WO1998013072A1 - Compositions et procedes destines au traitement de la resistance multiple aux anticancereux - Google Patents

Compositions et procedes destines au traitement de la resistance multiple aux anticancereux Download PDF

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WO1998013072A1
WO1998013072A1 PCT/US1997/017320 US9717320W WO9813072A1 WO 1998013072 A1 WO1998013072 A1 WO 1998013072A1 US 9717320 W US9717320 W US 9717320W WO 9813072 A1 WO9813072 A1 WO 9813072A1
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mdrl
oligonucleotide
cells
vinc
antisense
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PCT/US1997/017320
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Bruno Calabretta
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Thomas Jefferson University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/312—Phosphonates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates

Definitions

  • the invention relates to antisense oligonucleotides, in particular to antisense oligonucleotides which prevent expression of the mdr gene, and to compositions for and methods of using such oligonucleotides to eliminate multiple drug resistance and render cells that are associated with disease conditions more susceptible to chemotherapeutic treatments.
  • MDR mdrl gene product
  • P-glycoprotein a 170 KDA membrane glycoprotein that acts as an ATP- dependent efflux pump, increasing transport of various anticancer compounds out of cells and decreasing cellular accumulation of drugs and, thus, their efficacy.
  • Anticancer drugs that are associated with P-170-mediated drug resistance include Vinca alkaloids (vinblastine and vincristine), anthracyclines (adriamycin), taxol, actinomycin D and mitomycin. Since there is a well established correlation between the expression of the mdrl gene and the activity of the P-170-mediated transport mechanism in human tumors, compounds that can inhibit efflux by P-glycoprotein and enhance the accumulation of anticancer drugs might prove therapeutically useful.
  • a variety of pharmacological agents including verapamil, other calcium channel blockers, calmodulin inhibitors, cyclosporins, and steroid hormones, have been shown to interfere with P- glycoprotein function and to successfully reverse the MDR phenotype in vitro.
  • ODNs Antisense oligodeoxynucleotides
  • compositions that comprise a pharmaceutically acceptable carrier and an antisense oligonucleotides specific for mdrl as hereinafter defined.
  • the oligonucleotide has a nucleotide sequence capable of forming a stable duplex with a portion of an mRNA transcript of the mdrl gene.
  • the oligonucleotide is generally at least an 8-mer oligonucleotide, that is, the oligonucleotide is an oligomer containing at least 8 nucleotide residues, more preferably at least about 12 nucleotides.
  • the preferred maximum size of the oligonucleotide is about 60 nucleotides, more preferably about 50 nucleotides.
  • the oligomer is preferably an oligodeoxynucleotide. While oligonucleotides smaller than 12-mers may be utilized, they are statistically more likely to hybridize with non-targeted sequences, and for this reason may be less specific. In addition, a single mismatch may destabilize the hybrid.
  • oligonucleotides larger than 40-mers may be utilized, uptake may become more difficult without specialized vehicles or oligonucleotide carriers.
  • the oligonucleotide is a 15- to 40-mer oligodeoxynucleotide, more advantageously an 18- to 30-mer.
  • oligonucleotides having a sequence complementary to any region of the mdrl mRNA find utility in the present invention, preferred are oligonucleotides capable of forming a stable duplex with a portion of the transcript lying within about 50 nucleotides (preferably within about 40 nucleotides) upstream (the 5' direction), or about 50 (preferably 40) nucleotides downstream (the 3' direction) from the translation initiation codon. Also preferred are oligonucleotides which are capable of forming a stable duplex with a portion of a mdrl mRNA transcript including the translation initiation codon.
  • the invention is also a method for inhibiting expression of the mdrl gene product P-glycoprotein (P-l 70) in vivo.
  • Pharmaceutical compositions of the invention are administered to a patient and cells take up the antisense oligonucleotides.
  • the patient has cancer which has become drug resistant.
  • the invention thus provides a method of treating multiple drug resistant malignant disease in vivo comprising administering to an individual of mdrl antisense oligonucleotide sufficient to render the cancer cells susceptible to drug treatment, and administering an effective amount of a chemotherapeutic drug to eliminate cancer cells in the individual.
  • the malignant diseases treatable according to the invention is a blood disease such as leukemia.
  • Figure 1 A and Figure IB show the effect or mdrl [SJODNs on HL-60 Vinc cell proliferation.
  • the data in Figure 1 A refers to cells were treated with sense (• ) or antisense ( ⁇ ) mdrl [S]ODNs at a total dose of 200 ⁇ g/ml over 4 days (80 ⁇ g/ml at day 0 and 40 ⁇ g/ml from day 1 to day 3).
  • Control cells (A) were left untreated. Cell counts and viability were determined daily until the 8th day of culture. Representative of three different experiments with similar results.
  • IB shows data from cells were treated with scrambled (•) or antisense ( ⁇ ) mdrl [S]ODNs at a total dose of 360 ⁇ g/ml over 8 days (80 ⁇ g/ml at day 0 and 40 ⁇ g/ml from day 1 to day 7).
  • Control cells (A) were left untreated. Cell counts and viability were determined daily until the 8th day of culture. Representative of three different experiments with similar results. Both in (Fig. lA) and in (Fig. IB) each value is an average ⁇ standard error (S.E.) of four different determinations within the same experiment. When not shown, the standard error is smaller than the symbol.
  • Figures 2A and 2B are data on the effect of mdrl [S]ODNs plus vincristine on HL-60/Vinc cell proliferation Figure 2 A and cell survival (B).
  • (A) cells were treated with mdrl sense ( ⁇ ), scrambled ( ⁇ ) or antisense ( ⁇ ) [SJODNs at a total dose of 200 ⁇ g/ml fractioned in 4 days (80 ⁇ g/ml at day 0 and 40 ⁇ g/ml from day 1 to day 3) and then exposed to vincristine 0.01-1 ⁇ g/ml) for 72 hours.
  • Vincristine- treated cells ( ⁇ ) were exposed to VINC alone (same doses and exposure time). Control cells were left untreated.
  • Figures 3 A and 3B show data of expression of mdrl mRNA (Fig. 3 A) and gP-170 protein (Fig. 3B) in HL-60/Vinc cells treated with mdrl [SJODNs.
  • Fig. 3 A cells were exposed to mdrl S or AS [SJODNs (as reported in Fig. 2 legend), to 0.5 ⁇ g/ml vincristine (for 72 hours) and to mdrl S or AS [SJODNs followed by vincristine. Control cells were left untreated.
