EP0856052A1 - Chimiotherapie par les oligonucleotides antisens de l'hyperplasie ou du cancer de la prostate - Google Patents

Chimiotherapie par les oligonucleotides antisens de l'hyperplasie ou du cancer de la prostate

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Publication number
EP0856052A1
EP0856052A1 EP96931664A EP96931664A EP0856052A1 EP 0856052 A1 EP0856052 A1 EP 0856052A1 EP 96931664 A EP96931664 A EP 96931664A EP 96931664 A EP96931664 A EP 96931664A EP 0856052 A1 EP0856052 A1 EP 0856052A1
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Prior art keywords
oligonucleotides
oligonucleotide
seq
group
ofthe
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Paul A. Zamecnik
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University of Massachusetts UMass
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University of Massachusetts UMass
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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
    • C12N15/1137—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 enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y113/00—Oxidoreductases acting on single donors with incorporation of molecular oxygen (oxygenases) (1.13)
    • C12Y113/11—Oxidoreductases acting on single donors with incorporation of molecular oxygen (oxygenases) (1.13) with incorporation of two atoms of oxygen (1.13.11)
    • C12Y113/11012—Linoleate 13S-lipoxygenase (1.13.11.12)
    • 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
    • C12N2310/315—Phosphorothioates
    • 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
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • 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
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed

Definitions

  • the present invention relates to the field of chemotherapy for hyperplasias and cancers and, in particular, to chemotherapy for benign hyperplasia or cancer ofthe prostate.
  • the invention relates to the field of antisense oligonucleotides and their use in human hyperplasia and cancer therapy.
  • BPH benign prostatic hyperplasia
  • This condition may be a precursor to full blown prostatic cancer or may continue for decades without evolving into the deadly carcinoma.
  • treatment may range from "watchful waiting” to more aggressive approaches employing anti-androgen hormonal therapy, transurethral resection, or radical prostatectomy (see, e.g., Catalona (1994)).
  • Prostatic specific antigen was first described by Wang et al. (1979) as a specific marker for prostate tissue and was subsequently identified by Papsidero et al. (1980) as being present in the sera of prostate cancer patients. Since then, PSA in the sera has become the most prevalent diagnostic marker for cancer ofthe prostate (see, e.g., Gittes (1991); Catalona (1994); Oesterling (1995); and Pienta (1995)). Levels of serum PSA are also elevated in BPH but less so than in progressed prostatic cancer.
  • the complete amino acid sequence of PSA was disclosed by Watt et al. (1986) and the complete gene encoding PSA was disclosed by Lundwall (1989) and Klobeck et al. (1989).
  • PSA is a glycoprotein having a single polypeptide chain and a molecular mass of approximately 34kDa.
  • PSA is produced exclusively by epithelial cells ofthe prostate and is localized to the rough endoplasmic reticulum and associated vesicles until it is secreted into the acini and ducts ofthe prostate (see, e.g., Sinha, et al. (1987)).
  • PSA functions as a neutral serine protease which serves to liquefy the seminal coagulum by degrading seminal vesicle proteins including fibronectin and semenogelin (see, e.g., Lilja (1985); Warhol and Logtine (1985)).
  • Probasin is a prostate specific basic protein first isolated by Matuo et al. (1982) from rat dorsolateral prostate. A cDNA to the rat probasin was disclosed by Spence et al. (1989) and revealed two in-frame translation initiation codons which are believed to account for the secreted and nuclear forms ofthe protein.
  • probasin is a heparin binding protein that co-purifies with heparin binding growth factor- 1 (HBGF-1) and is positively regulated by androgen.
  • HBGF-1 heparin binding growth factor- 1
  • Probasin appears to have minor mitogenic activity (0.2-1% of HBGF-1) but this may be an artifact of its co-purification with HBGF-1 (Matuo et al. (1989)).
  • the present invention provides methods for treating a patient diagnosed as having benign prostatic hyperplasia or a prostatic cancer.
  • the methods include administering to the patient a therapeutically effective amount of a composition comprising an antisense oligonucleotide which selectively hybridizes to a PSA or probasin gene or mRNA sequence ofthe patient, thereby inhibiting the expression ofthe PSA or probasin gene or mRNA sequence.
