EP0667778A1 - Traitement d'un melanome au moyen d'oligonucleotides anti-codants par rapport au proto-oncogene c-myb - Google Patents

Traitement d'un melanome au moyen d'oligonucleotides anti-codants par rapport au proto-oncogene c-myb

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Publication number
EP0667778A1
EP0667778A1 EP92924379A EP92924379A EP0667778A1 EP 0667778 A1 EP0667778 A1 EP 0667778A1 EP 92924379 A EP92924379 A EP 92924379A EP 92924379 A EP92924379 A EP 92924379A EP 0667778 A1 EP0667778 A1 EP 0667778A1
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EP
European Patent Office
Prior art keywords
seq
oligonucleotide
mer
oligodeoxynucleotide
myb
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EP92924379A
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German (de)
English (en)
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EP0667778A4 (fr
Inventor
Alan M. Gewirtz
Bruno Calabretta
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University of Pennsylvania Penn
Temple University of Commonwealth System of Higher Education
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University of Pennsylvania Penn
Temple University of Commonwealth System of Higher Education
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Publication of EP0667778A1 publication Critical patent/EP0667778A1/fr
Publication of EP0667778A4 publication Critical patent/EP0667778A4/fr
Withdrawn legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • 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
    • 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
    • C12N15/1135—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 oncogenes or tumor suppressor genes
    • 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
    • C12N2310/3125—Methylphosphonates
    • 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 to proto-oncogenes, in particular antisense oligonucleotides to the c-myb gene, and the use 10 of such oligonucleotides as antineoplastic agents.
  • the proto-oncogene c-myb is the normal cellular 20 homologue of the avian myeloblastosis virus-transforming gene v-myb.
  • the c-myb gene codes for a nuclear protein expressed primarily in hematopoietic cells. It is a proto-oncogene, that is, it codes for a protein which is required for the survival of normal, non-tumor cells. 25 When the gene is altered in the appropriate manner, it has the potential to become an oncogene.
  • Oncogenes are genes whose expression within a cell provides some function in the transformation from normal to tumor cell.
  • the human c-myb gene has been isolated, cloned, 30 and sequenced. Majello et al. , Proc. Natl.
  • Antisense oligonucleotides to human c-myb mRNA that is, oligonucleotides having a nucleotide sequence complementary to the mRNA transcript of the c-myb gene, are disclosed in our allowed, co- pending application Serial No. 427,659, filed October 27, 1989, and corresponding international patent application WO90/05445, the entire disclosures of which are incorporated herein by reference.
  • C-myb antisense oligonucleotides are disclosed therein as being useful for the treatment of hematologic neoplasms, and for immunosuppression.
  • Melanoma also known as "malignant melanoma” or “cutaneous melanoma” is a neoplasm of melanocytes that has the potential for invasion and metastasis.
  • Melanocytes are elanosome-containing cells that specialize in the biosynthesis and transport of melanin pigment. Melanocytes reside in the skin at the basal layer of the epidermis. Under a variety of stimuli, they elaborate melanin pigment. Melanin synthesis occurs on the melanosome, a well-defined intracellular organelle within the melanosome.
  • the rate of increase in the incidence of melanoma is greater than for any other cancer, with the exception of bronchogenic carcinoma.
  • the incidence of melanoma is greatest among Caucasians, and is influenced by ultraviolet light exposure, and by geographical and occupational factors.
  • the incidence of melanoma is increasing rapidly in the United states and elsewhere, with an apparent doubling every ten to seventeen years.
  • melanoma accounts for roughly one percent of cancers in the United States, and about the same proportion of cancer deaths. While it represents only about three percent of cutaneous neoplasms, melanoma accounts for two thirds of all skin cancer fatalities. For the most part, melanoma first progresses through a radial growth phase at the site of the primary lesion.
  • This initial phase is characterized by little or no competence to metastasize.
  • Melanomas in this phase are generally treatable by surgical procedures.
  • a primary melanoma acquires competence to metastasize.
  • Surgery alone is ineffective in treating the melanoma, once metastasis has occurred.
  • Chemotherapy either alone or in combination with surgery, has been utilized in the treatment of melanoma.