  • Figures 4A and 4B show survival curves of SCID mice injected with HL- 60 Vinc cells and treated with mdrl [SJODNs and vincristine, given alone or in combination (details on the treatment schedules are in the Materials and Methods and Results sections).
  • Fig. 4A shows survival curves relative to the mice of the SCR-treated groups;
  • Fig. 4B shows survival curves of the mice in the AS-treated groups.
  • the survival curves of the untreated and of the vincristine treated-mice are the same in both panels.
  • Each experimental group consisted of 10 mice.
  • the survival curves are as follows: untreated; O — O vincristine; ⁇ — ⁇ scrambled; O — ⁇ scr.ambled plus vincristine; A — A antisense; ⁇ — ⁇ antisense plus vincristine.
  • VINC versus SCR .P 0.4 n.s.
  • VINC versus SCR+VINC P 0.2 n.s.
  • VINC versus AS+VINC .P 0.03 s.
  • an “antisense oligonucleotide specific for mdrl” or “mdrl antisense oligonucleotide” is meant an oligonucleotide having a sequence (I) capable of forming a stable triplex with a portion of the mdrl gene, or (ii) capable of forming a stable duplex with a portion of an mRNA transcript of the mdrl gene.
  • oligonucleotide includes linear oligomers of natural or modified monomers or linkages, including deoxyribonucleosides, ribonucleosides, ⁇ -anomeric forms thereof, polyamide nucleic acids, and the like, capable of specifically binding to a target polynucleotide by way of a regular pattern of monomer-to-monomer interactions, such as Watson-Crick type of base pairing, Hoogsteen or reverse Hoogsteen types of base pairing, or the like.
  • monomers are linked by phosphodiester bonds or analogs thereof to form oligonucleotides ranging in size from a few monomeric units, e.g., 3-4, to several hundreds of monomeric units.
  • nucleoside includes the natural nucleosides, including 2'-deoxy and 2'-hydroxyl forms, e.g., as described in Kornberg and Baker, DNA Replication, 2nd Ed. (Freeman, San Francisco, 1992).
  • nucleosides in reference to nucleosides includes synthetic nucleosides having modified base moieties and/or modified sugar moieties, e.g., described generally by Scheit, Nucleotide Analogs (John Wiley, New York, 1980). Such analogs include synthetic nucleosides designed to enhance binding properties, e.g., duplex or triplex stability, specificity, or the like.
  • phosphorothioate oligonucleotide means an oligonucleotide wherein one or more of the internucleotide linkages is a phosphorothioate group
  • alkylphosphonate oligonucleoside is meant an oligonucleotide wherein one or more of the internucleotide linkages is an alkylphosphonate group
  • R wherein R is an alkyl group, preferably methyl or ethyl.
  • “Stability” in reference to duplex or triplex formation roughly means how tightly an antisense oligonucleotide binds to its intended target sequence; more precisely, it means the free energy of formation of the duplex or triplex under physiological conditions. Melting temperature under a standard set of conditions, e.g., as described below, is a convenient measure of duplex and/or triplex stability.
  • antisense oligonucleotides of the invention are selected that have melting temperatures of at least 50°C under the standard conditions set forth below; thus, under physiological conditions and the preferred concentrations, duplex or triplex formation will be substantially favored over the state in which the antisense oligonucleotide and its target are dissociated.
  • a stable duplex or triplex may in some embodiments include mismatches between base pairs and/or among base triplets in the case of triplexes.
  • antisense oligonucleotides of the invention form perfectly matched duplexes and/or triplexes with their target polynucleotides.
  • downstream when used in reference to a direction along a nucleotide sequence means the 5' to 3' direction.
  • upstream means the 3' to 5' direction.
  • mdrl mRNA transcript means the presently known mRNA transcript of the mdrl gene and all variations thereof, or any further transcripts which may be elucidated.
  • mdrl gene inactivation is meant the interruption of expression of functional protein P- 170 which is the product of the "mdrl gene”.
  • MDR cancer is meant to refer to cancer cells which have a multiple drug resistant phenotype as a result of mdrl gene expression.
  • MDR leukemia is meant to refer to leukemia cells which have a multiple drug resistant phenotype as a result of mdrl gene expression.
  • the MDR phenotype associated with mdrl gene expression and production of P- 170 has been reversed in vivo using antisense oligonucleotides to inhibit mdrl gene expression.
  • the in vivo inhibition of P- 170 production in MDR cancer cells renders the cancer cells susceptible to chemotherapeutic elimination. Accordingly, the patient can be effectively treated to eliminate the cancer cells upon reversal of the MDR phenotype.
  • individuals suffering from MDR cancer can treated by administering antisense pharmaceutical compositions which comprise antisense oligonucleotides specific for mdrl to render the cancer susceptible to drugs.
  • the individual is also administered drugs which eliminate cancer cells.
  • the MDR cancer is a blood borne cancer such as MDR leukemia including chronic MDR leukemia and acute MDR leukemia, MDR lymphoma, or MDR myeloma (cancer of plasma cells).
  • the patient is administered the pharmaceutical composition that comprises the antisense oligonucleotides for at least 7 days, preferably for at least 7-14 days, more preferably 14-21 days or longer.
  • the antisense oligonucleotides are administered prior to and/or throughout anti-cancer chemotherapy.
  • the antisense oligonucleotide is administered at least 4-7 prior to and preferably throughout a chemotherapeutic cycle.
  • a chemotherapeutic cycle is three weeks (from Day 1 to Day 21) including weekly administration of anticancer drugs (on, for example, Day 1 , Day 8 and Day 15), the antisense oligonucleotides are administered starting on day -7 to -4 and preferably continuing until at least Day 15-21.
  • the patient is administered up to 1 gram/day of antisense oligonucleotide. Dosage varies depending upon the physical chemical characteristics of the antisense oligonucleotides, taking into account half life, activity and toxicity. Generally 10 mg to 1 gram are administered. In some embodiments, 70-700 mg of antisense oligonucleotide are administered per day. In some embodiments, 250-500 mg of antisense oligonucleotide are administered per day. In some embodiments, 10-100 mg of antisense oligonucleotide are administered per day.
  • Modes of administration include intravenous, intraperitoneal or subcutaneous. Administration is preferably continuous as opposed to bolus. According to some preferred embodiments, antisense oligonucleotides are administered with a continuous infusion pump.
  • antisense oligonucleotides are [SJOligonucleotides, i.e. phosphorothioates.
  • Anti-cancer drugs may be co-administered to the patient starting on day 1 or administration of such chemotherapeutics may be initiated on any day thereafter.