  • This inhibition of the PSA or probasin genes or mRNAs by antisense oligonucleotides results in a significant inhibition ofthe growth or survival of prostatic cells.
  • the methods provide a useful new means of treating benign prostatic hyperplasia and prostatic cancer.
  • the PSA antisense oligonucleotides may comprise at least 10 consecutive bases from SEQ ID NO.: 1, at least 10 consecutive bases from the joined exons of SEQ ID NO.: 1; or oligonucleotides that hybridize to the complements of these sequences under physiological conditions. More preferably, the antisense oligonucleotides comprise at least 15 consecutive bases, and most preferably, 20-30 consecutive bases from the above-described sequences.
  • the probasin antisense oligonucleotides may comprise at least 10 consecutive bases from SEQ ID NO.: 2, at least 10 consecutive bases from a genomic sequence corresponding to SEQ ID NO.: 2, or oligonucleotides that hybridize to the complements of these sequences under physiological conditions. More preferably, the antisense oligonucleotides comprise at least 15 consecutive bases, and most preferably, 20-30 consecutive bases from the above-described sequences.
  • sequences ofthe invention include, but are not limited to, those disclosed as SEQ ID NO.: 3, SEQ ID NO.: 4, SEQ ID NO.: 5, SEQ ID NO.: 6, SEQ ID NO.: 7, and SEQ ID NO.: 8.
  • all ofthe above-described oligonucleotides are modified oligonucleotides.
  • the modified oligonucleotide includes at least one synthetic internucleoside linkage such as a phosphorothioate, alkylphosphonate, phosphorodithioate, phosphate ester, alkylphosphonothioate, phosphoramidate, carbamate, carbonate, phosphate triester, acetamidate, or carboxymethyl ester.
  • the modified oligonucleotide has at least one low molecular weight organic group covalently bound to a phosphate group of said oligonucleotide.
  • the modified oligonucleotide has at least one low molecular weight organic group covalently bound to a 2' position of a ribose of said oligonucleotide.
  • low molecular weight organic groups include lower alkyl chains or aliphatic groups (e.g., methyl, ethyl, propyl, butyl), substituted alkyl and aliphatic groups (e.g., aminoethyl, aminopropyl, aminohydroxyethyl, aminohydroxypropyl), small saccharides or glycosyl groups.
  • the modified oligonucleotide has covalently attached thereto a prostate-targeting compound such as an androgen, androgen derivative, estrogen, estrogen derivative, estramustine, emcyt or estracyt.
  • a prostate-targeting compound such as an androgen, androgen derivative, estrogen, estrogen derivative, estramustine, emcyt or estracyt.
  • the antisense oligonucleotides are administered intravenously at a dosage between 1.0 ⁇ g and 100 mg per kg body weight ofthe patient.
  • the present invention also provides for any or all ofthe above-described antisense oligonucleotides, including the various modified oligonucleotides, in a pharmaceutical composition.
  • the antisense oligonucleotides are admixed with a sterile pharmaceutically acceptable carrier in a therapeutically effective amount such that the isolated antisense oligonucleotide selectively hybridizes to the PSA or probasin gene or mFtNA sequence when administered to a patient.
  • a pharmaceutical kit is also provided in which such a pharmaceutical composition is combined with a pharmaceutically acceptable carrier for intravenous administration.
  • the present invention provides new methods for the treatment of cancer ofthe prostate and pharmaceutical compositions useful therefor. It is now disclosed that antisense oligonucleotides complementary to genes which are expressed only or predominantly in prostatic cells are effective for inhibiting the growth of and/or killing hype ⁇ lastic or cancerous cells of prostatic origin.
  • the present invention provides oligonucleotides, including modified oligonucleotides, which have antisense homology to a sufficient portion of either the PSA or probasin gene such that they inhibit the expression of that gene. The expression of both of these genes is believed to be tissue specific to the prostate.
  • the antisense oligonucleotides ofthe invention can be administered systemically but selectively inhibit prostate cells, the present invention has particular utility in late stage prostate cancer which has metastasized.
  • PSA refers to the prostatic specific antigen well known in the art and described in the various references cited herein.
  • Genomic DNA sequences ofthe human PSA gene were disclosed in Lundwall (1989) and Klobeck et al. (1989).
