2′-methoxyethyl (2′-MOE) nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P═S) throughout the oligonucleotide. All 2′-MOE cytosines were 5-methyl-cytosines. These oligonucleotide sequences are shown in Table 1.
[0104] The human Jurkat T-cell line (American Type Culture Collection, Manassas, Va.) was maintained in RPMI 1640 growth media supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, Utah). HUVEC cells (Clonetics, San Diego, Calif.) were cultivated in endothelial basal media supplemented with 10% FBS (Hyclone, Logan, Utah).
[0105] Jurkat cells were grown to approximately 75% confluency and resuspended in culture media at a density of 1×10
[0106] HUVEC cells were allowed to reach 75% confluency prior to use. The cells were washed twice with warm (37° C.) OPTIMEM™ (Life Technologies). The cells were incubated in the presence of the appropriate culture medium, without the growth factors added, and the oligonucleotide formulated in LIPOFECTIN7 (Life Technologies), a 1:1 (w/w) liposome formulation of the cationic lipid N-[1-(2,3-dioleyloxy)propyl]-n,n,n-trimethylammonium chloride (DOTMA), and dioleoyl phosphotidylethanolamine (DOPE) in membrane filtered water. HUVEC cells were treated with 100 nM oligonucleotide in 10 μg/ml LIPOFECTIN7. Treatment was for four hours.
[0107] Total mRNA was isolated using the RNEASY7 Mini Kit (Qiagen, Valencia, Calif.; similar kits from other manufacturers may also be used), separated on a 1% agarose gel, transferred to HYBOND™-N+ membrane (Amersham Pharmacia Biotech, Piscataway, N.J.), a positively charged nylon membrane, and probed. p38 MAPK probes were made using the Prime-A-Gene7 kit (Promega Corporation, Madison, Wis.), a random primer labeling kit, using mouse p38α or p38β cDNA as a template. A glyceraldehyde 3-phosphate dehydrogenase (G3PDH) probe was purchased from Clontech (Palo Alto, Calif.), Catalog Number 9805-1. The fragments were purified from low-melting temperature agarose, as described in Maniatis, T., et al., TABLE 1 Nucleotide Sequences of Human p38α Chimeric (deoxy gapped) Phosphorothicate Oligonucleotides TARGET GENE SEQ NUCLEOTIDE GENE ISIS NUCLEOTIDE SEQUENCE ID CO- TARGET NO 5′ −> 3′) NO ORDINATES REGION 16486 3 0001-0020 5′-UTR 16487 4 0044-0063 5′-UTR 16488 5 0087-0106 5′-UTR 16489 6 0136-0155 5′-UTR 16490 7 0160-0179 5′-UTR 16491 8 0201-0220 5′-UTR 16492 9 0250-0269 5′-UTR 16493 10 0278-0297 AUG 16494 11 0286-0305 AUG 16495 12 0308-0327 coding 16496 13 0643-0662 coding 16497 14 0798-0817 coding 16498 15 0939-0958 coding 16499 16 1189-1208 coding 16500 17 1368-1387 stop 16501 18 1390-1409 3′-UTR 16502 19 1413-1432 3′-UTR 16503 20 1444-1463 3′-UTR 16504 21 1475-1494 3′-UTR 16505 22 1520-1539 3′-UTR
[0108] For an initial screen of human p38α antisense oligonucleotides, Jurkat cells were electroporated with 10 μM oligonucleotide. mRNA was measured by Northern blot. Results are shown in Table 2. Oligonucleotides 16496 (SEQ ID NO. 13), 16500 (SEQ ID NO. 17) and 16503 (SEQ ID NO. 20) gave 35% or greater inhibition of p38α mRNA.