  • the patient is treated with antisense oligonucleotides for at least 7 days prior to initiation of anti-cancer chemotherapy.
  • the cDNA nucleotide sequence of the mdrl gene and predicted amino acid poiypeptide are disclosed in Chen, C.J. et al., Internal duplication and homology with bacterial transport proteins in the mdrl (P-glycoprotein) gene from multidrug-resistant human cells, Cell 47:381-389, 1986, which is incorporated herein by reference).
  • the translation initiation codon ATG is preceded by a 5'-untranslated region.
  • the termination codon TGA is followed by a 3 '-untranslated region, including a polyadenylation sequence at the 3' end.
  • Whether or not a particular cancer is a MDR cancer may be ascertained by conventional molecular biological techniques, such as analysis of gene expression or protein production. Such assays to detect gene expression or protein production are well known. Patients may for example be identified by amplification of mdrl mRNA using standard procedures for amplification of mRNA sequences (reverse transcriptase, polymerase chain reaction; RT-PCR) or by immunoassay using anti-P170 antibodies.
  • target mdrl polynucleotides may be single-stranded or double-stranded DNA or RN A; however, single-stranded DNA or RNA targets are preferred.
  • the target to which the mdrl antisense oligonucleotides of the invention are directed include allelic forms of the mdrl gene and mRNA.
  • sequence of the target polynucleotide e.g., Peyman and Ulmann, Chemical Reviews, 90:543-584, 1990; Crooke, Ann. Rev. Pharmacal. Toxicol., 32:329-376 (1992); and Zamecnik and Stephenson, Proc. Natl. Acad. Sci., 75:280-284 (1974).
  • the sequences of mdrl antisense compounds are selected such that the G-C content is at least 60%.
  • Preferred mRNA targets include the 5' cap site, tRNA primer binding site, the initiation codon site, the mRNA donor splice site, and the mRNA acceptor splice site, e.g., Goodchild et al., U.S. patent 4,806,463.
  • oligonucleotides complementary to and hybridizable with any portion of the transcript are, in principle, effective for inhibiting translation, and capable of inducing the effects herein described. It is believed that translation is most effectively inhibited by blocking the mRNA at a site at or near the initiation codon. Thus, oligonucleotides complementary to the 5'-region of the mdrl mRNA transcript are preferred. Oligonucleotides complementary to the mdrl mRNA, including the initiation codon (the first codon at the 5' end of the translated portion of the mdrl transcript), or codons adjacent the initiation codon, are preferred.
  • antisense oligomers complementary to the 5'-region of the mdrl transcript are preferred, particularly the region including the initiation codon, it should be appreciated that useful antisense oligomers are not limited to those oligomers complementary to the sequences found in the translated portion of the mRNA transcript, but also includes oligomers complementary to nucleotide sequences contained in, or extending into, the 5 - and 3'-untranslated regions.
  • the antisense oligonucleotide is complementary to the mRNA transcript beginning with the initiation codon of the transcript and extending downstream thereof for a distance of 50 nucleotides.
  • oligomers based upon the 50-mer sequence, in particular, oligomers hybridizable to segments of the mdrl message containing the initiation codon, may be utilized.
  • particularly preferred are oligomers containing at least 12 nucleotides, more preferably 12-25 nucleotides, and in some embodiments, most preferably 18, 19 or 20 nucleotides.
  • Antisense oligonucleotides of the invention may comprise any polymeric compound capable of specifically binding to a target polynucleotide by way of a regular pattern of monomer-to-nucleoside interactions, such as Watson-Crick type of base pairing, Hoogsteen or reverse Hoogsteen types of base pairing, or the like.
  • Antisense compounds of the invention may also contain pendent groups or moieties, either as part of or separate from the basic repeat unit of the polymer, to enhance specificity, nuclease resistance, delivery, or other property related to efficacy, e.g., cholesterol moieties, duplex intercalators such as acridine, poly-L-lysine, "end-capping" with one or more nuclease-resistant linkage groups such as phosphorothioate, and the like.
  • pendent groups or moieties either as part of or separate from the basic repeat unit of the polymer, to enhance specificity, nuclease resistance, delivery, or other property related to efficacy, e.g., cholesterol moieties, duplex intercalators such as acridine, poly-L-lysine, "end-capping" with one or more nuclease-resistant linkage groups such as phosphorothioate, and the like.
  • alkylphosphonate oligonucleoside or alkylphosphotriester oligonucleotide For example, it is known that enhanced lipid solubility and/or resistance to nuclease digestion results by substituting an alkyl group or alkoxy group for a phosphate oxygen in the internucleotide phosphodiester linkage to form an alkylphosphonate oligonucleoside or alkylphosphotriester oligonucleotide.
  • Non-ionic oligonucleotides such as these are characterized by increased resistance to nuclease hydrolysis and/or increased cellular uptake, while retaining the ability to form stable complexes with complementary nucleic acid sequences.
  • the alkylphosphonates in particular, are stable to nuclease cleavage and soluble in lipid.
  • the preparation of alkylphosphonate oligonucleosides is disclosed in Tso et al., U.S. patent 4,469,863.
  • nuclease resistance is conferred on the antisense compounds of the invention by providing nuclease-resistant internucleosidic linkages.
  • nuclease-resistant internucleosidic linkages are known in the art, e.g., phosphorothioate: Zon and Geiser, Anti-Cancer Drug Design, 6:539-568 (1991); Stec et al., U.S. patent 5,151,510; Hirschbein, U.S. patent 5,166,387; Bergot, U.S.
  • Additional nuclease linkages include phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate, alkylphosphotriester such as methyl- and ethylphosphotriester, carbonate such as carboxymethyl ester, carbamate, morpholino carbamate, 3'-thioformacetal, silyl such as dialkyl(C,-C 6 )- or diphenylsilyl, sulfamate ester, and the like.
  • phosphorus analogs of the phosphodiester linkage are employed in the compounds of the invention, such as phosphorothioate, phosphorodithioate, phosphoramidate, or methylphosphonate. More preferably, phosphorothioate is employed as the nuclease resistant linkage.
  • Phosphorothioate oligonucleotides contain a sulfur-for-oxygen substitution in the internucleotide phosphodiester bond. Phosphorothioate oligonucleotides combine the properties of effective hybridization for duplex formation with substantial nuclease resistance, while retaining the water solubility of a charged phosphate analogue. The charge is believed to confer the property of cellular uptake via a receptor (Loke et al., Proc. Natl. Acad. Sci., 86, 3474-3478 (1989)).