  • the Klobeck et al. (1989) sequence is available on GenBank (Accession number X14810) and is reproduced here as SEQ. ID NO.: 1.
  • GenBank accesion number X14810
  • SEQ. ID NO.: 1 The translation initiation codon of this gene is found at base positions 401-403 and the stop codon is at positions 5566-5568 of SEQ ID NO.: 1.
  • the gene consists of five exons which are indicated on SEQ. ID NO.: 1.
  • a TATA signal is found at positions 332-338 and a transcriptional start region appears at positions 355-365.
  • PSA gene As will be obvious to one of ordinary skill in the art, other alleles ofthe PSA gene, including other human alleles and homologues from other mammalian species, encoding a PSA protein and hybridizing to SEQ ID NO.: 1 under stringent hybridization conditions, will exist in natural populations and are embraced by the term "PSA gene" as used herein.
  • a slightly different sequence for PSA is also available on GenBank (Acession number M27274). The PSA gene ofthe invention is intended to encompass all such sequences.
  • Probasin refers to the probasin protein known in the art and described in the various references cited herein.
  • a cDNA to one allele ofthe rat probasin gene was disclosed in Spence, et al. (1989).
  • the Spence et al. (1989) sequence is available on GenBank (Accession number M27156) and is reproduced here as SEQ. ID NO.: 2.
  • This gene has two potential translation initiation start codons which are in frame with each other. The first is at positions 41-43 of SEQ ID NO. 2 and the second is at positions 92-94. The stop codon is at positions 572-574.
  • the bases between the first and second initiation codons encode a hydrophobic sequence consistent with a secretory signal sequence.
  • the initiation of translation from the first start codon leads to production ofthe secreted form of probasin whereas translation from the second results in the nuclear form ofthe protein.
  • the term "probasin gene" is specifically intended to include a gene encoding either or both forms ofthe probasin protein.
  • probasin gene including other human alleles and homologues from other mammalian species, encoding a probasin protein and hybridizing to SEQ ID NO.: 2 under stringent hybridization conditions, will exist in natural populations and are embraced by the term "probasin gene" as used herein.
  • antisense oligonucleotide or “antisense” describes an oligonucleotide that is an oligoribonucleotide, oligodeoxyribonucleotide, modified oligoribonucleotide, or modified oligodeoxyribonucleotide which hybridizes under physiological conditions to DNA comprising a particular gene or to an mRNA transcript of that gene and, thereby, inhibits the transcription of that gene and/or the translation of that mRNA.
  • PSA-antisense oligonucleotide and by a “probasin-antisense oligonucleotide” are meant oligonucleotides which hybridize under physiological conditions to the PSA gene/mRNA or probasin gene/mRN A and, thereby, inhibit transcription/translation ofthe PSA and probasin genes/mRNAs, respectively.
  • the antisense molecules are designed so as to interfere with transcription or translation of PSA or probasin upon hybridization with the target.
  • the exact length of the antisense oligonucleotide and its degree of complementarity will depend upon the specific target selected, including the sequence ofthe target and the particular bases which comprise that sequence.
  • the antisense oligonucleotide be selected so as to hybridize selectively with the target under physiological conditions, i.e., to hybridize substantially more to the target sequence than to any other sequence in the target cell under physiological conditions.
  • Stringent hybridization conditions means hybridization conditions from 30°C-60°C and from 5x to 0.1 x SSC. Highly stringent hybridization conditions are at 45 °C and O.lx SSC. "Stringent hybridization conditions" is a term of art understood by those of ordinary skill in the art.
  • stringent hybridization conditions are those conditions of temperature and buffer solution which will permit hybridization of that nucleic acid sequence to its complementary sequence and not to substantially different sequences.
  • the exact conditions which constitute "stringent” conditions depend upon the length ofthe nucleic acid sequence and the frequency of occurrence of subsets of that sequence within other non-identical sequences.
  • antisense oligonucleotides Based upon SEQ ID NO.: 1 and SEQ ID NO.: 2, or upon allelic or homologous genomic or cDNA sequences, one of skill in the art can easily choose and synthesize any of a number of appropriate antisense molecules for use in accordance with the present invention.