  • Dimethyltriazeno-imidazole (DTIC) is the most active single agent for the treatment of metastatic melanoma, with an overall objective response rate of only 21%. DTIC can cause disturbances in liver function. It possesses modest hematologic toxicity.
  • nitrosoureas Somewhat less effective in treating malignant melanoma are the synthetic nitrosoureas, of which 1,3- bis(2-chloroethyl) -1-nitrosourea (BCNU) , l-(2- chloroethyl)-3-cyclohexyl-l-nitrourea (CCNU) , methyl-CCNU and chlorozotocin are best known.
  • BCNU 1,3- bis(2-chloroethyl) -1-nitrosourea
  • CCNU l-(2- chloroethyl)-3-cyclohexyl-l-nitrourea
  • chlorozotocin chlorozotocin
  • the invention provides a method for treating melanoma.
  • An effective amount of one or more c-myb antisense oligonucleotides is administered to an individual in need of such treatment.
  • Each oligonucleotide has a nucleotide sequence complementary to at least a portion of the mRNA transcript of the human c-myb gene.
  • the oligonucleotide is hybridizable to the mRNA transcript.
  • the oligonucleotide is at least a 12-mer oligonucleotide, that is, an oligomer containing at least 12 nucleotide residues.
  • the oligomer is advantageously a 12-mer to a 40-mer, 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. While oligonucleotides larger than 40-mers may be utilized, uptake may be more difficult. Moreover, partial matching of long sequences may lead to non ⁇ specific hybridization, and non-specific effects.
  • the oligonucleotide is a 15- to 30-mer oligodeoxynucleotide, more advantageously an 18- to 26- mer.
  • oligonucleotides having a sequence complementary to any region of the c-myb gene find utility in the present invention
  • oligodeoxynucleo ⁇ tides complementary to a portion of the c-myb mRNA trans ⁇ cript including the translation initiation codon are particularly preferred.
  • the invention is also a method for purging bone marrow of metastasized melanoma cells.
  • Bone marrow aspirated from amelanoma-inflicted individual is treated with an effective amount of c-myb antisense oligonucleotide, and the thus-treated cells are then returned to the body of the afflicted individual.
  • the bone marrow purging technique may be utilized for an autologous bone marrow rescue (transplantation) , in connection with a course of high dose chemotherapy.
  • the invention is also a composition for the treatment of melanoma comprising a pharmaceutically acceptable carrier and c-myb antisense oligonucleotide.
  • oligonucleotide includes both oligomers of ribonucleotides, i.e., oligoribonucleotides, and oligomers of deoxyribonucleotides, i.e., oligo- deoxyribonucleotides (also referred to herein as "oligo- deoxynucleotides”) . Oligodeoxynucleotides arepreferred. As used herein, unless otherwise indicated, the term “oligonucleotide” also includes oligomers which may be large enough to be termed "polynucleotides”.
  • oligonucleotide and “oligodeoxynuc- leotide” include not only oligomers and polymers of the common biologically significant nucleotides, i.e., the nucleotides adenine ("A") , deoxyadenine ("dA”) , guanine (“G”) , deoxyguanine (“dG”) , cytosine (“C”) , deoxycytosine (“dC”), thymine (“T”) and uracil (“U”) , but also include oligomers and polymers hybridizable to the c-myb mRNA transcript which may contain other nucleotides.
  • A adenine
  • dA deoxyadenine
  • G guanine
  • dG deoxyguanine
  • C cytosine
  • C deoxycytosine
  • T thymine
  • U uracil
  • oligonucleotide and “oligodeoxynucleotide” includes oligomers and polymers wherein one or more purine or pyrimidine moieties, sugar moieties or internucleotide linkages is chemically modified.
  • oligonucleotide is thus understood to also include oligomers which may properly be designated as “oligonucleosides" because of modification of the internucleotide phosphodiester bond.
  • modified oligonucleotides include, for example, the methylphosphonate oligonucleosides, discussed below.
