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    • 专利摘要:
    Disclosed are antisense oligonucleotides useful for treating disease states characterized by p53 induction, such as hypoxic conditions caused by ischemic attack such as proliferative cell disorders, e.g., cancer, or stroke. Antisense agents are preferably selected from the group consisting of a morpholino oligonucleotide, a peptide nucleic acid, a 2'0-allyl or 2'-O-alkyl modified oligonucleotide, or a "stereostructure" comprising an N3 '→ P5' Type oligonucleotides.
    公开号:KR20010089904A
    申请号:KR1020017005184
    申请日:1999-10-22
    公开日:2001-10-13
    发明作者:이버슨패트릭엘.
    申请人:추후보정；에이브이아이 바이오파마 인코포레이티드；
    IPC主号:
    专利说明:

    p53 morpholino-basal antisense {p53 MORPHOLINO-BASED ANTISENSE}
    [6] The importance of p53 in cell proliferation is evident from the observation that more than half of all human cancers exhibit p53 mutations (Levine, 1997). The induction of p53 expression results in a DNA alkylating agent, nitric oxide (Messmer, 1994), ionization and ultraviolet irradiation (Lu and Lane, 1993), restriction enzymes PvuII (Lu and Lane, 1993) (Nelson and Kastan, 1994). (Raafat, 1997), a model of epilepsy (Xiang, 1996), and hypoxia (Graeber, 1994) have also been shown to induce p53. As revealed in the present specification and in previous studies (Rininger, 1997), p53 protein levels are also highly upregulated during liver regeneration following partial hepatectomy, suggesting that damage to endogenous causes may also result in the induction of p53 in vivo Show.
    [7] The role for p53 has been required for cell cycle checkpoint activity, apoptosis, differentiation and DNA repair (Magnelli, 1997). Cell cycle checkpoints have two important functions. One is to ensure that events essential to the cell cycle are completed before a subsequent event; The other is to provide more time for the repair of damaged DNA before DNA replication and mitosis take place. (Hartwell, 1994). Checkpoint activity of p53 is predominant at the cell level entering the S-phase (DNA synthesis) of the cell cycle (Kastan, 1992). G 2 -M checkpoint activity for p53 was also reported due to p53 role as an upstream regulator of p21 waf-1 . In the absence of any protein, DNA-damaged cells stop at the G 2 -like state, but then proceed to the additional S phase without interfering with normal mitosis (M phase) to induce a nuclear anomaly, (Waldman, 1996).
    [8] Summary of the Invention
    [9] In one aspect, the invention relates to methods and compositions for treating disease states characterized by p53 induction in a subject mammal. This method is based on the base sequence identified as 5'-TCA GTC TGA GTC AGG CCC-3 'in a suitable pharmaceutical carrier, or a 5'-CCC TGC TCC CCC CTG GCT CC-3' Comprising administering to the subject a pharmaceutically effective amount of an antisense agent having the sequence SEQ ID NO: 2 wherein the antisense agent is selected from the group consisting of morpholino oligonucleotides, peptide nucleic acids, 2'-O-allyl or 2'-O-alkyl A modified oligonucleotide, an N3 'to P5' phosphoramidate oligonucleotide, or a C-5-propynopyrimidine-modified oligonucleotide. In a preferred embodiment, the antisense agent is a morpholino oligonucleotide having a morpholino subunit linked by a phosphorodiamidate backbone linkage. In another preferred embodiment, the antisense agent is a C-5-propynopyrimidine-modified oligonucleotide.
    [10] The present invention also includes compositions for use in the treatment of such disease states. Such compositions comprise the antisense agents described above in a suitable pharmaceutical carrier.
    [11] In a preferred use of the method of the present invention, the subject is a human subject. The disease state may be caused by an ischemia / resuspension injury such as ischemia or stroke or organ transplantation infertility. Alternatively, the disease state can be cancer, and preferred embodiments in this case further comprise the administration of a medicament effective to increase the radical oxygen species at the cellular level. Such agents include radiation sensitizers, ionizing radiation, high-pressure oxygen environments, and chemotherapeutic agents such as anthracycline or anthraquinone that increase radical oxygen species at the cellular level.
    [12] For cancer treatment, the method also from the G 2 period of the cell cycle include administration of a drug effective to interfere with progression of the M phase. Such agents include, but are not limited to, phosphokinase C (PKC) inhibitors, bis (chloroethyl) nitroso urea (BCNU), pentoxifylline, silymarin, staurosporine, phenylaustine, paclitaxel, retinoic acid, 2,5-dihydrocinnamate, herboside diene, 9-nitrocamptothecin, mitotoxin, apigenin, nocodazole, and callide.
    [13] In another aspect, the invention provides a 5'-TCA GTC TGA GTC AGG CCC-3 'and a 5'-CCC TGC TCC CCC CTG GCT CC-3' identified by SEQ ID NO: 2, wherein the oligonucleotide is selected from the group consisting of morpholino oligonucleotides, peptide nucleic acids, 2'-O-allyl or 2'-O-alkyl modified oligonucleotides, and oligonucleotides having a base sequence selected from the group consisting of N3 '- > P5 ' phosphoramidate oligonucleotides. In a preferred embodiment, the oligonucleotide is a morpholino oligonucleotide, preferably comprising a morpholino subunit linked by a phosphorodiamidate backbone linkage. The present invention also provides a 5'-TCA GTC TGA GTC AGG consisting of SEQ ID NO: 1 identified as AGG CCC-3 'and SEQ ID NO: 2 identified as 5'-CCC TGC TCC CCC CTG GCT CC-3' Wherein the oligonucleotide is a C-5-propynopyrimidine-modified oligonucleotide.
    [14] The objects and features of the present invention will become more apparent when the following detailed description of the present invention is read in conjunction with the accompanying drawings.
    [15] Brief Description of Drawings
    [16] Figure 1 shows the structure of a phosphorodiamidate-linked morpholino oligonucleotide analog; And
    [17] Figure 2 shows a partial hepatectomy and an antisense oligonucleotide with SEQ ID Nos. 1 and 2, namely phosphorothioate (S-ODN, 1 mg / 200 g); C-5-P modified phosphorothioate (C-5-P, 0.1 mg / 200 g); (In gm) between remnant regeneration in 24 hour-old rats following administration of a neutral skeleton (phosphodiamidate) morpholino oligonucleotide (3.4 mg / 200 g). Error bars represent a statistical difference of p < 0.05 for each individual group versus a saline-treated control.