TABLE 2 Inhibition of Human p38α mRNA expression in Jurkat Cells by Chimeric (deoxy gapped) Phosphoro- thioate Oligonucleotides SEQ GENE ISIS ID TARGET % mRNA % mRNA No: NO: REGION EXPRESSION INHIBITION control — 100% 0% 16486 3 5′-UTR 212% — 16487 4 5′-UTR 171% — 16488 5 5′-UTR 157% — 16489 6 5′-UTR 149% — 16490 7 5′-UTR 152% — 16491 8 5′-UTR 148% — 16492 9 5′-UTR 125% — 16493 10 AUG 101% — 16494 11 AUG 72% 28% 16495 12 coding 72% 28% 16496 13 coding 61% 39% 16497 14 coding 104% — 16498 15 coding 88% 12% 16499 16 coding 74% 26% 16500 17 stop 63% 37% 16501 18 3′-UTR 77% 23% 16502 19 3′-UTR 79% 21% 16503 20 3′-UTR 65% 35% 16504 21 3′-UTR 72% 28% 16505 22 3′-UTR 93% 7%
[0109] The most active human p38α oligonucleotides were chosen for dose response studies. Oligonucleotide 16490 (SEQ ID NO. 7) which showed no inhibition in the initial screen was included as a negative control. Jurkat cells were grown and treated as described above except the concentration of oligonucleotide was varied as indicated in Table 3. Results are shown in Table 3. Each of the active oligonucleotides showed a dose response effect with ICTABLE 3 Dose Response of p38α mRNA in Jurkat cells to human p38α Chimeric (deoxy gapped) Phosphorothioate Oligonucleotides SEQ ID ASO Gene % mRNA % mRNA ISIS # NO: Target Dose Expression Inhibition control — — — 100% 0% 16496 13 coding 2.5 nM 94% 6% ″ ″ ″ 5 nM 74% 26% ″ ″ ″ 10 nM 47% 53% ″ ″ ″ 20 nM 41% 59% 16500 17 stop 2.5 nM 82% 18% ″ ″ ″ 5 nM 71% 29% ″ ″ ″ 10 nM 49% 51% ″ ″ ″ 20 nM 31% 69% 16503 20 3′-UTR 2.5 nM 74% 26% ″ ″ ″ 5 nM 61% 39% ″ ″ ″ 10 nM 53% 47% ″ ″ ″ 20 nM 41% 59% 16490 7 5′-UTR 2.5 nM 112% — ″ ″ ″ 5 nM 109% — ″ ″ ″ 10 nM 104% — ″ ″ ″ 20 nM 97% 3%
[0110] Antisense oligonucleotides were designed to target human p38β. Target sequence data are from the p38β MAPK cDNA sequence; Genbank accession number U53442, provided herein as SEQ ID NO: 23. Oligonucleotides was synthesized as chimeric oligonucleotides (“gapmers”) 20 nucleotides in length, composed of a central “gap” region consisting of ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by five-nucleotide “wings.” The wings are composed of 2′-methoxyethyl (2′-MOE) nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P═S) throughout the oligonucleotide. All 2′-MOE cytosines were 5-methyl-cytosines. These oligonucleotide sequences are shown in Table 4.
TABLE 4 Nucleotide Sequences of Human p38β Phosphorothioate Oligonucleotides TARGET GENE SEQ NUCLEOTIDE GENE ISIS NUCLEOTIDE SEQUENCE ID CO- TARGET NO. (5′ −> 3′) NO: ORDINATES REGION 17891 25 0006-0025 AUG 17892 26 0041-0060 coding 17893 27 0065-0084 coding 17894 28 0121-0140 coding 17895 29 0240-0259 coding 17896 30 0274-0293 coding 17897 31 0355-0374 coding 17898 32 0405-0424 coding 17899 33 0521-0540 coding 17900 34 0635-0654 coding 17901 35 0731-0750 coding 17902 36 0935-0954 coding 17903 37 1005-1024 coding 17904 38 1126-1145 stop 17905 39 1228-1247 3′-UTR 17906 40 1294-1313 3′-UTR 17907 41 1318-1337 3′-UTR 17908 42 1581-1600 3′-UTR 17909 43 1753-1772 3′-UTR 17910 44 1836-1855 3′-UTR
[0111] For an initial screen of human p38β antisense oligonucleotides, HUVEC cells were cultured and treated as described in Example 2. mRNA was measured by Northern blot as described in Example 2. Results are shown in Table 5. Every oligonucleotide tested gave at least 50% inhibition. Oligonucleotides 17892 (SEQ ID NO. 26), 17893 (SEQ ID NO. 27), 17894 (SEQ ID NO. 28), 17899 (SEQ ID NO. 33), 17901 (SEQ ID NO. 35), 17903 (SEQ ID NO. 37), 17904 (SEQ ID NO. 38), 17905 (SEQ ID NO. 39), 17907 (SEQ ID NO. 41), 17908 (SEQ ID NO. 42), and 17909 (SEQ ID NO. 43) gave greater than approximately 85% inhibition and are preferred.