  • phosphorothioate oligonucleotide means an oligonucleotide wherein one or more of the internucleotide linkages is a phosphorothioate group, 0
  • methylphosphorateoligonucleoside ismeant an oligonucleotide wherein one or more of the internucleotide linkages is a methylphosphonate group
  • downstream when used in reference to a direction along a nucleotide sequence means the 5' ⁇ 3' direction.
  • upstream means the 3' ⁇ 5' direction.
  • c-myb mRNA transcript means the presently known mRNA transcript of the human c-myb gene, or any further transcripts which may be elucidated.
  • Fig. 1 is a graph of the effect of c-myb sense and antisense oligonucleotide on human melanoma cells
  • Control (ii) the indicated concentrations of an 18- er oligodeoxynucleotide complementary to codons 2-7 of the translated position of the c-myb mRNA transcript ("Antisense”) , or (iii) the corresponding sense 18-mer (“Sense”) .
  • Fig. 2 is similar to Fig. 1, except that the oligonucleotide treatment was extended for two days.
  • the oligonucleotide concentrations are cumulative of the two day treatment period.
  • Fig. 3 is similar to Fig. 2, except that the treatment was extended to five consecutive days. The oligonucleotide concentrations are cumulative.
  • Fig. 4 is similar to Fig. 1, except that another human melanoma cell line (SK MEL-37) was substituted for the CHP cells.
  • SK MEL-37 human melanoma cell line
  • Fig. 5 is a plot of the effect of c-myb sense and antisense oligomers on the growth of human melanoma
  • mice received 100 ⁇ g per day for 7 days of (i) a 24-mer phosphorothioate oligodeoxynucleotide complementary to codons 2-9 of the translated portion of the c-myb mRNA transcript
  • Antisense 2 mice
  • Sense 24- mer phosphorothioate oligodeoxynucleotide
  • Control no oligomer
  • the putative DNA sequence complementary to the mRNA transcript of the human c-myb gene has been reported in Majello et al. , Proc. Natl. Acad. Sci. U.S.A. 83, 9636-9640 (1986) .
  • Majello et aJL. further disclose the predicted 640 amino acid sequence of the putative c-myb protein.
  • the initiation codon ATG appears at position 114, preceded by a 5'-untranslated region.
  • the termination codon TGA at position 2034 is followed by a 3'-untranslated region spanning about 1200 nucleotides, which is followed by a poly(A) tail of about 140 nucleotides .
  • the antisense oligonucleotides of the invention may be synthesized by any of the known chemical oligonu- cleotide synthesis methods. Such methods are generally described, for example, in Winnacker, From Genes to Clones: Introduction to Gene Technology. VCH Verlagsges- ellschaft mbH (Ibelgaufts trans. 1987).
  • the antisense oligonucleotides are most advantageously prepared by utilizing any of the commercially available, automated nucleic acid synthesizers.
  • One such device, the Applied Biosyste s 380B DNA Synthesizer utilizes ?-cyanoethyl phosphoramidite chemistry.
  • an ⁇ tisense oligonucleotides hybridizable with any portion of the mRNA transcript may be prepared by oligonucleotide synthesis methods known to those skilled in the art.
  • oligonucleotide While any length oligonucleotide may be utilized in the practice of the invention, sequences shorter than 12 bases may be less specific in hybridizing to the target c-myb mRNA, may be more easily destroyed by enzymatic digestion, and may be destabilized by enzymatic digestion. Hence, oligonucleotides having 12 or more nucleotides are preferred.
  • oligomers of 12-40 nucleotides are preferred, more preferably 15-30 nucleotides, most preferably 18-26 nucleotides. While sequences of 18-21 nucleotides are most particularly preferred, for unmodified oligonucleotides, slightly longer chains of up to about 26 nucleotides, are preferred for modified oligonucleotides such as phosphorothioate oligonucleotides, which hybridize less strongly to mRNA than unmodified oligonucleotides.
  • Oligonucleotides complementary to and hybridizable with any portion of the c-myb mRNA transcript are, in principle, effective for inhibiting translation of the transcript, 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'-terminal region of the c-myb mRNA transcript are preferred. It is believed that secondary or tertiary structure which might interfere with hybridization is minimal in this region.