    [1] The invention relates to antisense agents and methods for the treatment of disease states characterized by the induction of p53. Such conditions include proliferative cell disorders such as cancer, restenosis, and psoriasis, and hypoxic conditions caused by ischemic attacks such as stroke.
    [2] references
    [3]
    [4]
    [5]
    [18] Justice
    [19] &Quot; Antisense oligonucleotide " or " antisense agent " refers to a molecule that contains a purine and pyrimidine heterocyclic base sequence supported by a backbone and is effective for hydrogen-bonding with a corresponding, contiguous base in a target nucleic acid sequence. The backbone comprises a subunit backbone that supports the purine and pyrimidine heterocyclic bases at positions permitting such hydrogen bonding. These backbone moieties are ring moieties of 5 to 7 atoms in length, linked together by phosphorus containing linkages of 1 to 3 atoms in length.
    [20] (I) the structure is such that the morpholino nitrogen of one subunit is linked together by a phosphorus-containing linkage of 1 to 3 atoms long connected to the 5 'uncharged carbon of the adjacent subunit, (ii) the morpholino oligonucleotide ) B is composed of a morpholino subunit structure of the type shown in Figure 1, which is a purine or pyrimidine base pair moiety effective to bind with a base of a polynucleotide in a base-specific hydrogen bond. Figure 1 illustrates these two subunits linked by phosphorodiamidate linkages.
    [21] The "N3'-P5'phosphoramidate" oligonucleotide is a 3'-oxygen of 2'-deoxyribose, for example as described in Gryaznov et al . And 3'-amine as described in Chen et al .
    [22] A "2'-O-allyl (or alkyl) modified oligonucleotide" is an oligonucleotide in which the 2'hydroxyl is converted to an allyl or alkyl ether. Alkyl ethers are typically methyl ethers.
    [23] &Quot; C-5-Propynylpyrimidine-modified oligonucleotide " is an oligonucleotide in which the C-5 methyl group of the thymidine base and / or the C-5 hydrogen of the cytidine base is replaced by a propyne group.
    [24] In " peptide nucleic acid ", the deoxyribose phosphate unit of the oligonucleotide backbone is replaced by a polyamide linkage. For example, Nielsen et al . And Hanvey et al . , The skeleton occupying an appropriate space is obtained by the use of a 2-aminoethylglycine unit having a nucleotide base attached to each 2-amino group by a methylene carbonyl group.
    [25] An " RNase-inactive " oligonucleotide or oligonucleotide analogue, unlike an RNase-active oligonucleotide, such as a phosphorothioate, acts by an RNase-independent mechanism. They are believed to function by sterically blocking target RNA formation, nuclear cytoplasmic transport or translation, and are therefore also referred to as " stere blockers ". This class includes, for example, methylphosphonates, morpholino oligonucleotides, peptide nucleic acid (PNA's), 2'-O-allyl or 2'-O-alkyl modified oligonucleotides as mentioned herein, → P5 'phosphoramidate.
    [26] " A condition characterized by the induction of p53 " is a disease state in which the level or expression of p53 is relatively increased in infected cells relative to the non-diseased state and / or the therapeutic effect of inhibiting p53 expression, . Examples are ischemia / ischemic injury, which is the result of cancer, myocardial infarction, or interstitial seizure, and the resulting hypoxic condition, which is the result of cancer, and organ transplantation.
    [27] Abbreviation:
    [28] ON = oligonucleotide
    [29] ODN = oligodeoxyribonucleotide
    [30] S-ODN = phosphorothioate oligonucleotide
    [31] C-5-P = C-5-propynylpyrimidine-modified oligonucleotide
    [32] II. Antisense oligonucleotides
    [33] A. Sequence
    [34] The antisense agents of the present invention comprise a nucleotide subunit linked by an internucleotide backbone linkage to provide a nucleotide base for hybridization with a target RNA sequence. The nucleotide sequence of these antisense agents is complementary (antisense) to the p53 mRNA region. The first base sequence corresponding to SEQ ID NO: 1 is complementary to a unique portion of rat p53 mRNA (residues 1182 to 1199; Genbank accession # X13058) and is referred to herein as p53T. This sequence is identical in rat, mouse, monkey, and human mRNA and is therefore ideally suited for in vivo testing in animal models.
    [35] The second sequence corresponding to SEQ ID NO: 2, named OL (1) p53, is an antisense to human p53 and thus is effective in inhibiting the expression of p53 in humans. (Iverson, U.S. Patent No. 5,641,754). Since this sequence has a rat sequence and four mismatches, it does not alter the expression of p53 in rats and is therefore a useful control in rat model studies.
    [36] B. Oligonucleotide Structure
    [37] A variety of oligonucleotide analogs known in the art provide advantages over " natural " polynucleotides in regions such as stability, particularly nuclease resistance, reduced non-specific binding, and bioavailability do. This structure can be modified in the framework, sugar moiety, or base itself. Such analogs include, for example, phosphorothioates (termed S-ODN), methylphosphonates, phosphotriesters, C-5-propynopyrimidine-modified oligonucleotides ), Morpholino oligonucleotides, peptide nucleic acid (PNA's), 2'-O-allyl or 2'-O-alkyl modified oligonucleotides, and N3 '→ P5' phosphoramidate.
    [38] Preparations of such antisense agents are well known in the art and are often easily performed in automated synthesizers. A general procedure for the synthesis of S-ODN, C-5-P, and non-hypomorpholino antisense oligonucleotides is shown in Example 1.
    [39] Two common mechanisms have been proposed to explain the inhibition of expression by antisense oligonucleotides (see, for example, Agrawal; Bonham; Boudvillain; and references cited therein). The first mechanism is that oligonucleotides and mRNA Lt; RTI ID = 0.0 > RNaseH < / RTI > to cause cleavage of mRNA. Oligonucleotides which belong to this class and which are proposed to belong also include phosphorothioates, phosphotriesters, and phosphodiester (i.e., unmodified "natural" oligonucleotides). Such compounds generally exhibit high activity, and phosphorothioates are the most widely used oligonucleotides for antisense use. However, such compounds tend to cause non-specific binding (Gee) with cellular proteins as well as undesirable side effects due to inadequate RNase cleavage (Giles) of non-target RNA variants.