  • compounds of the invention may comprise additional modifications, e.g., boronated bases, Spielvogel et al., 5,130,302; cholesterol moieties, Shea et al., Nucleic Acids Research, 18:3777-3783 (1990) or Letsinger et al., Proc. Natl. Acad. Sci., 86:6553-6556 (1989); and 5-propynyl modification of pyrimidines, Froehler et al., Tetrahedron Lett., 33:5307-5310 (1992).
  • additional modifications e.g., boronated bases, Eisenvogel et al., 5,130,302; cholesterol moieties, Shea et al., Nucleic Acids Research, 18:3777-3783 (1990) or Letsinger et al., Proc. Natl. Acad. Sci., 86:6553-6556 (1989); and 5-propynyl modification of pyrimidines, Fr
  • antisense compounds of the invention are synthesized by conventional means on commercially available automated DNA synthesizers, e.g., an Applied Biosystems (Foster City, CA) model 380B, 392 or 394 DNA/RNA synthesizer.
  • phosphoramidite chemistry is employed, e.g., as disclosed in the following references: Beaucage and Iyer, Tetrahedron, 48:2223-2311 (1992); Molko et al., U.S. patent 4,980,460; Koster et al., U.S. patent 4,725,677; Caruthers et al., U.S. patents 4,415,732; 4,458,066; and 4,973,679.
  • third strand association via Hoogsteen type of binding is most stable along homo- pyrimidine-homopurine tracks in a double stranded target.
  • base triplets form in T-A*T or C-G*C motifs (where "-" indicates Watson-Crick pairing and "*" indicates Hoogsteen type of binding); however, other motifs are also possible.
  • Hoogsteen base pairing permits parallel and antiparallel orientations between the third strand (the Hoogsteen strand) and the purine-rich strand of the duplex to which the third strand binds, depending on conditions and the composition of the strands.
  • nucleoside type e.g., whether ribose or deoxyribose nucleosides are employed
  • base modifications e.g., methylated cytosine, and the like
  • Roberts et al. Proc. Natl. Acad. Sci., 88:9397-9401 (1991); Roberts et al., Science, 258:1463-1466 (1992); Distefano et al., Proc. Natl. Acad.
  • oligonucleotide moieties is sufficiently large to ensure that specific binding will take place only at the desired target polynucleotide and not at other fortuitous sites, as explained in many references, e.g., Rosenberg et al., International application PCT/US92/05305; or Szostak et al., Meth. Enzymol, 68:419-429 (1979).
  • antisense compounds of the invention have lengths in the range of about 12 to 60 nucleotides. More preferably, antisense compounds of the invention have lengths in the r.ange of about 15 to 40 nucleotides; and most preferably, they have lengths in the range of about 18 to 30 nucleotides.
  • the antisense oligonucleotides used in the practice of the present invention will have a sequence which is completely complementary to a selected portion of the target polynucleotide. Absolute complementarity is not however required, particularly in larger oligomers. Thus, reference herein to a "nucleotide sequence complementary to" a target polynucleotide does not necessarily mean a sequence having 100% complementarity with the target segment. In general, any oligonucleotide having sufficient complementarity to form a stable duplex with the target (e.g. the mdrl mRNA) that is, an oligonucleotide which is "hybridizable", is suitable.
  • the target e.g. the mdrl mRNA
  • Stable duplex formation depends on the sequence and length of the hybridizing oligonucleotide and the degree of complementarity with the target polynucleotide. Generally, the larger the hybridizing oligomer, the more mismatches may be tolerated. More than one mismatch probably will not be tolerated for antisense oligomers of less than about 21 nucleotides. One skilled in the art may readily determine the degree of mismatching which may be tolerated between any given antisense oligomer and the target sequence, based upon the melting point, and therefore the thermal stability, of the resulting duplex.
  • the thermal stability of hybrids formed by the antisense oligonucleotides of the invention are determined by way of melting, or strand dissociation, curves.
  • the temperature of fifty percent strand dissociation is taken as the melting temperature, T m , which, in turn, provides a convenient measure of stability.
  • T m measurements are typically carried out in a saline solution at neutral pH with target and antisense oligonucleotide concentrations at between about 1.0-2.0 ⁇ M. Typical conditions are as follows: 150 mM NaCl and lOmM MgCl 2 in a 10 mM sodium phosphate buffer (pH 7.0) or in a lOmM Tris-HCl buffer (pH 7.0).
  • Data for melting curves are accumulated by heating a sample of the antisense oligonucleotide/target polynucleotide complex from room temperature to about 85-90°C. As the temperature of the sample increases, absorbance of 260 nm light is monitored at 1 °C intervals, e.g., using a Cary (Australia) model 1 E or a Hewlett-Packard (Palo Alto, CA) model HP 8459 UV/VIS spectrophotometer and model HP 89100A temperature controller, or like instruments. Such techniques provide a convenient means for measuring and comparing the binding strengths of antisense oligonucleotides of different lengths and compositions.
  • compositions of the invention include a pharmaceutical carrier that may contain a variety of components that provide a variety of functions, including regulation of drug concentration, regulation of solubility, chemical stabilization, regulation of viscosity, absorption enhancement, regulation of pH, and the like.
  • the pharmaceutical carrier may comprise a suitable liquid vehicle or excipient and an optional auxiliary additive or additives.
  • the liquid vehicles and excipients are conventional and commercially available. Illustrative thereof are distilled water, physiological saline, aqueous solutions of dextrose, and the like.
  • the pharmaceutical composition preferably includes a buffer such as a phosphate buffer, or other organic acid salt, preferably at a pH of between about 7 and 8.
  • a buffer such as a phosphate buffer, or other organic acid salt, preferably at a pH of between about 7 and 8.
  • micro-emulsions may be employed, for example by using a nonionic surfactant such as polysorbate 80 in an amount of
  • antioxidants such as ascorbic acid
  • hydrophilic polymers such as, monosaccharides, di saccharides, and other carbohydrates including cellulose or its derivatives, dextrins, chelating agents, such as EDTA, and like components well known to those in the pharmaceutical sciences, e.g., Remington's Pharmaceutical Science, latest edition (Mack Publishing Company, Easton, PA).
  • Antisense compounds of the invention include the pharmaceutically acceptable salts thereof, including those of alkaline earths, e.g., sodium or magnesium, ammonium or NX4+, wherein X is Cl -C4 alkyl.