  • sequences that are too distant in the 3'-direction from the initiation site may be less effective in hybridizing the mRNA transcripts because of a "read-through” phenomenon whereby the ribosome is postulated to unravel the antisense/sense duplex to permit translation of the message. See, e.g., Shakin, J. Biochemistry 261, 16018 (1986).
  • the antisense oligonucleotide is preferably directed to a site at or near the initiation codon for protein synthesis. Oligonucleotides complementary to the c-myb mRNA, including the initiation codon (the first codon at the 5' end of the translated portion of the c- myb transcript, comprising nucleotides 114-116 of the complete transcript) are preferred.
  • antisense oligomers complementary to the 5'-terminal region of the c-myb transcript are preferred, particularly the region including the initiation codon, it should be appreciated that useful antisense oligomers are not limited to those complementary to the sequences found in the translated portion (nucleotides 114 to 2031) of the mRNA transcript, but also includes oligomers complementary to nucleotide sequences contained in, or extending into, the 5'-and 3'-untranslated regions. Oligomers whose complementarity extends into the 5'- untranslated region of the c-myb transcript are believed particularly effective in inhibiting c-myb translation.
  • Preferredoligonucleotidescomplementarytothe 5 '-untranslated region of the transcript include molecules having a nucleotide sequence complementary to a portion of the c-myb mRNA transcript including the cap nucleotide, that is, the nucleotide at the extreme 5'-end of the transcript.
  • nucleotide sequence of the mRNA transcript 5'-terminus beginning with the cap nucleotide may be readily established, and antisense oligonuc ⁇ leotides complementary and hybridizable thereto may be prepared.
  • the following 40-mer oligodeoxynucleotide is complementary to the c-myb mRNA transcript beginning with the initiation codon of the transcript and extending downstream thereof (in the 3' direction): SEQ ID NO:l.
  • oligomers based upon the above sequence, in particular, oligomers hybridizable to segments of the c-myb message containing the initiation codon, may be utilized. Particularly preferred are the following 26- to 15-mers: SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6,
  • Oligodeoxynucleotides complementary to the c- myb mRNA transcript beginning with the second codon of the translated portion of the transcript are another group of preferred oligomers.
  • Such oligomers include, for example, the following 26- to 15-mers: SEQ ID NO: 14,
  • SEQ ID NO:20 SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24 and
  • oligonucleotide employed may represent an un ⁇ modified or modified oligonucleotide.
  • oligo- nucleotides hybridizable to the c-myb mRNA transcript finding utility according to the present invention include not only oligomers of the biologically sig- nificant native nucleotides, i.e.. A, dA, G, dG, C, dC, T and U, but also oligonucleotide species which have been modified for improved stability and/or lipid solubility.
  • alkylphosphonate oligonucleoside or allylphosphotriester 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 phos ⁇ phate oxygen in the internucleotide phosphodiester linkage to form an alkylphosphonate oligonucleoside or allylphosphotriester 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 U.S. Patent 4,469,863.
  • Methylphosphonate oligomers can be prepared by a variety of methods, both in solution and on insoluble polymer supports (Agrawal and Riftina, Nucl. Acids Res. , 6, 3009-3024 (1979); Miller et ______ , Biochemistry. 18,
  • methylphosphonate oligonucleosides The most efficient procedure for preparation of methylphosphonate oligonucleosides involves use of 5'-0- dimethoxytrityldeoxynucleoside-3 ' - 0_- diisopropylmethylphosphoramidite intermediates, which are similar to the methoxy or ⁇ -cyanoethyl phosphoramidite reagents used to prepare oligodeoxyribonucleotides.
  • the methylphosphonate oligomers can be prepared on controlled pore glass polymer supports using anautomated DNA synthesizer (Sarin et al . , Proc. Natl. Acad. Sci. USA, 85, 7448-7451 (1988)).
  • Resistance to nuclease digestion may also be achieved by modifying the internucleotide linkage at both the 5' and 3' termini with phosphoroamidites according to the procedure of Dagle et al., Nucl. Acids Res. 18, 4751-4757 (1990) .
  • 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 aJL. , Proc. Natl. Acad. Sci. U.S.A. 86, 3474-3478 (1989)). Phosphorothioate oligodeoxynucleotide are described by LaPlanche, et al.