    [40] A second class of oligonucleotide analogues referred to as " steric hindrance agents " or alternatively, " RNase inactive " or " RNase resistant " By acting as a three-dimensional block. This class includes methylphosphonates (Toulme) as well as morpholino oligonucleotides, peptide nucleic acid (PNA's), 2'-O-allyl or 2'-O-alkyl modified oligonucleotides (Bonham) 'Phosphoramidate (Gee). They are reported to be most effective when targeted to the AUG initiation codon, 5'-splice site, or 5'-untranslated region of mRNA. So far, when targeted at the downstream of the coding site, they were relatively low (Bonham), and did not show any activity more frequently. (See, for example, Toulme for methylphosphonate; Gambacorti for PNA's; Knudsen for double-stranded PNA's; and Gee for PNS's and N3 ' P5' phosphoramidates). It has been suggested that when directed to the coding site, the hybrid formed by ON's is not stable enough to avoid being displaced by the loosening action of the ribosome complex during translation. (Boudvillain, Gee) has linked or inserted ON into its target sequence as a covalent bond (Gee, Johansson) as an approach to solving the problem. Without this modification, however, the activity of stereoselective blocking of ON's has generally been limited to the above-mentioned initiation sites.
    [41] III. Induction of p53 in proliferating cells and inhibition by antisense p53
    [42] In the previously published paper (Arora, 1998), the antisense agents p53T and ON (1) p53 described above were administered to the immediate-onset rats after partial hepatectomy, as described in Example 2. [ Regenerating rat liver is a good in vivo model system for studying the mechanisms of growth regulation and cell proliferation in natural tissue environments. Cells that proliferate rapidly in regeneration are the most likely to become cancerous (Grisham, 1983) because they are available only for a minimum of time to restore DNA between cell cycles. As mentioned above, ON (1) p53 did not alter p53 expression in rats and was used as a control.
    [43] In the post-hepatectomy (PH) rats, a large amount of p53 was observed during the regeneration process. The result of antisense ON administration, i.e., the weight gain between regeneration; p53, PCNA, p21 and NADPH levels; DNA content of isolated cells; Microsomal protein content; Various microsomal enzyme assays; Oxidative stress as evidence of lipid peroxidation; And mitotic index were measured.
    [44] PCNA expression (Assy, 1998), which is an accurate marker of mitosis index and cell proliferation (Assy, 1998), and the 5-fold increase in G 1 cell population when treated with antisense p53T (SEQ ID NO: As evidenced by the reduction, the liver loses their G 1 -S cell cycle checkpoint activity. The p53T-treated PH rat liver also represents many polynuclear cells. As determined by enzyme assay, functional restoration between regeneration was also increased by p53 inhibition.
    [45] In summary, the data indicate that p53 expression is suppressed in the regenerating liver of rats after antisense p53T (SEQ ID NO: 1) treated hepatectomy, and that such inhibition is associated with increased mitosis, increased PCNA expression, 1 cells in the first cycle. The results are consistent with the G 1 -S cell cycle checkpoint activity of p53.
    [46] IV. Comparison results of S-ODN, C-5-P, and morpholino antisense drugs
    [47] A neutral-skeletal morpholino antisense drug with the nucleotide sequence given in SEQ ID NO: 1 and SEQ ID NO: 2 was prepared as described in Example 1. [ These morpholino oligomers exhibit high binding affinity to RNA targets, and the non-charged subunit is advantageous for absorption into cells and reduces non-specific binding interactions as compared to charge analogues such as phosphorothioates. The corresponding C-5-propyne cytosine modified (C-5-P) phosphorothioate was also prepared. As discussed above, the morpholino analogs are believed to act as " steric hindrance " agents. It is generally reported that C-5-P phosphorothioate belonging to phosphorothioate acts by the RNase-H dependent mechanism (Wagner).
    [48] With respect to wet weight gain between residual regeneration after 24 hours after PH as described in paragraph III and Example 2, the results of intraperitoneal administration of antisense agents were compared with those of unmodified phosphorothioate (S-ODNs) . The data shown in FIG. 2 show that the p53T sequence is an alternative analogue tested, and that for both OL (1) p53 sequences that remain inactive (as expected) with alternative analogs at the tested doses, Lt; / RTI >
    [49] Similar efficacy to the 1 mg / 200 gm dose of S-ODN p53T was achieved with 100 ug / 200 gm C-5-P modified S-ODN and 50 nM / 200 gm (approximately 3.7 mg / 200 g) morpholino anti- Nucleotide (with SEQ ID NO: 1). It is particularly valuable that the morpholino antisense oligomer is active when it is targeted to the exon 10 of the coding region of the gene rather than the 5'-untranslated region or the ANG initiation codon. As mentioned above, the " stereospermic " antisense agents generally did not show significant activity when targeted downstream from the AUG translation initiation time point. This coding sequence is particularly advantageous for antisense ON binding to form a double strand that is more stable to sterically block translation. In addition, as mentioned above, morpholino oligomers have proven to be particularly effective for RNA-binding molecules. Unless the invention is limited to a particular mechanism, it is expected that RNA helices are modified by the binding of the morpholino oligonucleotides, and this modification interferes with the binding of ribosomes.
    [50] Studies to support the present invention suggest that the propyne group of the C-5-P proline modified ODN may protrude into the major groove of the RNA double strand and may interfere with ribosome binding sterically. Thus, substitution with other sterically bulky groups, including groups with other electronic structures such as t-butyl of the propyne group, may exhibit similar inhibitory effects.
    [51] V. Therapy
    [52] The invention features p53 induction and provides a method of inhibiting p53 expression, particularly in cells where inhibition of p53 shows therapeutic benefit. The methods of the invention can be used to treat disease states characterized by p53 induction. The method of the present invention comprises administration of an oligonucleotide having a sequence corresponding to SEQ ID NO: 1 or SEQ ID NO: 2 to a subject having such a condition, or to a cell collected from such subject. Preferably, the subject is a human subject. (As noted above, SEQ ID NO: 2 is not effective for rat models.) Oligonucleotides are preferably selected from the group consisting of morpholino oligonucleotides, peptide nucleic acids, 2'-O-allyl or 2'- N3 ' to P5 ' phosphoramidate oligonucleotides, or C5-propynylpyrimidine-modified phosphorothioate oligonucleotides. Morpholino oligonucleotides are particularly preferred for both sequences. C-5-P modified morpholino oligonucleotides are also expected.
    [53] As demonstrated in the data presented here and in the data reported in Arora, 1998, anti (anti) -p53 ON inhibits the expression of p53 in rats following liver resection. Morpholino and C-5-P ON's were found to be more effective than equivalent amounts of unmodified phosphorothioate (S-ODN) as determined by weight gain between regeneration. Other than considering the fact that the morpholino oligomer is targeted to the coding region (exon 10) which is considerably downstream at the AUG initiation site which is a conventional target for RNAse-inactive (" steric hindrance ") antisense oligonucleotides.