  • antisense oligonucleotides should comprise at least 10 and, more preferably, at least 15 consecutive bases which are complementary to the PSA or probasin mRNA transcripts. Most preferably, the antisense oligonucleotides comprise a complementary sequence of 20-30 bases.
  • oligonucleotides may be chosen which are antisense to any region ofthe PSA or probasin genes or mRNA transcripts, in preferred embodiments the antisense oligonucleotides correspond to N- terminal or 5' upstream sites such as translation initiation, transcription initiation or promoter sites. In addition, 3'-untranslated regions or telomerase sites may be targeted. Targeting to mRNA splicing sites has also been used in the art but may be less preferred if alternative mRNA splicing occurs.
  • the PSA or probasin antisense is, preferably, targeted to sites in which mRNA secondary structure is not expected (see, e.g., Sainio et al.
  • SEQ ID NO.: 1 discloses a genomic DNA sequence
  • SEQ ID NO.: 2 discloses a cDNA sequence
  • one of ordinary skill in the art may easily derive the cDNA corresponding to the joined exons of SEQ ID NO.: 1 and may easily obtain the genomic DNA sequence corresponding to SEQ ID NO.: 2.
  • the present invention also provides for antisense oligonucleotides which are complementary to the cDNA corresponding to SEQ ID NO.: 1 and the genomic DNA corresponding to SEQ ID NO.: 2.
  • antisense to allelic or homologous cDNAs and genomic DNAs are enabled without undue experimentation.
  • the antisense oligonucleotides of the present invention need not be perfectly complementary to the PSA or probasin genes or mRNA transcripts in order to be effective. Rather, some degree of mismatches will be acceptable if the antisense oligonucleotide is of sufficient length.
  • the oligonucleotides should have sufficient length and complementarity so as to hybridize to a PSA or probasin transcript under physiological conditions. Preferably, of course, mismatches are absent or minimal.
  • the antisense oligonucleotides may have one or more non-complementary sequences of bases inserted into an otherwise complementary antisense oligonucleotide sequence. Such non-complementary sequences may "loop" out of a duplex formed by a PSA or probasin transcript and the bases flanking the non- complementary region. Therefore, the entire oligonucleotide may retain an inhibitory effect despite an apparently low percentage of complementarity.
  • oligonucleotides having a sequence at the 5' and/or 3' end which is capable of folding over and forming a duplex with itself.
  • the duplex region which is preferably at least 4-6 bases joined by a loop of 3-6 bases, stabilizes the oligonucleotide against degradation.
  • oligonucleotides are easily designed by adding the inverted complement of a 5' or 3' PSA or probasin sequence to the end ofthe oligonucleotide (see, e.g., Table 1, SEQ ID NO.: 5; Tang, J.-Y., et al. (1993) Nucleic Acids Res. 21 :2729-2735).
  • the PSA and probasin antisense oligonucleotides ofthe invention may be composed of "natural" deoxyribonucleotides, ribonucleotides, or any combination thereof.
  • oligonucleotides may be prepared by art recognized methods which may be carried out manually or by an automated synthesizer.
  • the antisense oligonucleotides ofthe invention also may include "modified" oligonucleotides. That is, the oligonucleotides may be modified in a number of ways which do not prevent them from hybridizing to their target but which enhance their stability or targeting to prostatic cells or which otherwise enhance their therapeutic effectiveness.
  • modified oligonucleotide as used herein describes an oligonucleotide in which (1) at least two of its nucleotides are covalently linked via a synthetic internucleoside linkage (i.e., a linkage other than a phosphodiester linkage between the 5' end of one nucleotide and the 3' end of another nucleotide) and/or (2) a chemical group not normally associated with nucleic acids has been covalently attached to the oligonucleotide.
  • a synthetic internucleoside linkage i.e., a linkage other than a phosphodiester linkage between the 5' end of one nucleotide and the 3' end of another nucleotide
  • Preferred synthetic internucleoside linkages are phosphorothioates, alkylphosphonates, phosphorodithioates, phosphate esters, alkylphosphonothioates, phosphoramidates, carbamates, carbonates, phosphate triesters, acetamidate, and carboxymethyl esters. Further, one or more of the 5'-3' phosphate group may be covalently joined to a low molecular weight (e.g., 15-500 Da) organic group.