TABLE 5 Inhibition of Human p38β mRNA expression in Huvec Cells by Chimeric (deoxy gapped) Phosphorothioate Oligonucleotides SEQ GENE ID TARGET % mRNA % mRNA ISIS No: NO: REGION EXPRESSION INHIBITION control — — 100% 0% 17891 25 AUG 22% 78% 17892 26 coding 10% 90% 17893 27 coding 4% 96% 17894 28 coding 13% 87% 17895 29 coding 25% 75% 17896 30 coding 24% 76% 17897 31 coding 25% 75% 17898 32 coding 49% 51% 17899 33 coding 5% 95% 17900 34 coding 40% 60% 17901 35 coding 15% 85% 17902 36 coding 49% 51% 17903 37 coding 11% 89% 17904 38 stop 9% 91% 17905 39 3′-UTR 14% 86% 17906 40 3′-UTR 22% 78% 17907 41 3′-UTR 8% 92% 17908 42 3′-UTR 17% 83% 17909 43 3′-UTR 13% 87% 17910 44 3′-UTR 26% 74%
[0112] Oligonucleotides 17893 (SEQ ID NO. 27), 17899 (SEQ ID NO. 33), 17904 (SEQ ID NO. 38), and 17907 (SEQ ID NO. 41) were chosen for dose response studies. HUVEC cells were cultured and treated as described in Example 2 except that the oligonucleotide concentration was varied as shown in Table 6. The Lipofectin7/Oligo ratio was maintained at 3 μg Lipofectin7/100 nM oligo, per ml. mRNA was measured by Northern blot as described in Example 2.
[0113] Results are shown in Table 6. Each oligonucleotide tested had an ICTABLE 6 Dose Response of p38β in Huvec cells to human p38β Chimeric (deoxy gapped) Phosphorothioate Oligonucleotides SEQ ID ASO Gene % mRNA % mRNA ISIS # NO: Target Dose Expression Inhibition control — — — 100% 0% 17893 27 coding 10 nM 37% 63% ″ ″ ″ 25 nM 18% 82% ″ ″ ″ 50 nM 16% 84% ″ ″ ″ 100 nM 19% 81% 17899 33 coding 10 nM 37% 63% ″ ″ ″ 25 nM 23% 77% ″ ″ ″ 50 nM 18% 82% ″ ″ ″ 100 nM 21% 79% 17904 38 stop 10 nM 31% 69% ″ ″ ″ 25 nM 21% 79% ″ ″ ″ 50 nM 17% 83% ″ ″ ″ 100 nM 19% 81% 17907 41 3′-UTR 10 nM 37% 63% ″ ″ ″ 25 nM 22% 78% ″ ″ ″ 50 nM 18% 72% ″ ″ ″ 100 nM 18% 72%
[0114] Antisense oligonucleotides were designed to target rat p38α. Target sequence data are from the p38 MAPK cDNA sequence; Genbank accession number U73142, provided herein as SEQ ID NO: 45. Oligonucleotides was synthesized as chimeric oligonucleotides (“gapmers”) 20 nucleotides in length, composed of a central “gap” region consisting of ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by five-nucleotide “wings.” The wings are composed of 2′-methoxyethyl (2′-MOE) nucleotides. The internucleoside (backbone) linkages in the wings are phosphodiester (P═O). Internucleoside linkages in the central gap are phosphorothioate (P═S). All 2′-MOE cytosines and 2′-OH cytosines were 5-methyl-cytosines. These oligonucleotide sequences are shown in Table 7.
[0115] bEND.3, a mouse endothelial cell line (gift of Dr. Werner Risau; see Montesano et al.,
[0116] After 4 hours at 37° C., the medium was replaced with high glucose DMEM medium containing 10% FBS and 1% Penicillin/Streptomycinin. The cells were typically allowed to recover overnight (about 18 to 24 hours) before RNA and/or protein assays were performed as described in Example 2. The p38α, p38β and G3PDH probes used were identical to those described in Example 2.
TABLE 7 Nucleotide Sequences of Rat p38α Phosphorothioate Oligonucleotides TARGET GENE NUCLEOTIDE GENE ISIS NUCLEOTIDE SEQUENCE CO- TARGET NO. (5′ −> 3′) SEQ ID NO ORDINATES REGION 21844 47 0001-0020 AUG 21845 48 0361-0380 coding 21846 49 0781-0800 coding 21871 50 0941-0960 coding 21872 51 1041-1060 coding 21873 52 1081-1100 stop 21874 53 1101-1120 3′-UTR 21875 54 1321-1340 3′-UTR 21876 55 1561-1580 3′-UTR 21877 56 1861-1880 3′-UTR 21878 57 2081-2100 3′-UTR 21879 58 2221-2240 3′-UTR 21880 59 2701-2720 3′-UTR 21881 60 3001-3020 3′-UTR
[0117] Rat p38α antisense oligonucleotides were screened in bEND.3 cells for inhibition of p38α and p38β mRNA expression. The concentration of oligonucleotide used was 100 nM. Results are shown in Table 8. Oligonucleotides 21844 (SEQ ID NO. 47), 21845 (SEQ ID NO. 48), 21872 (SEQ ID NO. 51), 21873 (SEQ ID NO. 52), 21875 (SEQ ID NO. 54), and 21876 (SEQ ID NO. 55) showed greater than approximately 70% inhibition of p38α mRNA with minimal effects on p38β mRNA levels. Oligonucleotide 21871 (SEQ ID NO. 50) inhibited both p38α and p38β levels greater than 70%.