    [54] A. Anti-proliferative therapy
    [55] As mentioned above, the induction of p53 expression is observed in cells exposed to various DNA-damaging agents. Damaged DNA is mostly restored by generally unplanned DNA synthesis. This restoration usually occurs within the G 1 sequence of the cell cycle preceding the S (synthesis) phase. However, if the altered DNA base remains untreated (cells immortalized to the S phase with the exclusion of the G 1 -S cell cycle checkpoint), the result is mutation due to incorrect replication of the damaged DNA nucleotide base template, And / or cell death due to its genomic cloning ability past the damaged DNA location of the cell.
    [56] Inhibition of p53 expression inhibits this cell cycle checkpoint and thus can be used to selectively kill DNA-damaged cells. Administration of antisense phosphorothioate oligonucleotides with the sequence OL (1) p53 (SEQ ID NO: 2) has been described in US Pat. No. 5,641,764 (Iverson) to produce apoptosis in vitro and in vivo in human liver cells . The effect was increased by exposing the cells to conditions with increased intracellular net reactive oxygen content. Since normal tissue has a greater oxygen scavenger activity than cancer cells, this treatment has little or no adverse effect on normal tissues. For example, most cells that contain one or more enzymes, such as SOD, catalase, or glutathione peroxidase, rapidly bind to oxygen species and inactivate excess reactive oxygen species. As described above, it is believed that inhibition of G 1 -S checkpoint by inhibition of p53 interferes with repair of damaged DNA, leading to mutagenesis and apoptosis.
    [57] Thus, in a preferred method, particularly useful in cancer cells of the present invention, the ON is administered in combination with a medicament capable of increasing cytoxicity-induced radical oxygen. The oligonucleotides and agents may be administered, in fact, either simultaneously or sequentially with any therapeutic agent administered first. However, the best results are obtained when oligonucleotides that allow therapeutic blood levels to be obtained are administered sufficiently beforehand.
    [58] Increased levels of radical oxygen include, for example, agents capable of capturing cytotoxic-induced radical oxygen such as radiation or anthracycline cytotoxic antibodies (eg, doxorubicin), BCNU, BSO (boutionin sulfoxamine), hydrogen peroxide , Or antisense oligonucleotide inhibitors of SOD (superoxide dismutase), catalase, GSH synthase, GSH reductase, or GSH peroxidase.
    [59] Agents capable of capturing cytotoxically induced radical oxygen include, for example, radiation sensitizers, chemotherapeutic agents that produce radical oxygen, or oligonucleotides capable of interacting with host cells causing hydroxy radical formation . (See, for example, Iversen, U.S. Ser. No. 07 / 735,067, filed July 25, 1991, entitled " Inhibition of Mutagenesis Induced by Cell Binding to Oligonucleotides, " Many cytotoxic agents that have a lethal effect that can be increased by one or more sensitizers on the target cells of a patient can be used in the methods of the invention. Radiation (radiation therapy is administered internally or by external means) and cytotoxic agents. Some examples of many such agents include anthracycline chemotherapeutic agents such as Nitrogen mustard, daunorubicin and doxorubicin, such as L-phenylalanine Nitrogen mustard (Melparan), and cis-diaminodichloro platinum Latin).
    [60] Internally delivered radiation includes therapeutically useful radioisotopes injected into a patient. These radioisotopes are radioisotope metals186Re,188Re,64Cu ,67Cu,109Pd,212Bi,203Pb,212Pb,211At,97Ru,105Rh,198Au,199Ag,131I, but are not limited thereto. Such a radioisotope, when administered to a patient, will generally bind to a carrier molecule (e.g., in the form of a chelate-antibody conjugate). Examples of internally delivered radiotherapeutic agents are described, for example, in EP Publication No. < RTI ID = 0.0 > 188,256. ≪ / RTI > Radiation administered by external means is external beam radiation, such as cobalt therapy.
    [61] The choice of sensitiser depends on such factors as the particular type of tumor to be treated and the cytotoxic agent to be administered. Preferably, the compound is selective for any target location in the body, such as a cancer cell. For example, US Patent No. 4,628,047 reports the use of diltiazem to increase the sensitivity of various forms of cancer cells to cytotoxic agents such as doxorubicin. The expected differences in sensitivity of different types of cancer cells to treatment with these agents, as well as other combinations of cytotoxic agents and sensitizers, are discussed in important advances in oncology . (DeVita et al ., Editiors, JB Lippincott Co., Philadelphia (1986), pp. 145-157)
    [62] A preferred sensitizer is a synthetic amino acid, BSO (butionon sulfoxamine, Chemical Dynamics Corporation, South Plainfield, NJ), which inhibits the gamma -glutamylcysteine synthase, resulting in a noticeable decrease in glutathione (GSH) . Thus, it is believed that BSO acts as a sensitizer for drugs with improved cytotoxic effects due to a decrease in glutathione levels in the cells. The S-isomer of BSO which completely removes glutathione in the cell is particularly preferred. (See U.S. Patent Nos. 5,171,885 and 5,245,077)
    [63] Additional combinations of sensitizers and cytotoxic agents may be identified by methods such as in vitro assays using cultured cells corresponding to the desired target cells (e.g., specific cancer cell lines). For example, Russo et al. Discloses an assay for determining whether BSO is effective in lowering the level of clathathione synthetase in certain types of cell lines.
    [64] In the above discussion of radical oxygen producing species, the cytotoxic agent and sensitizer are directed to a preferred target cell, i. E., A cancer cell characterized by p53 expression. Such cancers include bladder cancer, brain cancer, breast cancer, cervical cancer, colon cancer, esophageal cancer, laryngeal cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer and thyroid cancer.
    [65] In another preferred method, the administration of antisense ON involves the administration of a medicament that is effective in inhibiting the progression of the cell cycle from the G 2 phase to the M (mitotic) phase. Such agents include, for example, pentoxifylline (Russell, 1996), silymarin (Ahmad, 1998), staurosporine (Swe, 1997), phenylaustine (Kanoh, 1997), paclitaxel (Koch, 1996), herbicides (Horiguchi, 1996), 9-nitrocamptothecin (see, for example, Khooustov, 1995), mitotoxins (VanDolah, 1996), apigenin (Sato, 1994), nocodazole, and callemide (Zhang, 1998). A recent report (Cross, 1995) suggested that p53 is a tool of spindle checkpointing to ensure that a combination of spindle devices is completed during mitosis (M) of the cell cycle. In studies to support the present invention, the p21- deficient mice G 2 as listed in the above-showed a high degree of completion diploid in the infected cells when treated with M inhibitor. Inhibitors prevent cells from undergoing normal progression through mitosis and deficiency of mitosis (and G 1 -S group) checkpoints, and cells that have undergone a continuous synthesis (S) eventually produce isozyme Is assumed. Since p53 is required for the transcriptional activity of p21, cells in which p53 is inhibited by the antisense agent of the present invention show similar results.