  • oligoribonucleotide analogues mean that other agents may also be used for the purposes described here, e.g., 2'-0- methylribonucleotides (Inove et al. , Nucleic Acids Res. 15, 6131 (1987) and chimeric oligonucleotides that are composite RNA-DNA analogues (Inove et aJL. , FEBS Lett. 215, 327 (1987) .
  • oligodeoxyribonucleotides are preferred in the practice of the present invention. Oligodeoxyribonucleotides are further preferred because, upon hybridization with c-myb mRNA, the resulting DNA-RNA hybrid duplex is a substrate for RNase H, which specifically attacks the RNA portion of DNA-RNA hybrid. Degradation of the mRNA strand of the duplexreleases the antisense oligodeoxynucleotide strand for hybridization with additional c-myb messages.
  • the antisense oligonucleotides used in the method of the present invention will have a sequence which is completely complementary to the target portion of the c-myb message. Absolute complementarity is not however required, particularly in larger oligomers.
  • reference herein to a "nucleotide sequence complementary to at least a portion of the mRNA transcript" of c-myb does not necessarily mean a sequence having 100% complementarity with the transcript. In gene- ral, any oligonucleotide having sufficient com ⁇ plementarity to form a stable duplex with c-myb mRNA is suitable.
  • Stable duplex formation depends on the sequence and length of the hybridizing oligonucleotide and the degree of complementarity with the target region of the c-myb message. 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 c-myb message sequence, based upon the melting point, and therefore the stability, of the resulting duplex. Melting points of duplexes of a given base pair composition can be readily determined from standard texts, such as Molecular Cloning: A Laboratory Manual. (2nd edition, 1989), J. Sambrook et al. , eds.
  • oligonucleotides capable of stable hybridiza ⁇ tion with any region of the c-myb message are within the scope of the present invention, oligonucleotides complementary to a region including the initiation codon are believed particularly effective. Particularly preferred are oligonucleotides hybridizable to a region of the c-myb mRNA up to 40 nucleotides upstream (in the 5' direction) of the initiation codon or up to 40 nucleotides downstream (in the 3' direction) of that codon.
  • the antisense oligonucleotides may be combined with a pharmaceutical carrier, such as a suitable liquid vehicle or excipient and an optional auxiliary additive or additives.
  • a suitable liquid vehicle or excipient such as distilled water, physiological saline, aqueous solution of dextrose, and the like.
  • the c-myb mRNA antisense oligonucleotides are preferably administeredparenterally, mostpreferably intravenously.
  • the vehicle is designed accordingly.
  • oligonucleotide may be administered subcutaneously via controlled release dosage forms.
  • the oligonucleotides may be conjugated to poly(L- lysime) 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.
  • the antisense oligonucleotides may be administered by a variety of specialized oligonucleotide delivery techniques.
  • oligonucleotides may be encapsulated in for therapeutic delivery.
  • 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.
  • Oligonucleotides have been successfully encapsulated in unilameller liposomes. Reconstituted Sendaivirus envelopes havebeen suc ⁇ cessfully used to deliver RNA and DNA to cells. Arad et al., Biochem. Biophy. Acta. 859, 88-94 (1986).
  • the c-myb antisense oligonucleotides may be administered de novo as the primary therapy.
  • the oligonucleotides may be administered as an adjuvant following surgical removal of a melanoma to patients who may be disease-free but at high risk of recurrence.
  • a preferred method of administration of oligonucleotide for treatment of melanoma comprises either regional or systemic 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 sys ⁇ temic 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.
  • 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.
  • High dose chemotherapy has been coupled with autologous bone marrow rescue (transplantation) in an attempt to treat melanoma. While high dose chemotherapy has resulted in significantly higher therapeutic responses, patient survival is not generally prolonged: Jones et al . . , Cancer Chemother. Pharmacol.
  • c-myb antisense oligonucleotides may be used as bone marrow purging agents for the in vitro cleansing of bone marrow of melanoma cells in conjunction with high dose conventional chemotherapy. While normal hematopoietic cells are sensitive to c-myb antisense, they are less sensitive than malignant cells expressing c-myb, as taught in our copending patent application Serial No.