TABLE 8 Inhibition of Mouse p38 mRNA expression in bEND.3 Cells by Chimeric (deoxy gapped) Mixed Backbone p38α Antisense Oligonucleotides SEQ GENE % p38α ID TARGET mRNA % p38β mRNA ISIS No: NO: REGION INHIBITION INHIBITION control — — 0% 0% 21844 47 AUG 81% 20% 21845 48 coding 75% 25% 21871 50 coding 90% 71% 21872 51 coding 87% 23% 21873 52 stop 90% 3% 21874 53 3′-UTR 38% 21% 21875 54 3′-UTR 77% — 21876 55 3′-UTR 69% — 21877 56 3′-UTR 55% 13% 21878 57 3′-UTR 25% 10% 21879 58 3′-UTR — — 21881 60 3′-UTR — —
[0118] Several of the most active oligonucleotides were selected for dose response studies. bEND.3 cells were cultured and treated as described above, except that the concentration of oligonucleotide was varied as noted in Table 9. Results are shown in Table 9.
TABLE 9 Dose Response of bEND.3 cells to rat p38β Chimeric (deoxy gapped) Phosphorothioate Oligonucleotides % p38α % p38β SEQ ID ASO Gene mRNA mRNA ISIS # NO: Target Dose Inhibition Inhibition control — — — 100% 0% 21844 47 AUG 1 nM — — ″ ″ ″ 5 nM — — ″ ″ ″ 25 nM 36% 8% ″ ″ ″ 100 nM 80% 5% 21871 50 coding 1 nM 1% — ″ ″ ″ 5 nM 23% 4% ″ ″ ″ 25 nM 34% 24% ″ ″ ″ 100 nM 89% 56% 21872 51 stop 1 nM — — ″ ″ ″ 5 nM — — ″ ″ ″ 25 nM 35% — ″ ″ ″ 100 nM 76% 1% 21873 52 stop 1 nM — 53% ″ ″ ″ 5 nM — 31% ″ ″ ″ 25 nM 54% 28% ″ ″ ″ 100 nM 92% 25% 21875 54 3′-UTR 1 nM — 11% ″ ″ ″ 5 nM — 16% ″ ″ ″ 25 nM 33% 2% ″ ″ ″ 100 nM 72% 4%
[0119] Antisense oligonucleotides were designed to target mouse p38β. Target sequence data are from a mouse EST sequence; Genbank accession number AI119044, provided herein as SEQ ID NO: 61. Oligonucleotides was synthesized as chimeric oligonucleotides (“gapmers”) 20 nucleotides in length, composed of a central “gap” region consisting of ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by five-nucleotide “wings.” The wings are composed of 2′-methoxyethyl (2′-MOE) nucleotides. The internucleoside (backbone) linkages in the wings are phosphodiester (P═O). Internucleoside linkages in the central gap are phosphorothioate (P═S). All 2′-MOE cytosines and 2′-OH cytosines were 5-methyl-cytosines. These oligonucleotide sequences are shown in Table 10.
TABLE 10 Nucleotide Sequences of Mouse p38β Chimeric (deoxy gapped) Phosphorothioate Oligonucleotides TARGET GENE NUCLEOTIDE NUCLEOTIDE SEQUENCE CO- ISIS NO. (5′ −> 3′) SEQ ID NO: ORDINATES 100800 63 0051-0070 100801 64 0119-0138 100802 65 0131-0150 100803 66 0171-0190 100804 67 0211-0230 100805 68 0261-0280 100806 69 0341-0360 100807 70 0441-0460 100808 71 0521-0540 100809 72 0551-0570 100810 73 0617-0636 100811 74 0644-0663 100812 75 0686-0705 100813 76 0711-0730 101123 77 21871 mismatch
[0120] Mouse p38β antisense sequences were screened in bEND.3 cells as described in Example 4. Results are shown in Table 11.