    [66] Nochodazole or Cross, Zhang spasm inhibitors can also be used in combination with anti-p53 antisense agents. In the absence of p53-dependent speckle checkpoints, such cells are expected to carry out a continuous cell cycle with abnormal or incomplete mitotic division, and also induce polyhydramides or diploids.
    [67] Other hyperproliferative disorders can also be treated. Examples of hyperproliferative skin disorders include, but are not limited to, human papillomavirus (HPV) -infected cells commonly associated with warts, superficial tumors of skin such as melanoma, pre-malignant and malignant carcinomas, UV keratitis, It is psoriasis. Hyperproliferative diseases of other epithelial tissues (e.g., endothelium, mesothelioma) include reversible obstructive airways diseases such as asthma and bronchitis. A variety of hyperproliferative disorders of the eye, including conical corneas, angular conjunctivitis, spring conjunctivitis, and keratitis albedo, can also be treated. This method can be used to treat hyperplastic vascular disease such as early smooth muscle cell proliferation, vascular occlusion, and restenosis.
    [68] B. Treatment of hypoxia or ischemic conditions
    [69] Other disease states characterized by p53 induction include hypoxic conditions such as those resulting from an ischemic attack such as stroke. For example, it has been observed that p53 is preferentially expressed in apoptotic cells following ischemia in injured neurons in cerebral (Li), and ischemia and epilepsy (Xiang) models. Raafat observed an increased level of p53 after ischemia / resuspension of the kidney, suggesting that the increase could promote apoptosis. Heat shocks have also been reported to induce p53. (Graeber) Therefore, in another aspect, the present invention provides a method for the treatment of heat shock, interstitial seizures, stroke, and ischemia and / Or other occurrences of resuspension (eg, following tracheal transplantation).
    [70] VI. Preparations and administration
    [71] A. Therapeutic preparations
    [72] For administration according to the therapeutic methods of the present invention, the antisense oligonucleotides preferably bind to a pharmaceutically acceptable carrier such as suitable liquid excipients or excipients, and optional supplemental excipients. Liquid excipients and excipients are conventional commercially available. Examples thereof include distilled water, physiological saline solution, aqueous solution of dextrose, and others.
    [73] In general, in addition to the active compounds, the pharmaceutical compositions of the present invention may include suitable excipients and adjuvants that facilitate processing of the active compounds into pharmaceutically usable formulations. In particular, suitable excipients include, for example, excipients such as lactose or sucrose, mannitol or sorbitol, additives such as sugars, such as sugars, and / or calcium phosphate, and starch, gelatin, gum tragacanth, , Hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidone. If desired, a disintegrating agent such as carboxymethyl starch, cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, as well as the above-mentioned starches may be added. Adjuvants include, for example, silica, talc, stearic acid or salts thereof, and / or flow-adjusting agents such as polyethylene glycol and lubricants.
    [74] The core may be provided with suitable coatings resistant to stomach fluids, if desired. For this purpose, concentrated sugar solutions which may optionally comprise gum arabic, talc, polyvinylpyrrolidone, polyethylene glycol, and / or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures may be used . To produce a gastric resistant coating, a suitable solution of cellulose preparation, such as acetylcellulose phthalate or hydroxypropylmethyl-cellulose phthalate, is used. Dyes and dyes may be added to the tablets of the glyphosate to characterize or differentiate the dose of active compound.
    [75] Other pharmaceutical formulations that can be used for oral use include soft, sealed capsules made of gelatin, and plasticizers such as glycerol or sorbitol, as well as push-fit capsules made of gelatin. Press fit capsules may contain the active compound in granular form which may be mixed with additives such as lactose, a binder such as starch, and / or a lubricant such as talc or magnesium stearate and optionally a stabilizer. In soft capsules, the active compound is preferably dissolved or suspended in a suitable liquid such as fatty oil, liquid paraffin, or liquid polyethylene glycol. In addition, stabilizers may be added.
    [76] Pharmaceutical preparations which can be used in rectal applications may, for example, comprise a suppository consisting of a combination of an active compound and a suppository base. Suitable suppository bases include natural or synthetic triglycerides, paraffin hydrocarbons, polyethylene glycols, or higher alkanols. In addition, it is possible to use a gelatin rectal administration capsule consisting of a combination of a base and an active compound. Possible base materials include liquid triglycerides, polyethylene glycols, or paraffin hydrocarbons.
    [77] Suitable liquid compositions for parenteral administration include aqueous solutions of the active compounds in water-soluble or water-dispersible form. In addition, the active saline solution of the active compound can be administered as a suitable oil-in-water emulsion. Suitable lipophilic solvents or excipients include, for example, fatty oils such as sesame oil or synthetic fatty acid esters such as, for example, ethyl olefins or triglycerides. The aqueous injection suspensions may contain materials such as sodium carboxymethyl cellulose, sorbitol, and / or dextran that increase the viscosity of the present liquor. The present liquor may also contain stabilizers.
    [78] In addition to administration by conventional carriers, the active ingredient may be administered by a variety of specialized delivery techniques. For example, a compound of the present invention may be encapsulated in liposomes and administered. Active ingredients that are dependent on solubility can be present in both the aqueous phase and the lipid layer (s), or what is commonly referred to as liposomal entrained liquid. The lipid layer generally comprises phospholipids such as lectin or sphingomyelin, steroids such as cholesterol, ionic surfactants such as diacetyl phosphate, stearylamine, or acetic acid, and / or other hydrophobic materials. The diameter of the liposome is generally in the range of about 15 nm to about 5 mu.
    [79] Methods for preparing such sugar forms are known or will be apparent to those skilled in the art. See, for example, Remington's Pharmaceutical Science (19 th., Ed., William & Wilkins, 1995). Pharmaceutical preparations are prepared according to processes well known in the art. For example, by conventional mixing, granulation, sugar-making, dissolving, or lyophilizing processes. The process used will depend on the physical properties of the active ingredient ultimately used.
    [80] B. Treatment of tumors
    [81] The composition may be administered orally to the subject, or percutaneously or non-intravaginally, for example, by intravenous, subcutaneous, intraperitoneal, or intramuscular injection. For in vivo antitumor use, the p53 mRNA antisense oligonucleotide is preferably administered intravenously.