  • a low molecular weight e.g. 15-500 Da
  • Such low molecular weight organic groups include lower alkyl chains or aliphatic groups (e.g., methyl, ethyl, propyl, butyl), substituted alkyl or aliphatic groups (e.g., aminoethyl, aminopropyl, aminohydroxyethyl, aminohydroxypropyl), a small saccharides or glycosyl groups.
  • Other low molecular weight organic modifications include additions to the internucleoside phosphate linkages such as cholesteryl or diamine compounds with varying numbers of carbon residues between the amino groups and terminal ribose.
  • Oligonucleotides with these linkages or other modifications can be prepared according to known methods (see, e.g., Agrawal and Goodchild (1987); Agrawal et al. (1988); Uhlmann et al. (1990); Agrawal et al. (1992); Agrawal (1993); and U.S. Pat. No. 5,149,798).
  • modified oligonucleotide also encompasses oligonucleotides with a covalently modified base and/or sugar.
  • modified oligonucleotides include oligonucleotides having backbone sugars which are covalently attached to low molecular weight organic groups other than a hydroxyl group at the 3' position and other than a phosphate group at the 5' position.
  • modified oligonucleotides may include a 2'-O-alkylated ribose group such as a 2'-O-methylated ribose.
  • modified oligonucleotides may include sugars such as arabinose instead of ribose.
  • the modified oligonucleotides may be branched oligonucleotides.
  • Unoxidized or partially oxidized oligonucleotides having a substitution in one or more nonbridging oxygen per nucleotide in the molecule are also considered to be modified oligonucleotides.
  • modified oligonucleotides are oligonucleotides having prostate- targeting, nuclease resistance-conferring, or other bulky substituents and/or various other structural modifications not found in vivo without human intervention.
  • the androgen receptor and other hormonal receptor sites on prostate cells allow for targeting antisense oligonucleotides specifically or particularly to prostatic cells.
  • Attachment ofthe antisense oligonucleotides by a molecular "tether" e.g., an alkyl chain
  • estramustine emcyt or estracyt (Sheridan and Tew (1991)
  • Estramustine targets particularly to the ventral prostate (Forsgren, et al. (1979)).
  • chemotherapeutic agents e.g., dexamethasone, vinblastine, etoposide
  • modified oligonucleotides are hybrid or chimeric oligonucleotides in which some but not all ofthe phosphodiester linkages, bases or sugars have been modified.
  • the currently most preferred modified oligonucleotides are 2'-O- methylated hybrid oligonucleotides. Since degradation occurs mainly at the 3' end, secondarily at the 5' end, and less in the middle, unmodified oligonucleotides located at this position can activate RNase H. and yet are degraded slowly. Furthermore, the T m of such a 27-mer is approximately 20 °C higher than that of a 27-mer all phosphorothioate oligodeoxynucleotide. This greater affinity for the targeted genomic area can result in greater inhibiting efficacy.
  • the number of synthetic linkages at the termini need not be ten and synthetic linkages may be combined with other modifications, such as alkylation of a 5' or 3' phosphate, or 2'-O- alkylation.
  • one may produce a modified oligonucleotide with the following structure, where B represents any base, R is an alkyl, aliphatic or other substituent, the subscript S represents a synthetic (e.g. phosphorothioate) linkage, and each n is an independently chosen integer from 1 to about 20:
  • the methods ofthe present invention represent new and useful additions to the field of benign prostate hype ⁇ lasia or prostate cancer therapy.
  • the methods ofthe present invention are especially useful for late stage prostate cancer in which metastases have occurred and in which the cells have become resistant to estrogen or anti-androgen therapy.
  • the methods may, however, also be used in benign prostate hype ⁇ lasia or early stage prostate cancer and may provide a substitute for more radical procedures such as transurethral resection, radical prostatectomy, or physical or chemical castration.
  • the products ofthe present invention include the isolated antisense oligonucleotides described above.
  • the term "isolated" as applied to an antisense oligonucleotide means not covalently bound to and physically separated from the 5' and 3' sequences which flank the corresponding antisense sequence in nature.
  • Admimstration ofthe PSA or probasin antisense oligonucleotides may be oral, intravenous, parenteral, cutaneous or subcutaneous.
  • the admimstration also may be localized to the prostate or to the region ofthe tumor by injection to or perfusion of the site.