[0121] Oligonucleotides 100800 (SEQ ID NO. 63), 100801 (SEQ ID NO. 64), 100803 (SEQ ID NO. 66), 100804 (SEQ ID NO. 67), 100805 (SEQ ID NO. 68), 100807 (SEQ ID NO. 70), 100808 (SEQ ID NO. 71), 100809 (SEQ ID NO. 72), 100810 (SEQ ID NO. 73), 100811 (SEQ ID NO.74), and 100813 (SEQ ID NO. 76) resulted in at least 50% inhibition of p38β mRNA expression. Oligonucleotides 100801 (SEQ ID NO.64), 100803 (SEQ ID NO. 66), 100804 (SEQ ID NO. 67), 100805 (SEQ ID NO. 68), 100809 (SEQ ID NO. 72), and 100810 (SEQ ID NO. 73) resulted in at least 70% inhibition and are preferred. Oligonucleotides 100801 (SEQ ID NO. 64), 100805 (SEQ ID NO. 68), and 100811 (SEQ ID NO. 74) resulted in significant inhibition of p38α mRNA expression in addition to their effects on p38β.
TABLE 11 Inhibition of Mouse p38 mRNA expression in bEND.3 Cells by Chimeric (deoxy gapped) Mixed Backbone p38β Antisense Oligonucleotides SEQ ID % p38β mRNA % p38α mRNA ISIS No: NO: INHIBITION INHIBITION control — 0% 0% 100800 63 51% — 100801 64 74% 31% 100802 65 35% — 100803 66 74% 18% 100804 67 85% 18% 100805 68 78% 58% 100806 69 22% 3% 100807 70 64% — 100808 71 53% 13% 100809 72 84% 14% 100810 73 72% 1% 100811 74 60% 43% 100812 75 36% 17% 100813 76 54% —
[0122] p38 MAPK antisense oligonucleotides were tested for their ability to reduce IL-6 secretion. bEND.3 cells were cultured and treated as described in Example 4 except that 48 hours after oligonucleotide treatment, cells were stimulated for 6 hours with 1 ng/mL recombinant mouse IL-1 (R&D Systems, Minneapolis, Minn.). IL-6 was measured in the medium using an IL-6 ELISA kit (Endogen Inc., Woburn, Mass.).
[0123] Results are shown in Table 12. oligonucleotides targeting a specific p38 MAPK isoform were effective in reducing IL-6 secretion greater than approximately 50%.
TABLE 12 Effect of p38 Antisense Oligonucleotides on IL-6 secretion SEQ ID GENE DOSE % IL-6 ISIS No: NO: TARGET (μM) INHIBITION control — — 0% 21873 52 p38α 100 49% 100804 67 p38β 100 57% 21871 50 p38α 200 23% and p38β
[0124] Rat p38α antisense oligonucleotides were screened in Rat A-10 cells. A-10 cells (American Type Culture Collection, Manassas, Va.) were grown in high-glucose DMEM (Life Technologies, Gaithersburg, Md.) medium containing 10% fetal calf serum (FCS). Cells were treated with oligonucleotide as described in Example 2. Oligonucleotide concentration was 200 nM. mRNA was isolated 24 hours after time zero and quantitated by Northern blot as described in Example 2.
[0125] Results are shown in Table 13. Oligonucleotides 21845 (SEQ ID NO. 48), 21846 (SEQ ID NO. 49), 21871 (SEQ ID NO. 50), 21872 (SEQ ID NO. 51), 21873 (SEQ ID NO. 52), 21874 (SEQ ID NO. 53), 21875 (SEQ ID NO. 54), 21877 (SEQ ID NO. 56), 21878 (SEQ ID NO. 57), 21879 (SEQ ID NO. 58), and 21881 (SEQ ID NO. 60) inhibited p38β mRNA expression by 65% or greater in this assay. Oligonucleotides 21846 (SEQ ID NO. 49), 21871 (SEQ ID NO. 50), 21872 (SEQ ID NO. 51), 21877 (SEQ ID NO. 56), and 21879 (SEQ ID NO. 58) inhibited p38α mRNA expression by greater than 85% and are preferred.
TABLE 13 Inhibition of Rat p38α mRNA expression in A-10 Cells by Chimeric (deoxy gapped) Mixed Backbone p38α Antisense Oligonucleotides SEQ % p38α % p38α ID GENE mRNA mRNA ISIS No: NO: TARGET REGION EXPRESSION INHIBITION control — — 100% 0% 21844 47 AUG 75% 25% 21845 48 coding 25% 75% 21846 49 coding 8% 92% 21871 50 coding 12% 88% 21872 51 coding 13% 87% 21873 52 stop 19% 81% 21874 53 3′-UTR 22% 78% 21875 54 3′-UTR 26% 74% 21876 55 3′-UTR 61% 39% 21877 56 3′-UTR 12% 88% 21878 57 3′-UTR 35% 65% 21879 58 3′-UTR 11% 89% 21881 60 3′-UTR 31% 69%
[0126] The most active oligonucleotide in this screen (SEQ ID NO. 49) was used in rat cardiac myocytes prepared from neonatal rats (Zechner, D., et. al.,
[0127] Results are shown in Table 14. Oligonucleotide 21846 (SEQ ID NO. 49) was able to reduce p38α expression in rat cardiac myocytes by nearly 60%. The JNK-2 antisense oligonucleotide had little effect on p38α expression.