  • Other pharmaceutically acceptable salts include organic carboxylic acids such as acetic, lactic, tartaric, malic, isethionic, lactobionic, and succinic acids; organic sulfonic acids such as methanesulfonic, ethanesulfonic, and benzenesulfonic; and inorganic acids such as hydrochloric, sulfuric, phosphoric, and sulfamic acids.
  • Pharmaceutically acceptable salts of a compound having a hydroxyl group include the anion of such compound in combination with a suitable cation such as Na+, NH4+, or the like.
  • the mdrl antisense oligonucleotides are preferably administered parenterally, most preferably intravenously.
  • the vehicle is designed accordingly.
  • oligonucleotide may be administered subcutaneously via controlled release dosage forms.
  • the antisense oligonucleotides may be administered by a variety of specialized oligonucleotide delivery techniques.
  • Sustained release systems suitable for use with the pharmaceutical compositions of the invention include semi-permeable polymer matrices in the form of films, microcapsules, or the like, comprising polylactides, copolymers of L-glutamic acid and gamma-ethyl-L-glutamate, poly(2-hydroxyethyl methacrylate), and like materials, e.g., Rosenberg et al., International application PCT US92/05305.
  • the oligonucleotides may be encapsulated in liposomes for therapeutic delivery, as described for example in Liposome Technology, Vol. II, Incorporation of Drugs, Proteins, and Genetic Material, CRC Press.
  • the oligonucleotide depending upon its solubility, may be present both in the aqueous layer and in the lipidic layer, or in what is generally termed a liposomic suspension.
  • the hydrophobic layer generally but not exclusively, comprises phospholipids such as lecithin .and sphingomyelin, steroids such as cholesterol, ionic surfactants such as diacetylphosphate, stearylamine, or phosphatidic acid, and/or other materials of a hydrophobic nature.
  • liposomes to introduce nucleotides into cells is described in U.S. Pat. Nos. 4,897,355 and 4,394,448, for example.
  • For general methods of preparing liposomes comprising biological materials see, for example, U.S. Pat. Nos. 4,235,871, 4,231,877, 4,224,179, 4,753,788, 4,673,657, 4,247,411 and 4,814,270.
  • the oligonucleotides may be conjugated to poly(L-lysine) to increase cell penetration.
  • Such conjugates are described by Lemaitre et al., Proc. Natl. Acad. Sci. USA, 84, 648-652 (1987).
  • the procedure requires that the 3'-terminal nucleotide be a ribonucleotide.
  • the resulting aldehyde groups are then randomly coupled to the epsilon-amino groups of lysine residues of poly(L-lysine) by Schiff base formation, and then reduced with sodium cyanoborohydride. This procedure converts the 3'-terminal ribose ring into a morpholine structure antisense oligomers.
  • Antisense compounds of the invention also include conjugates of such oligonucleotides with appropriate ligand-binding molecules.
  • the oligonucleotides may be conjugated for therapeutic administration to ligand-binding molecules which recognize cell-surface molecules, such as according to International Patent Application WO 91/04753.
  • the ligand-binding molecule may comprise, for example, an antibody against a cell surface antigen, an antibody against a cell surface receptor, a growth factor having a corresponding cell surface receptor, an antibody to such a growth factor, or an antibody which recognizes a complex of a growth factor and its receptor.
  • Methods for conjugating ligand-binding molecules to oligonucleotides are detailed in WO 91/04753.
  • the growth factor to which the antisense oligonucleotide may be conjugated may comprise transferrin or folate.
  • Transferrin-polylysine-oligonucleotide complexes or folate-polylysine-oligonucleotide complexes may be prepared for uptake by cells expressing high levels of transferrin or folate receptor.
  • the preparation of transferrin complexes as carriers of oligonucleotide uptake into cells is described by Wagner et al., Proc. Natl. Acad. Sci. USA 87, 3410-3414 (1990).
  • a preferred method of administration of oligonucleotide comprises either systemic or regional perfusion, as is appropriate.
  • the afferent and efferent vessels supplying the extremity containing the lesion are isolated and connected to a low-flow perfusion pump in continuity with an oxygenator and a heat exchanger.
  • the iliac vessels may be used for perfusion of the lower extremity.
  • the axillary vessels are cannulated high in the axilla for upper extremity lesions.
  • Oligonucleotide is added to the perfusion circuit, and the perfusion is continued for an appropriate time period, e.g., one hour.
  • Perfusion rates of from 100 to 150 ml/minute may be employed for lower extremity lesions, while half that rate should be employed for upper extremity lesions.
  • Systemic heparinization may be used throughout the perfusion, and reversed after the perfusion is complete. This isolation perfusion technique permits administration of higher doses of chemotherapeutic agent than would otherwise be tolerated upon infusion into the arterial or venous systemic circulation.
  • the oligonucleotides are preferably delivered via a central venous catheter, which is connected to an appropriate continuous infusion device.
  • Indwelling catheters provide long term access to the intravenous circulation for frequent administration of drugs over extended time periods. They are generally surgically inserted into the external cephalic or internal jugular vein under general or local anesthesia.
  • the subclavian vein is another common site of catheterization.
  • the infuser pump may be external, or may form part of an entirely implantable central venous system such as the INFUSAPORT system available from Infusaid Corp., Norwood, MA and the PORT-A-CATH system available from Pharmacia Laboratories, Piscataway, NJ. These devices are implanted into a subcutaneous pocket under local anesthesia.
  • a catheter, connected to the pump injection port, is threaded through the subclavian vein to the superior vena cava.
  • the implant contains a supply of oligonucleotide in a reservoir which may be replenished as needed by injection of additional drug from a hypodermic needle through a self-sealing diaphragm in the reservoir.
  • Completely implantable infusers are preferred, as they are generally well accepted by patients because of the convenience, ease of maintenance and cosmetic advantage of such devices.
  • antisense oligonucleotide synthesis may be induced in situ by local treatment of the targeted cells with a vector containing an artificially-constructed gene comprising a transcriptional promotor and mdrl DNA in inverted orientation.
  • the mdrl DNA for insertion into the artificial gene in inverted orientation comprises cDNA which may be prepared, for example, by reverse transcriptase polymerase chain reaction from RNA using primers derived from the published mdrl cDNA sequence.
  • the inverted mdrl gene segment which is complementary to the mdrl mRNA, is produced in situ in the targeted cell.
  • the endogenously produced RNA hybridizes to mdrl mRNA, resulting in interference with mdrl function and inhibition of the proliferation of the targeted cell.
  • the promotor segment of the artificially-constructed gene serves as a signal conferring expression of the inverted mdrl sequence which lies downstream thereof. It will include all of the signals necessary for initiating transcription of the sequence.