  • bone marrow is harvested from a donor by standard operating room procedures from the iliac bones of the donor.
  • Methods of aspirating bone marrow from donors are well- known in the art. Examples of apparatus and processes for aspirating bone marrow from donors are disclosed in U.S. Patents 4,481,946 and 4,486,188, incorporated herein by reference.
  • Sufficient marrow is withdrawn so that the recipient, who is either the donor (autologous transplant) or another individual (allogeneic transplant) , may receive from about 4 x 10 8 to about 8 x 10 8 processed marrow cells per kg of bodyweight. This generally requires aspiration of about 750 to about 1000 ml of marrow. The aspirated marrow is filtered until a single cell suspension, known to those skilled in the art as a "buffy coat" preparation, is obtained. This suspension of leukocytes is treated with c-myb antisense oligonucleotides in a suitable carrier, advantageously in a concentration of about 8 mg/ml.
  • the leucocyte suspension may be stored in liquid nitrogen using standard procedures known to those skilled in the art until purging is carried out.
  • the purged marrow can be stored frozen in liquid nitrogen until ready for use. Methods of freezing bone marrow and biological substances are disclosed, for example, in U.S. Patents 4,107,937 and 4,117,881.
  • bone marrow for treat- ment with c-myb antisense may be utilized, which methods may result in even more purified preparations of hemato ⁇ poietic cells than the aforesaid buffy coat preparation.
  • One or more growth factors may be added to the aspirated marrow or buffy coat preparation to stimulate growth of neoplasms, and thereby increase their sensi ⁇ tivity to the toxicity of the c-myb antisense oligonucleotides.
  • the cells to be transferred are washed with auto- logous plasma or buffer to remove unincorporated oligomer.
  • the washed cells are then infused back into the patient.
  • the c-myb antisense oligonucleotides may be administered in a dosage effective for inhibiting the proliferation of melanoma cells in the afflicted individual, while maintaining the viability of normal cells. Such amounts may vary depending on the nature and extent of the neoplasm, the particular oligonucleotide utilized, and other factors.
  • the actual dosage admin ⁇ istered may take into account the size and weight of the patient, whether the nature of the treatment is prophy- lactic or 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. Inhibition of melanoma cell proliferation has been observed at antisense concentrations of as low as 10 ⁇ g/ml. At 20 ⁇ g/ml, inhibition was profound. Concen ⁇ trations of from about 1 to about 100 ⁇ g/ml may be employed, preferably from about 10 ⁇ g/ml to about 100 ⁇ g/ml, most preferably from about 20 ⁇ g/ml to about 60 ⁇ 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 0.1 to 1,000 mg oligonucleotide per day, preferably from about 10 to about 700 mg per day. Greater or lesser amounts of oligonucleotide may be administered, as required.
  • a course of treatment may advantageously comprise infusion of the recommended daily dose of oligonucleotide for a period of from about 3 to about 28 days, more preferably from about 7 to about 10 days.
  • a daily dose of about 350 mg oligonucleotide is believed sufficient, to achieve an effective intercellular concentration of 20 ⁇ g.
  • the c-myb antisense oligonucleotides may be administered in amounts effective to kill neoplastic cells while maintaining the viability of normal hematologic cells. Such amounts may vary depending on the extent to which melanoma cells may have metastasized to the bone marrow, the particular oligonucleotide utilized, the relative sensitivity of the neoplastic cells to the oligo ⁇ nucleotide, and other factors. Concentrations from about 10 to 200 ⁇ g/ml per 10 5 cells may be employed, preferably from about 40 to 150 ⁇ g/ml per 10 5 cells.
  • Supplemental dosing of the same or lesser amounts of oligonucleotide are advantageous to optimize the treatment.
  • dosages of from about 2 to 40 mg antisense per ml of marrow may be effectively utilized, preferably from about 8 to 24 mg/ml.
  • Greater or lesser amounts of oligonucleotide may be employed.