TABLE 14 Inhibition of Rat p38α mRNA expression in Rat Cardiac Myocytes by A Chimeric (deoxy gapped) Mixed Backbone p38α Antisense Oligonucleotide SEQ GENE % p38α ID TARGET mRNA % p38α mRNA ISIS No: NO: REGION EXPRESSION INHIBITION control — — 100% 0% 21846 49 coding 41% 59%
[0128] Additional antisense oligonucleotides were designed to target human p38β based on active rat sequences. Target sequence data are from the p38 MAPK cDNA sequence; Genbank accession number L35253, provided herein as SEQ ID NO: 1. Oligonucleotides was synthesized as chimeric oligonucleotides (“gapmers”) 20 nucleotides in length, composed of a central “gap” region consisting of ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by five-nucleotide “wings.” The wings are composed of 2′-methoxyethyl (2′-MOE) nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P═S) throughout the oligonucleotide. All 2′-MOE cytosines and 2′-OH cytosines were 5-methyl-cytosines. These oligonucleotide sequences are shown in Table 15.
TABLE 15 Additional Nucleotide Sequences of Human p38α Chi- meric (deoxy gapped) Phosphorothioate Oligonucleo- tides TARGET GENE SEQ NUCLEOTIDE GENE ISIS NUCLEOTIDE SEQUENCE ID CO- TARGET NO. (5′ −> 3′) NO: ORDINATES REGION 100860 78 0284-0303 Start 100861 79 0344-0363 coding 100862 80 0439-0458 coding 100863 81 0464-0483 coding 100864 82 0538-0557 coding 100865 83 0644-0663 coding 100866 84 0704-0723 coding 100867 85 0764-0783 coding 100868 86 0824-0843 coding 100869 87 0907-0926 coding 100870 88 0960-0979 coding 100871 89 1064-1083 coding 100872 90 1164-1183 coding 100873 91 1224-1243 coding 100874 92 1258-1277 coding 100875 93 1324-1343 coding 100876 94 1364-1383 stop 100877 95 1452-1471 3′-UTR
[0129] For an initial screen of human p38α antisense oligonucleotides, T-24 cells, a human transitional cell bladder carcinoma cell line, were obtained from the American Type Culture Collection (ATCC) (Manassas, Va.). T-24 cells were routinely cultured in complete McCoy's 5A basal media (Gibco/Life Technologies, Gaithersburg, Md.) supplemented with 10% fetal calf serum (Gibco/Life Technologies, Gaithersburg, Md.), penicillin 100 units per mL, and streptomycin 100 micrograms per mL (Gibco/Life Technologies, Gaithersburg, Md.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence. Cells were seeded into 96-well plates (Falcon-Primaria #3872) at a density of 7000 cells/well for use in RT-PCR analysis. A control oligonucleotide ISIS 118965 (TTATCCTAGCTTAGACCTAT, herein incorporated as SEQ ID NO: 96) was synthesized as chimeric oligonucleotide (“gapmer”) 20 nucleotides in length, composed of a central “gap” region consisting of ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by five-nucleotide “wings.” The wings are composed of 2′-methoxyethyl (2′-MOE) nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P═S) throughout the oligonucleotide. All 2′-MOE cytosines and 2′-OH cytosines were 5-methyl-cytosines.
[0130] For Northern blotting or other analysis, cells may be seeded onto 100 mm or other standard tissue culture plates and treated similarly, using appropriate volumes of medium and oligonucleotide. mRNA was measured by Northern blot. Results are shown in Table 16. Oligonucleotides 100861 (SEQ ID NO. 79), 100862 (SEQ ID NO. 80), 100863 (SEQ ID NO. 81), 100866 (SEQ ID NO. 84), 100867 (SEQ ID NO. 85), 100868 (SEQ ID NO. 86), 100870 (SEQ ID NO. 88), 100871 (SEQ ID NO. 89), 100872 (SEQ ID NO. 90), 100873 (SEQ ID NO. 91), and 100874 (SEQ ID (NO. 92), 100875 (SEQ ID NO. 93) and 100877 (SEQ ID NO. 95) gave than approximately 40% inhibition and are preferred.