    [82] According to the present invention, oligonucleotides that inhibit the expression of p53 and function as oxidative restoration path downregulators are administered to patients prior to or in conjunction with the administration of agents capable of capturing radical oxygen induced by cytotoxicity. When administered simultaneously, the two active agents may be in a combined form or in an unconjugated form. In the combined form, it is preferred that the conjugate comprises a cleavable bond such that the oligonucleotide and the cytotoxic agent are dissociated at the target location.
    [83] The amount of each drug to be administered is the combination of two pharmaceutically effective forms of the drug. The dosage will vary depending on factors such as age, health, sex, height and weight of the subject, route of administration, drug toxicity, and relative sensitivity of the cancer to oligonucleotides and cytotoxic agents. Recommended dosage and dosage forms for most cytotoxic agents have been established and are available from Medical Economics Company, Inc., Oradell, N.J. Such as the Physicians Desk Reference published by the Physicians & If necessary, these indicators can be determined for each system by well-established procedures and analyzes such as, for example, clinical trials.
    [84] For in vivo use, a preferred dosage of an oligonucleotide of the invention is a typical amount necessary to reach a concentration in blood of about 0.01 to 1 [mu] mol / l. This concentration can be obtained in a variety of ways; Doses within the range of about 0.05 to about 0.2 mg / kg / hour by continuous IV infusion have been found acceptable. More or less amounts of oligonucleotides can be administered in the required amount.
    [85] The following Example 3 demonstrates the use of the invention in the treatment of human B-cell lymphoma.
    [86] C. Treatment of ischemic injury
    [87] The ischemic condition may be caused by intermittent cerebral circulation, such as caused by heart failure, or other conditions causing total loss of blood supply to the brain, or local intervention in blood haemorrhage, such as due to cerebral hemorrhage, Or a malformed event, or head trauma. The ischemic condition to be treated is usually associated with a stroke which is defined as a gradual decrease or loss of nervous system function caused by occlusion or rupture of blood vessels in the brain. Secondary brain injury as a result of an intrinsic ischemic event typically occurs in the area surrounding ischemic injury, in the case of localized ischemia and / or in selective vulnerable areas of the hippocampus or cerebral basal ganglia, in grobal ischemia Of brain cells, or lesions. Secondary impairment can often be manifested by functional disturbances such as short-term or long-term memory loss.
    [88] ON is prepared with a suitable inert carrier such as sterile physiological saline solution for non-invasive administration. The dosage administered will depend on the route of administration. One suitable route is intracerebroventicular (ICV) at a dose level of about 50 μg-5 mg ON / kg body weight. Effective doses to exhibit significant reductions in the pharmacologically effective dose, i. E. Anatomical and / or functional impairment, can be determined from the dose / response seen in the model system for ischemia and stroke injury. This model system includes an ischemic and rat 4-vessel occlusion model that is produced by the instantaneous occlusion of the carotid artery of the neck of a Gerbil-Russisch model. Tissue is examined for anatomical damage, and functional impairment can be assessed by observing the effect on hyperactivity or short term memory, which is a common result of cerebral ischemia in animals. Such processes are known in the art; For example, U.S. Pat. Patent No. 5,051,403.
    [89] D. Restenosis
    [90] An important aspect of the successful treatment of anti-restenosis, particularly in connection with antisense agents, is the effective delivery of antisense oligomers to infected cells. Systemically administered anti-restonocis drugs typically fail to obtain an effective concentration at the site of vascular injury unless an unacceptable high concentration of drug is used. &Quot; Method for Treating Restonosis by Antisense Targeting of CMV ", Shared & A delivery device such as that disclosed in Application Serial No. 09 / 062,160 may be used to deliver the oligomer to the site of the patient's vasodilation. The oligomer is preferably a morpholino oligomer and is preferably contained in a pharmaceutically acceptable carrier. Oligomers may also be contained in the biocompatible polymeric carrier also disclosed in the reference application.
    [91] Preferably, the oligomer is delivered simultaneously with the angioplasty procedure. As reported in Farrell, when a compound is administered to balloon-injured arteries, the uptake of oligonucleotides by cells may be significantly increased compared to the uptake by normal arteries. Pharmaceutical treatments using the formulations of the present invention can be combined with radiation or photodynamic therapy.
    [92] The dosage will depend on the size of the subject, the route of administration, and the extent of the infected tissue according to standard pharmaceutical experience. The desired level of drug is a level effective to inhibit p53 expression and reduce or prevent restenosis, without unacceptable side effects. In the case of an adult, the recommended dosage is an antisense oligomer in the range of 1 to 25 mu mol, preferably 2 to 15 mu mol. The optimal dose for the route of administration can be determined by routine experimentation according to methods known in the art. For example, in the case of administration to a venous injury site, in vivo models such as those described in Edelman and Rosenberg are available.
    [93] When the oligomer is combined with a weak delivery device, as described above, the device is effective to deliver the appropriate dosage of the drug. With respect to the surface area of the tissue to be treated, an effective dose is typically in the range of 30 to 3000 μg per cm 2 of vessel wall, more preferably about 300 to 1500 μg / cm 2. The patient can also be administered the composition at dosage levels sufficient to further inhibit restenosis on a cyclic basis after angioplasty.
    [94] While the invention has been described with reference to particular methods and embodiments, various modifications may be made without departing from the spirit of the invention.
    [95] Example 1
    [96] Oligonucleotide synthesis
    [97] Applied Biosystems Model 380B DNA Synthesizer (Foster City, Calif.) And ABI User Bulletin, no. Lt; RTI ID = 0.0 > phosphoramidate < / RTI > The synthesizer was programmed with the protocol of the user manual.