  • PSA or probasin antisense oligonucleotides may be administered as part of a pharmaceutical composition.
  • a pharmaceutical composition may include the antisense oligonucleotides in combination with any standard physiologically and/or pharmaceutically acceptable carriers which are known in the art.
  • the compositions should be sterile and contain a therapeutically effective amount ofthe antisense oligonucleotides in a unit of weight or volume suitable for administration to a patient.
  • pharmaceutically acceptable means a non ⁇ toxic material that does not interfere with the effectiveness ofthe biological activity ofthe active ingredients.
  • physiologically acceptable refers to a non-toxic material that is compatible with a biological system such as a cell, cell culture, tissue, or organism.
  • the characteristics ofthe carrier will depend on the route of administration.
  • Physiologically and pharmaceutically acceptable carriers include diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials which are well known in the art.
  • the pharmaceutical composition ofthe invention may also contain other active factors and/or agents which inhibit prostate cell growth or increase cell death. Such additional factors and/or agents may be included in the pharmaceutical composition to produce a synergistic effect or to minimize side- effects caused.
  • the pharmaceutical composition ofthe invention may be in the form of a liposome in which the PSA or probasin antisense oligonucleotides are combined, in addition to other pharmaceutically acceptable carriers, with amphipathic agents such as lipids which exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers which are in aqueous solution.
  • Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like. Preparation of such liposomal formulations is within the level of skill in the art, as disclosed, for example, in U.S. Pat. No.
  • the pharmaceutical composition ofthe invention may further include compounds such as cyclodextrins and the like which enhance delivery of oligonucleotides into cells.
  • cationic detergents e.g. Lipofectin
  • the oligonucleotides When a therapeutically effective amount of PSA or probasin antisense oligonucleotides is administered orally, the oligonucleotides will be in the form of a tablet, capsule, powder, solution or elixir. When administered in tablet form, the pharmaceutical composition ofthe invention may additionally contain a solid carrier such as a gelatin or an adjuvant.
  • the tablet, capsule, and powder may contain from about 5 to 95% ofthe PSA and/or probasin antisense oligonucleotides and preferably from about 25 to 90% ofthe oligonucleotides.
  • a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, sesame oil, or synthetic oils
  • the liquid form ofthe pharmaceutical composition may further contain physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol.
  • the pharmaceutical composition may contain from about 0.5 to 90% by weight of a PSA and/or probasin antisense oligonucleotide and preferably from about 1 to 50% ofthe oligonucleotide.
  • the oligonucleotides When a therapeutically effective amount of a PSA or probasin antisense oligonucleotide is administered by intravenous, cutaneous or subcutaneous injection, the oligonucleotides will be in the form of a pyrogen-free, parenterally acceptable aqueous solution.
  • parenterally acceptable solutions having due regard to pH, isotonicity, stability, and the like, is within the skill in the art.
  • a preferred pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection should contain, in addition to the antisense oligonucleotides, an isotonic vehicle such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection, or another vehicle as known in the art.
  • an isotonic vehicle such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection, or another vehicle as known in the art.
  • the pharmaceutical composition ofthe present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those of skill in the art.
  • administration of the antisense oligonucleotides is localized to the region ofthe targeted cells in order to maximize the delivery ofthe antisense and to minimize the amount of antisense needed per treatment.
  • administration is by direct injection at or perfusion ofthe site ofthe targeted cells, such as a tumor.
  • the antisense oligonucleotides may be adhered to small particles (e.g., microscopic gold beads) which are impelled through the membranes ofthe target cells (see, e.g., U.S. Pat. No. 5,149,655).
  • a recombinant gene is constructed which encodes a PSA or probasin antisense oligonucleotide and this gene is introduced within the targeted cells on a vector.
  • a PSA or probasin antisense gene may, for example, consist of the normal PSA or probasin sequence, or a subset ofthe normal sequences, operably joined in reverse orientation to a promoter region.
  • An operable antisense gene may be introduced on an integration vector or may be introduced on an expression vector. In order to be most effective, it is preferred that the antisense sequences be operably joined to a strong eukaryotic promoter which is inducible or constitutively expressed.
  • the PSA and/or probasin antisense oligonucleotides are administered in therapeutically effective amounts.