TABLE 16 Inhibition of Human p38α mRNA expression in T-24 Cells by Chimeric (deoxy gapped) Phosphorothioate Oligonucleotides SEQ % P38α % P38β ID GENE TARGET mRNA mRNA ISIS No: NO: REGION EXPRESSION EXPRESSION 100860 78 0284-0303 73% 71% 100861 79 0344-0363 60% 47% 100862 80 0439-0458 56% 45% 100863 81 0464-0483 49% 67% 100864 82 0538-0557 66% 70% 100865 83 0644-0663 64% 63% 100866 84 0704-0723 55% 65% 100867 85 0764-0783 58% 33% 100868 86 0824-0843 47% 60% 100869 87 0907-0926 61% 100% 100870 88 0960-0979 51% No data 100871 89 1064-1083 57% 96% 100872 90 1164-1183 37% 77% 100873 91 1224-1243 34% 70% 100874 92 1258-1277 42% 76% 100875 93 1324-1343 39% 90% 100876 94 1364-1383 77% 93% 100877 95 1452-1471 47% 95%
[0131] Oligonucleotides 100872 (SEQ ID NO. 90), 100873 (SEQ ID NO. 91), 100874 (SEQ ID NO. 92), and 100875 (SEQ ID NO. 93) were chosen for dose response studies.
[0132] Results are shown in Table 17. The effect of these oligonucleotides on human p38β was also determined.
TABLE 17 Dose Response of p38α in T-24 cells to human p38α Chimeric (deoxy gapped) Phosphorothioate Oligonucleotides % p38α % p38β SEQ ID ASO Gene mRNA mRNA ISIS # NO: Target Dose Expression Inhibition Control 96 — — 94% 80% 118965 100872 90 coding 50 nM 45% 108% ″ ″ ″ 100 nM 18% 91% ″ ″ ″ 200 nM 17% 92% 100873 91 coding 50 nM 19% 90% ″ ″ ″ 100 nM 12% 78% ″ ″ ″ 200 nM 8% 44% 100874 92 coding 50 nM 47% 107% ″ ″ ″ 100 nM 27% 101% ″ ″ ″ 200 nM 13% 51% 100875 93 coding 50 nM 30% 105% ″ ″ ″ 100 nM 13% 92% ″ ″ ″ 200 nM 8% 69%
[0133] Additional antisense oligonucleotides were designed to target human p38β based on active rat sequences. Target sequence data are from the p38 MAPK cDNA sequence; Genbank accession number U53442, provided herein as SEQ ID NO: 23.
[0134] Oligonucleotides was synthesized as chimeric oligonucleotides (“gapmers”) 20 nucleotides in length, composed of a central “gap” region consisting of ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by five-nucleotide “wings.” The wings are composed of 2′-methoxyethyl (2′-MOE) nucleotides. The internucleoside (backbone) linkages in the wings are phosphodiester (P═O). Internucleoside linkages in the central gap are phosphorothioate (P═S). All 2′-MOE cytosines and 2′-OH cytosines were 5-methyl-cytosines. These oligonucleotide sequences are shown in Table 18. A control oligonucleotide ISIS 118966 (GTTCGATCGGCTCGTGTCGA), herein incorporated as SEQ ID NO: 107) was synthesized as chimeric oligonucleotide (“gapmer”) 20 nucleotides in length, composed of a central “gap” region consisting of ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by five-nucleotide “wings.” The wings are composed of 2′-methoxyethyl (2′-MOE) nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P═S) in the gap and phosphodiester in the wings. All 2′-MOE cytosines and 2′-OH cytosines were 5-methyl-cytosines.
TABLE 18 Additional Nucleotide Sequences of Human p38β Chi- meric (deoxy gapped) Mixed-Backbone Phosphorothio- ate Oligonucleotides TARGET GENE SEQ NUCLEOTIDE GENE ISIS NUCLEOTIDE SEQUENCE ID CO- TARGET NO. (5′ −> 3′) NO. ORDINATES REGION 107869 97 117-136 coding 107870 98 156-175 coding 107871 99 207-226 coding 107872 100 287-306 coding 107873 101 414-433 coding 107874 102 491-510 coding 107875 103 587-606 coding 107876 104 614-633 coding 107877 105 649-668 coding 107878 106 682-701 coding
[0135] For an initial screen of human p38β antisense oligonucleotides, T-24 cells, a human transitional cell bladder carcinoma cell line, were obtained from the American Type Culture Collection (ATCC) (Manassas, Va.). T-24 cells were routinely cultured in complete McCoy's 5A basal media (Gibco/Life Technologies, Gaithersburg, Md.) supplemented with 10% fetal calf serum (Gibco/Life Technologies, Gaithersburg, Md.), penicillin 100 units per mL, and streptomycin 100 micrograms per mL (Gibco/Life Technologies, Gaithersburg, Md.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence. Cells were seeded into 96-well plates (Falcon-Primaria #3872) at a density of 7000 cells/well for use in RT-PCR analysis. A control oligonucleotide ISIS 118966 (TTATCCTAGCTTAGACCTAT, herein incorporated as SEQ ID NO: 106) was synthesized as chimeric oligonucleotide (“gapmer”) 20 nucleotides in length, composed of a central “gap” region consisting of ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by five-nucleotide “wings.” The wings are composed of 2′-methoxyethyl (2′-MOE) nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P═S) in the gap and phosphodiester in the wings. All 2′-MOE cytosines and 2′-OH cytosines were 5-methyl-cytosines.