  • CHP melanoma cells (Children's Hospital of Philadelphia, Philadelphia, PA) were grown in RPMI culture medium containing 2% oxalophosphate and 5% bovine calf serum at 37°C in 5% C0 2 . Cells (5,000/ml) were seeded in 200 ⁇ l volumes into individual wells of a 96 well Costar plate at day -3, and allowed to grow for three days.
  • the unmodified antisense 18-mer oligodeoxynucleotide (SEQ ID NO:22) complementary to codons 2-7 of the translated portion of the c-myb mRNA, or the corresponding sense 18-mer (SEQ ID NO:26) were added to the cultures at concentrations of 0, 10, 20, 50 and 100 ⁇ g/ml for 1, 2 or 5 consecutive days. On day 7, 100 ⁇ l of fresh medium was added to the cultures. Cell viability/proliferation was determined on day 8 utilizing a commercially available kit (CELLTITER 96TM Promega, Madison, WI) .
  • the assay is based on the ability of viable cells to convert a tetrazolium salt into a blue formazan product which can be quantified by measuring the absorbance at 570 nm with a conventional microplate reader.
  • the extent of absorbance at 570 nm is proportional to the amount of formazan produced, and thus the number of viable cells remaining. Accordingly, 10 ⁇ l of 3-(4,5-dimethylthiazol-2-yl) -2,5 diphenyltetrazolium bromide solution (5 mg/ml) was added to 110 ⁇ l of cell suspension and incubated for 4 hours at 37 ⁇ C.
  • the results of the viability assay are set forth in Figures 1 (one-day treatment) , 2 (two-day treatment) and 3 (five-day treatment) .
  • the oligonucleotide concentrations indicated in Figures 2 and 3 are cumulative dosages for the two- and five-day treatments, respectively. It may be appreciated from the figures that treatment of the melanoma cells with antisense oligomer resulted in sequence-specific killing. The effect was most pronounced when the melanoma cells were treated for one day with an antisense oligomer concen ⁇ tration of at least 50 ⁇ g/ml, or when the cells were treated on consecutive days with as little as 20 ⁇ g/ml oligomer.
  • SK MEL-37 250,000 melanoma cells
  • C.B-17/LCRTAC-SCID DF from Fox Chase Cancer Institute, Philadelphia PA
  • the tumors were allowed to grow to a size of approximately 5 mm in diameter.
  • ALZET constant infusion pumps Alza Corporation, Palo Alto, CA
  • delivering a total dosage of 700 ⁇ g oligomer over 7 days were surgically implanted into the mice.
  • the tumor area was then monitored daily.
  • Another mouse received a 24-mer "sense" phosphorothioate oligonucleotide (corresponding to codons 2-9 of the translated c-myb mRNA) .
  • Two mice received a 24-mer anti- sense phosphorothioate oligonucleotide having the nucleotide sequence of SEQ ID NO: 16 (TATGCTGTGC CGGGGGT- CTTC GGGC) , complementary to c-myb mRNA codons 2-9.
  • the results are shown in Figure 5. Each curve represents one animal. In the two antisense-treated animals, the tumor size stayed the same or regressed. In contrast, the tumors continued to grow in the sense-treated and control animals. The animals were sacrificed on day 19.
  • the tumors were removed and weighed.
  • the weights of the tumors in the control and sense-treated animals were 9.9 and 9.6 grams, respectively.
  • the tumor weights in the two anti- sense treated animals were only 0.1 and 0.2 grams.
  • the following non-limiting example illustrates one methodology for purging bone marrow of metastatic melanoma.
  • Bone marrow is harvested from the iliac bones of a donor under general anesthesia in an operating room using standard techniques. Multiple aspirations are taken into heparinized syringes. Sufficient marrow is withdrawn so that the marrow recipient will be able to receive about 4 x 10 8 to about 8 x 10 8 processed marrow cells per kg of body weight. Thus, about 750 to 1000 ml of marrow is withdrawn. The aspirated marrow is transferred immediately into a transport medium (TC-199, Gibco, Grand Island, New York) containing 10,000 units of preservative-free heparin per 100 ml of medium.
  • TC-199 Gibco, Grand Island, New York
  • the aspirated marrow is filtered through three progressively finer meshes until a single cell suspension results, i.e., a suspension devoid of cellular aggregates, debris and bone particles.