  • therapeutically effective amount means that amount of antisense which, under the conditions of administration, including mode of administration and presence of other active components, is sufficient to result in a meaningful patient benefit, i.e., the killing or inhibition ofthe growth of target cells.
  • the amount of PSA and/or probasin antisense oligonucleotides in the pharmaceutical composition ofthe present invention will depend not only upon the potency ofthe antisense but also upon the nature and severity ofthe condition being treated, and on the nature of prior treatments which the patient has undergone. Ultimately, the attending physician will decide the amount of antisense with which to treat each individual patient. Initially, the attending physician will administer low doses ofthe inhibitor and observe the patient's response. Larger doses of antisense may be administered until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not increased further. In preferred embodiments, it is contemplated that the various pharmaceutical compositions used to practice the method ofthe present invention should contain about 1.0 ⁇ g to about 100 mg of oligonucleotide per kg body weight.
  • the duration of intravenous therapy using the pharmaceutical compositions ofthe present invention will vary, depending on the severity ofthe disease being treated and the condition and potential idiosyncratic response of each individual patient. Because a bolus of oligonucleotides, particularly highly negatively-charged phosphorothioate modified oligonucleotides, may have adverse side effects (e.g., rapid lowering of blood pressure), slow intravenous administration is preferred. Thus, intravenous administration of therapeutically effective amounts over a 12-24 hour period are contemplated. Ultimately the attending physician will decide on the appropriate duration of intravenous therapy using the pharmaceutical composition of the present invention.
  • antisense oligonucleotides substantially complementary to subsets of SEQ ID NO.: 1 or SEQ ID NO.: 2, and chemical modifications ofthe same which do not prevent hybridization under physiological conditions, are contemplated as equivalents ofthe examples presented below.
  • the use of prostate specific antisense oligonucleotides is contemplated as a method of selectively inhibiting the growth of or killing prostatic cells.
  • LNCaP LNCaP, PC3-1435, and DU145, all obtained from the American Type Culture Collection.
  • the LNCaP cells grow as stellate cells in a monolayer, retain hormone sensitivity and, of particular importance, secrete PSA into the tissue culture medium (Oesterling (1995)).
  • Cells were grown in Dulbecco's medium supplemented with 10 percent fetal calf serum, glutamate, pyruvate, penicillin and streptomycin, in 25-150 cm flasks, incubated at 37 °C in 6 percent CO -air.
  • SEQ ID NO. : 3 is antisense to positions 92- 118 of the probasin gene (SEQ ID NO.: 1).
  • SEQ ID NO.: 4 is antisense to a region upstream ofthe probasin gene at positions 76-99.
  • SEQ ID NO.: 5 is a self-stabilized or hai ⁇ in oligonucleotide. The first 21 bases are complementary to positions 80-100 ofthe probasin gene.
  • SEQ ID NO.: 6 is another self-stabilized antisense oligonucleotide. The first 21 bases of this oligonucleotide are complementary to positions 92-112 ofthe probasin gene. The remaining eight are identical to positions 96-103 ofthe gene, allowing for formation of a 3' hai ⁇ in.
  • SEQ ID NO.: 7 and SEQ ID NO.: 8 are antisense sequences corresponding to positions 401-427 and 384-410 ofthe PSA gene. Table 1 shows some ofthe antisense oligonucleotides tested. The numbers at the left of each sequence correspond to the sequence numbers in the sequence listing.
  • modified oligonucleotides were tested in which just the terminal two phosphodiester linkages at each end had been replaced by phosphorothioate synthetic linkages (shown as a subscript S between nucleotides in Table 1) and/or in which small aliphatic chemical groups (e.g., 2-hydroxy-3- amino-propyl) were added to the 3' terminal phosphate.
  • PSA protein was measured in the tissue culture medium in which LNCaP cells were grown in multi-well plates. This provided a quantitative assay, using a l25 I-anti-PSA labeled antibody sandwich assay.
  • the labeled antibody is available from Hybritech (San Diego, CA).
  • Table 5 shows the results from two negative controls (no treatment), two positive controls employing an arbitrary oligonucleotide which is antisense to a portion ofthe HIV genome, and the SEQ ID NO.: 8 modified oligonucleotide of Table 1.