[0136] For Northern blotting or other analysis, cells may be seeded onto 100 mm or other standard tissue culture plates and treated similarly, using appropriate volumes of medium and oligonucleotide. mRNA was measured by Northern blot. Results are shown in Table 19. For comparison, ISIS 17893 and ISIS 17899, both targeting human p38β (SEQ ID NO: 27) and ISIS 100802 targeting mouse p38β (SEQ ID NO: 65) described in Examples 3 and 5 above, respectively, were included in the screen.
[0137] Oligonucleotides 107869 (SEQ ID NO. 97), 107871 (SEQ ID NO. 99), 107872 (SEQ ID NO. 100), 107873 (SEQ ID NO. 101), 107878 (SEQ ID NO.106), 17893 (SEQ ID NO. 27), 17899 (SEQ ID NO. 33) and 100802 (SEQ ID NO.65, targeted to mouse p38β) gave greater than approximately 40% inhibition and are preferred.
TABLE 19 Inhibition of Human p38β mRNA expression in T-24 Cells by Chimeric (deoxy gapped) Mixed-Backbone Phosphorothioate Oligonucleotides SEQ ID GENE TARGET % p38β mRNA % p38α mRNA ISIS No: NO: REGION EXPRESSION EXPRESSION 107869 97 Coding 60% 93% 107870 98 Coding 74% 97% 107871 99 Coding 60% 111% 107872 100 Coding 57% 123% 107873 101 Coding 58% 120% 107874 102 Coding 61% 100% 107875 103 Coding 92% 112% 107876 104 Coding 127% 137% 107877 105 Coding No data No data 107878 106 Coding 54% 112% 17893 27 Coding 31% 61% 17899 33 Coding 56% 117% 100802 65 Coding 47% 78%
[0138] Oligonucleotides 107871, 107872, 107873, 107874, 107875, 107877, 107878, 17893 and 17899 were chosen for dose response studies.
[0139] Results are shown in Table 20. The effect of these oligonucleotides on human p38α was also determined.
TABLE 20 Dose Response of p38β in T-24 cells to human p38β Chimeric (deoxy gapped) Mixed-backbone Phosphorothioate Oligonucleotides % p38β % p38α SEQ ID ASO Gene mRNA mRNA ISIS # NO: Target Dose Expression Inhibition Control 107 — — 100% 100% 118966 107871 99 coding 50 nM 41% 105% ″ ″ ″ 100 nM 42% 132% ″ ″ ″ 200 nM 10% 123% 107872 100 coding 50 nM 71% 124% ″ ″ ″ 100 nM 13% 84% ″ ″ ″ 200 nM 22% 102% 107873 101 coding 50 nM 69% 132% ″ ″ ″ 100 nM 41% 119% ″ ″ ″ 200 nM 23% 131% 107874 102 coding 50 nM 75% 109% ″ ″ ″ 100 nM 34% 99% ″ ″ ″ 200 nM 23% 87% 107875 103 coding 50 nM 82% 93% ″ ″ ″ 100 nM 38% 101% ″ ″ ″ 200 nM 40% 91% 107877 105 coding 50 nM 50% 127% ″ ″ ″ 100 nM 34% 125% ″ ″ ″ 200 nM 22% 106% 107878 106 coding 50 nM 70% 110% ″ ″ ″ 100 nM 43% 109% ″ ″ ″ 200 nM 27% 116% 17893 27 coding 50 nM 28% 88% ″ ″ ″ 100 nM 27% 115% ″ ″ ″ 200 nM 16% 108% 17899 33 coding 50 nM 89% 87% ″ ″ ″ 100 nM 36% 104% ″ ″ ″ 200 nM 15% 80%
[0140] These data show that the oligonucleotides designed to target human p38β, do so in a target-specific and dose-dependent manner.