  • the promotor may be of any origin as long as it specifies a rate of transcription which will produce sufficient antisense mRNA to inhibit the expression of the mdrl gene, and therefore the proliferation of the targeted cells.
  • a highly efficient promotor such as a viral promotor is employed.
  • Other sources of potent promoters include cellular genes that are expressed at high levels.
  • the promotor segment may comprise a constitutive or a regulatable promotor.
  • the artificial gene may be introduced by any of the methods described in U.S. Patent 4,740,463, incorporated herein by reference.
  • One technique is transfection, which can be done by several different methods.
  • One method of transfection involves the addition of DEAE-dextran to increase the uptake of the naked DNA molecules by a recipient cell. See McCutchin, J.H. and Pagano, J.S., J. Natl. Cancer Inst. 41, 351-7 (1968).
  • Another method of transfection is the calcium phosphate precipitation technique which depends upon the addition of Ca ++ to a phosphate-containing DNA solution. The resulting precipitate apparently includes DNA in association with calcium phosphate crystals.
  • Transfection may also be carried out by cationic phospholipid-mediated delivery.
  • polycationic liposomes can be formed from N-[l-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA).
  • DOTMA N-[l-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride
  • the artificially-constructed gene can be introduced in to cells, in vitro or in vivo, via a transducing viral vector.
  • a transducing viral vector See Tabin et al., Mol. Cel. Biol. 2, 426-436 (1982).
  • Use of a retrovirus will infect a variety of cells and cause the artificial gene to be inserted into the genome of infected cells.
  • Such infection could either be accomplished with the aid of a helper retrovirus, which would allow the virus to spread through the organism, or the antisense retrovirus could be produced in a helper-free system, such as ⁇ 2-like cells (See Mann et al., Cell 33, 153-160, 1983) that package amphotropic viruses.
  • a helper-free virus might be employed to minimize spread throughout the organism.
  • Viral vectors in addition to retroviruses can also be employed, such as papovaviruses, SV40-like viruses, or papilloma viruses.
  • retroviruses for gene transfer has been reviewed by Eglitis and .Anderson, BioTechniques 6, 608-614 (1988).
  • Vesicle fusion could also be employed to deliver the artificial gene.
  • Vesicle fusion may be physically targeted to the malignant cells if the vesicle were approximately designed to be taken up by those cells.
  • Such a delivery system would be expected to have a lower efficiency of integration and expression of the artificial gene delivered, but would have a higher specificity than a retroviral vector.
  • a combination strategy of targeted vesicles containing papilloma virus or retrovirus DNA molecules might provide a method for increasing the efficiency of expression of targeted molecules.
  • Particulate systems and polymers for in vitro and in vivo delivery of polynucleotides were extensively reviewed by Feigner in Advanced Drug Delivery Reviews 5, 163-187 (1990). Techniques for direct delivery of purified genes in vivo, without the use of retroviruses, has been reviewed by Feigner in Nature 349, 351-352 (1991). Such methods of direct delivery of polynucleotides may be utilized for local delivery of either exogenous mdrl antisense oligonucleotide or artificially-constructed genes producing mdrl antisense oligonucleotide in situ. Recently, Wolf et al. demonstrated that direct injection of non-replicating gene sequences in a non-viral vehicle is possible.
  • DNA injected directly into mouse muscle did not integrate into the host genome, and plasmid essentially identical to the starting material was recovered from the muscle months after injection. Interestingly, no special delivery system is required. Simple saline or sucrose solutions are sufficient to delivery DNA and RNA.
  • the amount of antisense oligonucleotide may vary depending on the nature and extent of the neoplasm, the particular oligonucleotide utilized, and other factors.
  • the actual dosage administered may take into account the size and weight of the patient, whether the nature of the treatment is therapeutic in nature, the age, health and sex of the patient, the route of administration, whether the treatment is regional or systemic, and other factors.
  • Intercellular concentrations of from about 1 to about 200 ⁇ g/ml at the target polynucleotide may be employed, preferably from about 10 ⁇ g/ml to about 100 ⁇ g/ml.
  • the patient should receive a sufficient daily dosage of antisense oligonucleotide to achieve these intercellular concentrations of drug.
  • the daily dosage may range from about 0.2 mg, more preferably from about 25 mg, to about 2 grams per day, with at least about 250 mg per day being preferred.
  • An effective human continuous intravenous infusion dosage based upon animal studies and Phase I clinical trials employing antisense oligonucleotides targeting other genes in antileukemic therapy, is about 250 mg/day. Greater or lesser amounts of oligonucleotide may be administered, as required.
  • Those skilled in the art should be readily able to derive appropriate dosages and schedules of administration to suit the specific circumstance and needs of the patient.
  • a course of treatment may advantageously comprise infusion of the recommended daily dose as a continuous intravenous infusion over 7 days.
  • the oligonucleotide may be given for a period of from about 3 to about 28 days, more preferably from about 7 to about 10 days.
  • the treatment regimen may comprise dosing on alternate days.
  • the effectiveness of the treatment may be assessed by routine methods which are used for determining whether or not remission has occurred. Such methods generally depend upon some combination of morphological, cytochemical, cytogenetic, immunologic and molecular analyses. In addition, remission can be assessed genetically by probing the level of expression of one or more relevant oncogenes.
  • the reverse transcriptase polymerase chain reaction methodology can be used to detect even very low numbers of mRNA transcript. For example, RT-PCR has been used to detect and genotype the three known bcr-abl fusion sequences in Phi leukemias. See PCT US92/05035 and Kawasaki et al., Proc. Natl. Acad. Sci.
  • the present invention may be used ex vivo to reverse MDR in cells removed form the patient which are to be reintroduced.
  • bone marrow may be removed from the patient, cultured in the presence of antisense oligonucleotides of the invention and then exposed to convential cytostatic compositions prior to return into the patient.
  • the ex vivo culturing of the bone marrow according to the invention will reverse MDR of cancer cells present in the cultured bone marrow.
  • the HL-60 Vinc human promyelocitic cell line isolated from parental HL- 60 cells for resistance to vincristine (Krishnamachary, N. et al., The MRP gene associated with non-P-glycoprotein Multidrug Resistance encodes a 190-KDa membrane bound glycoprotein, Cancer Res. 53:3658-3661, 1993, which is incorporated herein by reference) was kindly provided by Dr. Melvin Center. Cells were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS; Sigma Chemical Co., St. Louis, MO), 102 ⁇ g/ml L-glutamine, in a humid, 5% CO 2 incubator at 37°C.