    [98] A typical synthesis outline is: The 3 'base (i.e., unmodified nucleotide, C-5-propyne nucleotide, or morpholinonucleotide analogue) of the ODN linked by the 3' hydroxyl group was inserted into a 1 mu mol silica gel support column, 'One by one depending on the type of base. All liquid reagents are supplied by Applied Biosystems, Inc. Argon gas is supplied by Air Products (Omaha). The first step is flushing the column with acetone nitrile, then argon gas to remove any residues in the column. Next, the tricholaiacetic acid quantizes dimethoxytrityl (DMT) groups that cap to the 5 'oxygen of the nucleotides during ODN synthesis. Next, the quantized DMT is removed from the base. The column was flushed again with acetone nitrile and argon. The DMT residue was collected into a DMT residue port and transferred to a test tube to check for a bright orange color. The color check is performed to verify that the diteritization is successful. The DMT group can be spectrophotometrically quantified by reading absorbance at 498 nm. If the color is not a bright orange then the base dityrylation will not be successful and synthesis of impure ODNs may occur. The next step in the synthesis is the binding of the next base of the ODN sequence. Suitable nucleotide bases modified with cyanoethylphosphoramidate are dissolved in acetone nitrile and carried on a tetrazolone column. The base reacts to form a phosphite bond between the nucleotides. The column is flushed with acetone nitrile and argon. The column is flushed with acetic anhydride and 1-methylimidizole to cap any unconjugated 5'hydroxy groups with an acetyl group. The cap will prevent unconjugated bases from any sequencing by binding during synthesis and reduce the length of the impurities. The column is again flushed and the trivalent phosphite is oxidized to pentavalent phosphorothioate triester by tetraethylthiuram disulfide and acetone nitrile. The column is flushed with acetone nitrile and argon. Then, starting from the trimerization step, the synthesis for the next base in the sequence is repeated again. The aluminum hydroxide is passed through the column and the eluate is collected to cut the S-ODNs from the column. Aluminum hydroxide and cyanoethyl (from the phosphate linkage) are removed by distillation of the S-ODN solution with a vacuum centrifuge overnight. Dilute the dried ODN with sterile saline. The purity of the S-ODN is checked by treating a sample of S-ODN diluted with water on a 10% polyacrylamide gel. The concentration of S-ODN is determined by reading at 260 nm absorbance and increasing absorbance at extinction ratio.
    [99] Further details in the preparation of morpholino oligonucleotides are described, for example, in Summerton and Weller, Antisense and Nucleic Acid Drug Development . 7 : 187-195 (1997).
    [100] Example 2
    [101] Partial hepatectomy
    [102] Partial liver resection studies were performed in male Sprague Dawley rats (Sasco, Omaha NE) weighing 200-220 grams. The animals were housed in clean plastic casings at UNMC's AAALAC-approved facility on a 12-hour day / night cycle and allowed access to Purina rat meals and unlimited fresh water. Animal protocols were approved by the Institutional Animal Care and Use Committee.
    [103] Procedures were performed as described in Higgins and Anderson (1931). Sterile surgical techniques were used. The rats were anesthetized with methoxyflurane (Mallinckrodt Veterinary, Mundelin, IL) and placed so that the abdominal surface of the rats was exposed. A 3 to 4 cm long section was shaved along the midline just behind the dentate gyrus and swabbed with Betadine. The liver was exposed by incising along the right median line. The central and left side lobes were securely connected and then incised. This resulted in a removal of approximately 65 to 70% of the total. The abdominal incision was closed in two layers. The control rats that did not undergo partial hepatectomy were the same but underwent false surgical procedures that only exposed their liver and did not incise partially.
    [104] Oligonucleotide administration
    [105] ODNs were injected intraperitoneally into a dose of 1 mg / 200 gm / day S-ODN, 0.1 mg / 220 g / day C-5-P S-ODN, and 50 nM (approximately 3.4 mg) 200 g / day morpholino . All rats were dosed with ODNs immediately after receiving post-surgical consciousness, and after 24 hours, depending on the length of the experiment. Next, the rats were allowed to rest for 1, 2, 5, or 7 days after surgery. The wet weight gain was determined 24 hours after administration of the ODN indicating the data shown in Fig.
    [106] Example 3
    [107] Treatment of human B-cell lymphoma
    [108] Medication regimen: Starting at day 0, the patient was administered a morpholino antisense oligonucleotide with SEQ ID NO: 1 at a dose of 0.2 mg / kg / hr as a continuous IV infusion over a 7 day period via a vein access device Receive. The MINE chemotherapy is started on the 4th day according to the following schedule.
    [109] Mesna Injection (MESNEX ™ , Bristol-Meyers Oncology): 500 mg / M 2 IV for 30 minutes before Ifosfamide, 250 mg / M 2 IV for 4 hours after Ifosfamide, 4 to 7 days after Ifosfamide, 250 mg / M 2 .
    [110] Ifosfamide (IFEX ™ , Bristol-Meyers Oncology): 1.33 g / M 2 daily for 4-7 days IV.
    [111] Mitoxantrone hydrochloride (NOVANTRONE TM , Lederle Laboratories): 10 mg / M 2 on day 4 IV.
    [112] Etoposide (VEPESID ™ , Bristol-meyers Oncology): 80 mg / M 2 daily for 4-7 days IV.
    [113] Patients are regularly assessed for toxicity and response to their disease. Patients receive weekly complete blood count (CBC) and platelet counts. Repeat the cycle once every four weeks for up to six times. Patients are assessed for each cycle of response to measurable disease by a health check. Classify patients according to the response (complete response, partial response, stable response, or progressive disease).
    [114] The patient is expected to show improved clinical response to the treatment of the present invention, which is contrary to traditional MINE therapy, which does not involve the use of antisense oligonucleotide SEQ ID NO: 1 (OL (1) p53). OL (1) p53 is expected to preferentially kill cancer cells by p53-independent apoptosis by increasing or decreasing the effect of MINE on cancer cells by inhibiting the cell cycle repair mechanism.
    权利要求:
    Claims (30)
    [1" claim-type="Currently amended] The nucleotide sequence SEQ ID NO: 1 identified as 5'-TCA GTC TGA GTC AGG CCC-3 'in a suitable pharmaceutical carrier, or the nucleotide sequence SEQ ID NO: 1 identified as 5'-CCC TGC TCC CCC CTG GCT CC-3' 1. A method for treating a disease state characterized by the induction of p53 in a mammalian subject comprising administering to the subject a pharmaceutically effective amount of an antisense agent having a NO: 2, wherein the antisense agent is selected from the group consisting of morpholino oligonucleotides, 2'-O-allyl or 2'-O-alkyl modified oligonucleotides, N3'-P5 'phosphoramidate oligonucleotides, or C-5-propynopyrimidine-modified oligonucleotides.
    [2" claim-type="Currently amended] 2. The method of claim 1, wherein the antisense agent is a morpholino oligonucleotide.
    [3" claim-type="Currently amended] 3. The method of claim 2, wherein the morpholino oligonucleotide comprises a morpholino subunit linked by a phosphorodiamidate backbone bond.
    [4" claim-type="Currently amended] 2. The method of claim 1, wherein the oligonucleotide is a C-5-propynopyrimidine-modified oligonucleotide.