  • the filtered marrow is then processed further into an automated cell separator (e.g.,
  • Cobe 2991 Cell Processor which prepares a "buffy coat" product, (i.e., leukocytes devoid of red cells and platelets) .
  • the buffy coat preparation is then placed in a transfer pack for further processing and storage.
  • the purging procedure may be carried out as follows.
  • Cells in the buffy coat preparation are adjusted to a cell concentration of about 2 x 10 7 / ⁇ LL in TC-199 containing about 20% autologous plasma.
  • C-myb antisense oligodeoxynucleotide for example, in a concentration of about 8 mg/ml, is added to the transfer packs containing the cell suspension.
  • Recombinant human hematopoietic growth factors e.g. , rH IL-3 or rH GM-CSF, may be added to the suspension to stimulate growth of neoplasms and thereby increase their sensitivity c-myb antisense oligonucleotide toxicity.
  • the transfer packs are then placed in a 37°C waterbath and incubated for 18 - 24 hours with gentle shaking.
  • the cells may then either be frozen in liquid nitrogen or washed once at 4°C in TC-199 containing about 20% autologous plasma to remove unincorporated oligomer. Washed cells are then infused into the recipient. Care must be taken to work under sterile conditions wherever possible and to maintain scrupulous aseptic techniques at all times.

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Abstract

On traite un mélanome en administrant des oligonucléotides contenant une séquence nucléotidique complémentaire à au moins une partie du transcrit ARNm du gène c-myb humain. Ces oligonucléotides 'anti-codants' peuvent être hybridés au transcrit ARNm c-myb.
EP92924379A 1991-11-15 1992-11-12 Traitement d'un melanome au moyen d'oligonucleotides anti-codants par rapport au proto-oncogene c-myb. Withdrawn EP0667778A4 (fr)

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US79299991A 1991-11-15 1991-11-15
PCT/US1992/009656 WO1993009789A1 (fr) 1991-11-15 1992-11-12 Traitement d'un melanome au moyen d'oligonucleotides anti-codants par rapport au proto-oncogene c-myb
US792999 1997-02-03

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US5749847A (en) * 1988-01-21 1998-05-12 Massachusetts Institute Of Technology Delivery of nucleotides into organisms by electroporation
US5989849A (en) * 1991-05-09 1999-11-23 Temple University Of The Commonwealth System Of Higher Education Antisense of oligonucleotides to c-kit proto-oncogene and in vitro methods
US5646042A (en) * 1992-08-26 1997-07-08 Ribozyme Pharmaceuticals, Inc. C-myb targeted ribozymes
US5658780A (en) * 1992-12-07 1997-08-19 Ribozyme Pharmaceuticals, Inc. Rel a targeted ribozymes
AU7983294A (en) * 1993-10-19 1995-05-08 Regents Of The University Of Michigan, The P53-mediated apoptosis
US5618709A (en) * 1994-01-14 1997-04-08 University Of Pennsylvania Antisense oligonucleotides specific for STK-1 and method for inhibiting expression of the STK-1 protein
US6207646B1 (en) 1994-07-15 2001-03-27 University Of Iowa Research Foundation Immunostimulatory nucleic acid molecules
US5994320A (en) * 1995-02-06 1999-11-30 Regents Of The University Of Minnesota Antisense oligonucleotides and methods for treating central nervous system tumors

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WO1990005445A1 (fr) * 1988-11-07 1990-05-31 Temple University Of The Commonwealth System Of Higher Education Oligonucleotides non codants du proto-oncogene c-myb et leurs emplois

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WO1990005445A1 (fr) * 1988-11-07 1990-05-31 Temple University Of The Commonwealth System Of Higher Education Oligonucleotides non codants du proto-oncogene c-myb et leurs emplois

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, vol. 85, January 1988, WASHINGTON US, pages 74-78, XP002025496 LINNENBACH, A. ET AL.: "Structural alteration in the MYB protooncogene and deletion within the gene encoding alpha-type protein kinase C in human melanoma cell lines" *
See also references of WO9309789A1 *

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JPH07501525A (ja) 1995-02-16

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