  • FBS heat-inactivated fetal bovine serum
  • HL-60 Vinc cells were not maintained in the presence of vincristine because mdrl is constitutively overexpressed as confirmed by routinely checking mdrl mRNA and P-170 glycoprotein levels.
  • fresh aliquots of HL-60/ Vine cells and HL-60 parental cells were defrosted from liquid nitrogen stocks every 2 months during the study, and analyzed for mdrl expression and drug-response before use in each experiment.
  • Drug and oligodeoxynucleotides ODNs
  • VINC Vincristine
  • HBSS Hank's Balanced Salt solution
  • GIBCO BRL Grand Island, NY
  • SJODNs Phosphorothioate oligodeoxynucleotides
  • Applied Biosystems Foster City, CA
  • DNA automated synthesizer model 380B The sequences of the mdrl antisense and sense [SJODNs are 5'-GTCCCCTTCAAGATCCAT-3' (SEQ ID NO:l) and 5 ⁇ TGGATCTTGAAGGGGAC-3' (SEQ ID NO:2), respectively. They are complementary (or corresponding) to codons 1-6 of the published human mdrl cDNA sequence (Chen, C.J.
  • Control cells were grown in the same conditions without [SJODNs and drug. At the end of the combination treatment, quadruplicate samples from each group were harvested, counted and assayed for viability (trypan blue dye exclusion). Colony assay 2x10" HL-60/Vinc cells, seeded in 24-well plates, were treated with mdrl
  • RNA extraction and reverse transcription-polymerase chain reaction (RT-PCR) analysis Total RNA was extracted from cells by the acid quanidinium thiocyanate- phenol-chloroform technique (Chomczynski, P. et al., Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction, Anal. Biochem., 162:156-159, 1987, which is incorporated herein by reference), mdrl and ⁇ -actin mRNA transcripts were detected by RT-PCR, mdrl expression was detected with 3' and 5' primers corresponding to nucleotides 535-556 and 361-381, respectively, of the published cDNA sequence.
  • PCR yields a 196-base-pair (bp) product (Noonan, K.E. et al., Quantitative analysis of MDR1 (multidrug resistance) gene expression in human tumors by polymerase chain reaction., Proc. Natl. Acad. Sci. USA, 87:7160-7164, 1990 which is incorporated herein by reference).
  • RNA from each sample was divided into two aliquots (0.5 ⁇ g each) that were separately reverse-transcribed using 400 U of Moloney murine leukemia virus reverse transcriptase (Mo-MLV-RT, BRL, Gaithersburg, MD) and 100 ng or mdrl or ⁇ - actin 3' primer for 1 hour at 37°C.
  • Mo-MLV-RT Moloney murine leukemia virus reverse transcriptase
  • PCR reaction The resulting cDNA fragments were amplified with 5' U or Taq polymerase (Boehringer Mannheim) and 200 ng of mdrl or ⁇ -actin and 5' primers in a GencAmp PCR System 9600 (Perkin-Elmer, Norwalk, CT) for 35 cycles or sequential denaturation (at 94 °C for 45 seconds), annealing (at 55%C for 45 seconds), and extension (at 72 °C for 30 seconds). After the last cycle, all PCR products were subjected to a final extension of 10 minutes at 72 °C.
  • SCID mice severe combined immunodeficient mice were purchased from Taconic Farms (Germantown, NY) and maintained under sterile conditions. SCID mice are excellent hosts for human hematopoietic cells, because defective T- and B-lymphocyte development makes these animals severely immunocompromised. Nevertheless, the presence of residual immune cells usually requires that such animals be irradiated prior to engrafting of human hematopoietic tissue.
  • Male SCID mice (6-8 weeks old) were irradiated (200 cGy total body irradiation), and the following day (day 1) injected intravenously (i.v.) with 5 x 10 ? HL-60 Vinc cells in 0.2 mi of RPMI 1640 medium.
  • mdrl AS [SJODNs] The ability of mdrl AS [SJODNs to increase vincristine cytotoxicity in HL- 60 Vinc resistant cells and thus to reverse multidrug resistance was assessed in combination experiments in which HL-60 Vinc cells were first exposed to mdrl AS [SJODNs and then to VINC (see the Materials and Methods section for details) (Fig. 2A). Treatment with vincristine alone had modest effects on HL-60/Vinc cell proliferation; only at the highest dose (1 ⁇ g/ml) the inhibition of cell proliferation, expressed as percentage of the untreated control, reach 48%.
  • IC 50 values were calculated from the percent growth inhibition caused by the different treatments on HL-60/Vinc cells.
  • HL-60 parental cells and HL-60 Vinc cells showed a marked difference in the IC 50 value following exposure to VINC (0.008 and 0.95 ⁇ g/ml, respectively), with a resistance index of about 120.
  • Treatment of HL-60 Vinc cells with mdrl S or SCR [SJODNs followed by vincristine did not significantly change the IC 50 and .85 ⁇ g/ml, respectively.
  • the IC 50 value of the resistant cells exposed to AS+VINC was essentially identical to that of HL-60 parental cells exposed to the drug alone, consistent with a complete restoration of the in vitro sensitivity to vincristine in HL-60/Vinc cells.
  • the HL-60 Vine human leukemia cell line selected in vitro for resistance to vincristine exhibits cross-resistance to other MDR related drugs commonly used as antineoplastic agents.
  • Figure 2B reports the survival curves from methycellulose colony assays performed on HL-60/Vinc cells treated with mdrl [SJODNs and vincristine (see Materials and Methods section).
  • SJODNs and vincristine see Materials and Methods section.
  • the combinations S+VINC and SCR+VINC did not cause any increase in the lethal effect or VINC since the survival curves of cells treated with VINC alone, or S+VINC or SCR+VINC are superimposable and the surviving fractions have similar values (about 90% at 0.01 ⁇ g/ml and about 50% at 1 ⁇ g/ml).
  • gP-170 protein levels there was no difference in gP-170 protein levels in control, S-or SCR-treated cells (lanes 1, 2 and 3, respectively).
  • a decrease in gP-170 protein level was detected after exposure of HL-60 Vinc cells to mdrl AS [SJODNs (lane 4) at the same dose used to assess the effects on cell proliferation (Fig. 1 and 2).
  • the levels of HSP 72/73 protein and MYB protein did not diminish in AS [SJODNs-treated cells, suggesting that the down-regulation of P- 170 glycoprotein expression was a specific effect of the marl AS [SJODNs treatment.