    [5" claim-type="Currently amended] 2. The method of claim 1, wherein the antisense agent has the sequence SEQ ID NO: 1.
    [6" claim-type="Currently amended] 2. The method of claim 1, wherein the antisense agent has the sequence SEQ ID NO: 2.
    [7" claim-type="Currently amended] 2. The method of claim 1, wherein the subject is a human subject.
    [8" claim-type="Currently amended] 2. The method of claim 1, wherein the disease state is cancer.
    [9" claim-type="Currently amended] 9. The method of claim 8, further comprising treatment of said subject with a medicament effective to increase a radical oxygen species at the cellular level.
    [10" claim-type="Currently amended] 10. The method of claim 9, wherein the agent is selected from a radiation sensitizer, ionizing radiation, a high-pressure oxygen environment, and a chemotherapeutic agent that increases radical oxygen species at the cellular level.
    [11" claim-type="Currently amended] 11. The method of claim 10, wherein the chemotherapeutic agent is an anthracycline or anthraquinone.
    [12" claim-type="Currently amended] The method of claim 8, wherein G 2 from the group of the cell cycle method further comprising the treatment of the target by the drug effective to interfere with progression of the M phase.
    [13" claim-type="Currently amended] 13. The method of claim 12, wherein the agent is selected from the group consisting of a phosphokinase C (PKC) inhibitor, bis (chloroethyl) nitroso urea (BCNU), pentoxifylline, silymarin, staurosporine, phenylaxystine, paclitaxel, retinoic acid, Characterized in that it is selected from the group consisting of bipyridol, methyl-2,5-dihydrocinnamate, herboxydiene, 9-nitrocamptothecin, mitotoxin, apigenin, .
    [14" claim-type="Currently amended] 2. The method of claim 1, wherein said disease state is caused by ischemia or ischemia / resuspension injury.
    [15" claim-type="Currently amended] 2 identified from the group consisting of SEQ ID NO: 1 identified as 5'-TCA GTC TGA GTC AGG CCC-3 'and 5'-CCC TGC TCC CCC CTG GCT CC-3' Wherein the antisense agent comprises a morpholino oligonucleotide, a peptide nucleic acid, a 2'-O-allyl or 2'-O-alkyl modified oligonucleotide, and a N3 'to P5 Or a C5-propynylpyrimidine-modified phosphorylated oligonucleotide, or a C5-propynylpyrimidine-modified phosphorylated oligonucleotide, or a C5-propynylpyrimidine-modified oligonucleotide.
    [16" claim-type="Currently amended] 16. The composition of claim 15, wherein the antisense agent is a morpholino oligonucleotide.
    [17" claim-type="Currently amended] 17. The composition of claim 16, wherein the morpholino oligonucleotide comprises a morpholino subunit linked by a phosphorodiamidate backbone bond.
    [18" claim-type="Currently amended] 16. The composition of claim 15, wherein the oligonucleotide is a C-5-propynopyrimidine-modified oligonucleotide.
    [19" claim-type="Currently amended] 16. The composition of claim 15, wherein the antisense agent has the sequence SEQ ID NO:
    [20" claim-type="Currently amended] 16. The composition of claim 15, wherein the antisense agent has the sequence SEQ ID NO: 2.
    [21" claim-type="Currently amended] A nucleotide sequence selected from the group consisting of SEQ ID NO: 1 identified as 5'-TCA GTC TGA GTC AGG CCC-3, and SEQ ID NO: 2 identified as 5'-CCC TGC TCC CCC CTG GCT CC- Wherein the oligonucleotide is selected from the group consisting of morpholino oligonucleotides, peptide nucleic acids, 2'-O-allyl or 2'-O-alkyl modified oligonucleotides, and N3 '- P5' phosphoramidate oligonucleotides Lt; / RTI > oligonucleotides.
    [22" claim-type="Currently amended] 23. The oligonucleotide of claim 21, having the nucleotide sequence SEQ ID NO: 1.
    [23" claim-type="Currently amended] 29. The oligonucleotide of claim 21, having the nucleotide sequence SEQ ID NO: 2.
    [24" claim-type="Currently amended] 24. The oligonucleotide of claim 23, wherein the oligonucleotide is a morpholino oligonucleotide.
    [25" claim-type="Currently amended] 25. The morpholino oligonucleotide of claim 24, comprising a morpholino subunit linked by a phosphorodiamidate backbone bond.
    [26" claim-type="Currently amended] 24. The oligonucleotide of claim 23, wherein the oligonucleotide is a morpholino oligonucleotide.
    [27" claim-type="Currently amended] 27. The morpholino oligonucleotide of claim 26 comprising a morpholino subunit linked by a phosphorodiamidate backbone bond.
    [28" claim-type="Currently amended] 2 identified from the group consisting of SEQ ID NO: 1 identified as 5'-TCA GTC TGA GTC AGG CCC-3 'and 5'-CCC TGC TCC CCC CTG GCT CC-3' An oligonucleotide having a sequence, wherein the oligonucleotide is a C-5-propynopyrimidine-modified oligonucleotide.
    [29" claim-type="Currently amended] 29. The oligonucleotide of claim 28, having the nucleotide sequence SEQ ID NO: 1.
    [30" claim-type="Currently amended] 29. The oligonucleotide of claim 28, having the nucleotide sequence SEQ ID NO: 2.
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    同族专利:
    公开号 | 公开日
    CA2347905A1|2000-05-04|
    AU1221700A|2000-05-15|
    WO2000024885A2|2000-05-04|
    JP2002528464A|2002-09-03|
    DE69932346D1|2006-08-24|
    EP1124950A2|2001-08-22|
    EP1124950B1|2006-07-12|
    AU771579B2|2004-03-25|
    US6365577B1|2002-04-02|
    AT332969T|2006-08-15|
    JP2011079865A|2011-04-21|
    WO2000024885A3|2000-07-20|
    ES2268903T3|2007-03-16|
    JP4777515B2|2011-09-21|
    DE69932346T2|2007-07-05|
    KR100675123B1|2007-01-29|
    CA2347905C|2009-12-15|
    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    法律状态:
    1998-10-26|Priority to US10569598P
    1998-10-26|Priority to US60/105,695
    1999-10-22|Application filed by 추후보정, 에이브이아이 바이오파마 인코포레이티드
    2001-10-13|Publication of KR20010089904A
    2007-01-29|Application granted
    2007-01-29|Publication of KR100675123B1
    优先权:
    申请号 | 申请日 | 专利标题
    US10569598P| true| 1998-10-26|1998-10-26|
    US60/105,695|1998-10-26|
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