  • the PSA output into the tissue culture medium of LNCaP cells was reduced 51 percent at the 5 ⁇ M level, and 45 percent at the 2.5 ⁇ M level at a 48 hour time period when the PSA gene was targeted by antisense oligonucleotides.
  • PSA gene (P S) 4+ 3+ 2-1/2+ 1 +
  • ADDRESSEE WOLF, GREENFIELD & SACKS, P.C.
  • MOLECULE TYPE DNA (genomic)
  • CTTTGTCCCC TAGATGAAGT CTCCATGAGC TACAAGGGCC TGGTGCATCC AGGGTGATCT 180
  • CTTATCCCCT CAGAATCCAC AACTTGTACC AAGTTTCCCT TCTCCCAGTC CAAGACCCCA 1366
  • CTGTGTCCCT CTATGTCTCT TTGTGTCGCT TTCATTATGT CTCTTGGTAA CTGGCTTCGG 2097
  • AAA AGC GTG ATC TTG CTG GGT CGG CAC AGC CTG TTT CAT CCT GAA GAC 3525 Lys Ser Val He Leu Leu Gly Arg His Ser Leu Phe His Pro Glu Asp 70 75 80 85
  • CAGTG'1_____' GCCTGGCCCG TAG TC TTG ACC CCA AAG AAA CTT CAG TGT 3932
  • TTGTCCTTAG GTGTGAGGTC (_AGGGTTGCT AGGAAAAGAA ATCAGCAGAC ACAGGTGTAG 5715
  • AAG AAT CTC AAA AAG AAG ATT GAA GGG AAT TGG AGA ACC GTT TAC TTA 151 Lys Asn Leu Lys Lys Lys He Glu Gly Asn Trp Arg Thr Val Tyr Leu 5 10 15 20
  • ORGANISM SYNTHETIC OLIGONUCLEOTIDE
  • ix FEATURE

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Abstract

L'invention porte sur un procédé sélectif inhibant la croissance des cellules prostatiques ou les tuant à l'aide d'oligonucléotides antisens de gènes spécifiques de la prostate. Lesdits oligonucléotides peuvent présenter la structure d'acides nucléiques naturels ou d'oligonucléosides modifiés à stabilité renforcée ou ciblant des tissus spécifiques. Les gènes spécifiques de la prostate vers lesquels les antisens peuvent être dirigés comportent les gènes PSA et de la probasine. Elle porte également sur des préparations pharmaceutiques comprenant lesdits oligonucléotides antisens utilisées dans ces procédés. Lesdits procédés et produits s'avèrent notamment utiles pour le traitement de l'hyperplasie prostatique bénigne du cancer de la prostate.
EP96931664A 1995-09-20 1996-09-20 Chimiotherapie par les oligonucleotides antisens de l'hyperplasie ou du cancer de la prostate Withdrawn EP0856052A1 (fr)

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US404495P 1995-09-20 1995-09-20
PCT/US1996/015123 WO1997011172A1 (fr) 1995-09-20 1996-09-20 Chimiotherapie par les oligonucleotides antisens de l'hyperplasie ou du cancer de la prostate
US4044P 1997-03-12

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US6100444A (en) * 1997-02-11 2000-08-08 University Of Rochester Medical Center Prostate specific regulatory nucleic acid sequences and transgenic non-human animals expressing prostate specific antigen
JP2002516261A (ja) * 1998-05-22 2002-06-04 エンターメッド インコーポレイテッド セリンプロテアーゼを利用して内皮細胞増殖を阻害し血管新生を調節するための組成物と方法
US6413513B1 (en) 1998-05-22 2002-07-02 Entremed, Inc. Compositions and methods for inhibiting endothelial cell proliferation and regulating angiogenesis using cancer markers

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IL108367A0 (en) * 1993-01-27 1994-04-12 Hektoen Inst For Medical Resea Antisense polynzcleotide inhibition of human growth factor-sensitive cancer cells
EP0652014A1 (fr) * 1993-11-10 1995-05-10 National Institute Of Immunology Traitement d'hypertrophie prostatique
US6468981B1 (en) * 1994-07-29 2002-10-22 Emory University Compositions and methods for targeting pharmaceutically active materials to cells containing androgen receptors

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