  • Figures 4A and 4B reports the survival curves of HL-60 Vine SCID mice treated with [SJODNs and vincristine, individually or in combination.
  • the curves relative to the mice in the SCR- treated groups were separated from those of the mice of the AS-treated groups (Fig. 4B).
  • the survival curves of the untreated and the vincristine-treated mice are the same in each panel.
  • the administration of vincristine alone did not prolong the survival of HL-60/Vinc SCID mice, consistent with the in vitro resistance of the leukemic cells to the drug.
  • mice were all dead 84 days after implant of HL-60 Vinc cells, while the mice in the vincristine-treated group were all dead 91 days after leukemia inoculation.
  • the median survival times were 56 and 70 days for the control and the vincristine group, respectively, such difference was not statistically significant).
  • SCR [SJODNs alone or SCR [SJODNs plus vincristine did not affect SCID mice survival, both curves being very similar to that of the control mice (untreated and vincristine-treated).
  • mice in the SCR-treated group were all dead within 85 days, and those in the SCR plus vincristine- treated group within 92 days from leukemic cells inoculation, with a median survival time of 71 and 77 days, respectively.
  • the treatment with mdrl AS [SJODNs was not able by itself to affect SCID mice survival (Fig. 4B).
  • the mice were all dead 100 days after leukemic cells injection, with a median survival time of 57 days (the median survival time of untreated mice was 56 days).
  • the statistical analysis of the control versus the AS curve gave a non-significant P value (0.45) indicating that the observed delay in the death of the antisense-treated mice compared with controls (about 20 days) was only apparent.
  • MDR multidrug resistance
  • the present invention modulated and reversed multidrug resistance in a human leukemia resistant cell line, both in vitro and in vivo, using AS [SJONDs targeted to the mdrl mRNA.
  • HL-60 Vinc cells that were selected from HL-60 parental cells for resistance to vincristine overexpressed the p-170KDa mdrl gene product and exhibited cross-referenced to other antineoplastic drugs related to the MDR phenotype, most likely as consequence or increased transport or anticancer agents out of cells and/or decreased intracellular accumulation.

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Abstract

L'invention concerne des oligonucléotides antisens spécifiques contre le gène 1 de résistance multiple aux anticancéreux, utilisés pour inverser la résistance multiple aux anticancéreux dans des cellules cancéreuses in vivo. Les oligonucléotides sont administrés en tant que partie d'un schéma de traitement comprenant une chimiothérapie anticancéreuse.
PCT/US1997/017320 1996-09-24 1997-09-24 Compositions et procedes destines au traitement de la resistance multiple aux anticancereux WO1998013072A1 (fr)

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WO2000035422A2 (fr) * 1998-12-18 2000-06-22 Hadasit Medical Research Services & Development Ltd. Procede d'administration d'un compose a des cellules resistant a plusieurs medicaments
WO2001023565A1 (fr) * 1999-09-28 2001-04-05 Gentest Corporation P-glycoproteines provenant de macaca fascicularis et utilisations correspondantes
US6790621B2 (en) 2001-01-12 2004-09-14 Washington State University Research Foundation Method of detecting ivermectin sensitivity in a canine subject by identifying a mutation in a mdr1-encoding sequence
US6855812B2 (en) 2001-03-19 2005-02-15 Becton, Dickinson And Company P-glycoproteins and uses thereof
WO2018013993A1 (fr) * 2016-07-14 2018-01-18 University Of Southern California Procédés et composition pour produire et utiliser des cellules immunitaires et des cellules souches pour des thérapies à base de cellules

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WO1996002556A2 (fr) * 1994-07-18 1996-02-01 Hybridon, Inc. Oligonucleotide a activite anti-gene mdr-1

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 15 February 1993, Vol. 190, No. 3, THIERRY et al., "Overcoming Multiple Drug Resistance in Human Tumor Cells Using Free and Liposomally Encapsulated Antisense Oligonucleotides", pages 952-960. *
CLINICAL SCIENCE, 1996, Vol. 91, LIU et al., "Modulation of Multidrug Resistance Gene (mdr-I) with Antisense Oligonucleotides", pages 93-98. *
JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS, 1996, Vol. 276, No. 3, NAKAI et al., "Cellular Uptake Mechanism for Oligonucleotides: Involvement of Endocytosis in the Uptake of Phosphodiester Oligonucleotides by a Human Colorectal Adenocarcinoma Cell Line, HCT-15", pages 1362-1372. *
PROCEEDINGS OF THE AMERICAN ASSOCIATION FOR CANCER RESEARCH, March 1995, Volume 36, RAMACHANDRAN et al., "Reversal of Multidrug Resistance by MDR-1 Antisense Phosphorothioate Oligodeoxy Nucleotides in SW620 Ad300 Human Colon Carcinoma Cells In Vitro in Xenografts", page 412, Abstract 2456. *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000035422A2 (fr) * 1998-12-18 2000-06-22 Hadasit Medical Research Services & Development Ltd. Procede d'administration d'un compose a des cellules resistant a plusieurs medicaments
WO2000035422A3 (fr) * 1998-12-18 2000-10-12 Hadasit Med Res Service Procede d'administration d'un compose a des cellules resistant a plusieurs medicaments
WO2001023565A1 (fr) * 1999-09-28 2001-04-05 Gentest Corporation P-glycoproteines provenant de macaca fascicularis et utilisations correspondantes
US6617450B1 (en) 1999-09-28 2003-09-09 Becton Dickinson And Company P-glycoproteins and uses thereof
US6790621B2 (en) 2001-01-12 2004-09-14 Washington State University Research Foundation Method of detecting ivermectin sensitivity in a canine subject by identifying a mutation in a mdr1-encoding sequence
US7393643B2 (en) 2001-01-12 2008-07-01 Washington State University Research Foundation Method of detecting ivermectin sensitivity in a canine subject by identifying a mutation in a MDR1-encoding sequence
US7776588B2 (en) 2001-01-12 2010-08-17 Washington State University Research Foundation MDR1 variants and methods for their use
US6855812B2 (en) 2001-03-19 2005-02-15 Becton, Dickinson And Company P-glycoproteins and uses thereof
WO2018013993A1 (fr) * 2016-07-14 2018-01-18 University Of Southern California Procédés et composition pour produire et utiliser des cellules immunitaires et des cellules souches pour des thérapies à base de cellules
US11382931B2 (en) 2016-07-14 2022-07-12 University Of Southern California Methods and composition for producing and using immune cells and stem cells for cell-based therapies

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