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WO1993000909A1 - Procede et systeme de determination de l'activite de la neurotrophine - Google Patents

Procede et systeme de determination de l'activite de la neurotrophine Download PDF

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Publication number
WO1993000909A1
WO1993000909A1 PCT/US1992/003392 US9203392W WO9300909A1 WO 1993000909 A1 WO1993000909 A1 WO 1993000909A1 US 9203392 W US9203392 W US 9203392W WO 9300909 A1 WO9300909 A1 WO 9300909A1
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Prior art keywords
cell
bdnf
oligonudeotide
daim
tumor
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PCT/US1992/003392
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English (en)
Inventor
Stephen P. Squinto
Steven H. Nye
George D. Yancopoulos
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Regeneron Pharmaceuticals, Inc.
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Priority to AU23223/92A priority Critical patent/AU2322392A/en
Priority to EP92915861A priority patent/EP0593663A4/en
Priority to JP5502196A priority patent/JPH06509333A/ja
Priority to CA002112799A priority patent/CA2112799A1/fr
Publication of WO1993000909A1 publication Critical patent/WO1993000909A1/fr

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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
    • 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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • 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
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/142—Toxicological screening, e.g. expression profiles which identify toxicity

Definitions

  • This invention relates to pharmaceutical compositions and methods for the treatment of mammals bearing tumor cells which express neurotrophic factors and which utilize an autocrine loop mechanism for survival. More specifically, this invention relates to methods of interrupting the signal transduction pathway of the brain-derived neurotrophic factor ("BDNF”) and causing cell death in BDNF-expressing cells.
  • BDNF brain-derived neurotrophic factor
  • neuroblastoma is used to designate the spectrum of neurogenic neoplasms derived from embryonic sympathetic neuroblasts, neural crest cells and the mantle layer of the neural tube.
  • Neuroblastoma tumors the most commonly diagnosed neoplasms in infants under 1 year of age, occur with a frequency of 1 out of 10,000 live births.
  • Neuroblastoma is considered the third most common malignancy of childhood, accounting for approximately 10% of all pediatric neoplasms, and at least 15% of all cancer- related deaths in children.
  • neuroblastomas often contain elements of both adrenergic and cholinergic neurotransmitter pathways. They express neuron- specific enolase and neurofilament proteins and exhibit substrate adherent cell growth in culture with neurite formation.
  • human neuroblastoma Perhaps the most salient feature of human neuroblastoma is amplification of the N-myc oncogene, which has been identified in 19 of 22 neuroblastoma cell lines and in approximately 31% of tumor tissues from patients with stage Dl and stage IV neuroblastoma [Kohl et al. Science.226:1335 (1984)].
  • the human neuroblastoma cell line SK-N-SH and its derivative SH-SY5Y are of thoracic origin rather than neural crest, and do not express amplified N-myc but do express N-ras.
  • SCLC small cell lung carcinoma
  • Small cell lung carcinoma represents approximately one third of all lung cancers. While several neuroblastomas are known to express amplified levels of N-myc, SCLC are generally characterized by activated levels of either N-myc or L- myc. Small cell carcinomas represent approximately 20-25% of all pulmonary malignancies, yielding an incidence of approximately 25,000- 30,000 cases per year in the United States, and are by far the most aggressive of pulmonary malignancies. SCLC is frequently (70%-90%) metastatic at presentation. Small cell carcinomas are neuroectodermal in origin. These tumors possess properties of amine precursor uptake and decarboxylation (APUD), and other neuronal characteristics, such as the production of neuroactive peptides. Paraneoplastic syndromes, such as subacute sensory neuropathy, occur with greater frequency among victims of small cell than other lung cancers.
  • APUD amine precursor uptake and decarboxylation
  • Paraneoplastic syndromes such as subacute sensory neuropathy, occur with greater frequency among victims of small cell than other lung cancers
  • neurotrophic factors proteins, termed neurotrophic factors, originally defined by their ability to support the survival of neuronal cells. In addition to promoting neuronal survival, these factors can influence proliferative and differentiative processes within the nervous system and may also have actions outside the nervous system.
  • NNF nerve growth factor
  • BDNF brain-derived neurotrophic factor
  • NT-3 neurotrophin-3
  • These three proteins along with the recently identified neurotrophin-4 [Hollbook et al. Neuron. 6:845-58 (1991)] comprise a family (designated "neurotrophins"), each member of which shares about 55% to 60% amino acid sequence identity with the others. Understanding the biological roles of these neurotrophic factors requires characterization of the receptor and signal transduction pathways they use to exert their effects.
  • BDNF protein tyrosine kinase receptors
  • BDNF is a neuronal survival molecule which is capable of binding to the trk cell- surface receptor known as trkB, which has intrinsic protein kinase activity and is mainly expressed in the nervous system [Kaplan et al, Nature. 350:158
  • the trkB gene encodes protein that binds and mediates functional responses to both BDNF and NT-3 [PCT International Application WO91/03568; Squinto et al. Cell, 65:885 (1991); Glass et al. Cell, 66:405-413 (1991); Soppet et al. Cell, 65:895 (1991)].
  • trk receptors into fibroblasts have enabled the creation of cells that display biological responses to neurotrophic factors that mimic the actions of traditional growth factors.
  • FGF or PDGF growth factors
  • Such models provide a powerful assay system that can be used to detect and /or measure neurotrophin activity, to identify agents that exhibit neurotrophin-like activity and to identify antagonists which block binding of ligands to neurotrophin receptors [Glass et al, supra! .
  • BDNF and NT-3 might serve as survival and /or mitogenic factors for neuronal precursors that have not yet achieved a post-mitotic phenotype.
  • ERK kinases represent a recently identified and molecularly cloned family of extracellular signal-regulated protein kinases [Boulton et al. Cell, 65:663-75 (1991)]. ERK activity is rapidly activated in response to growth factor (i.e., insulin and NGF) stimulation of cells and represents a class of intracellular growth factor (i.e., insulin and NGF) stimulation of cells and represents a class of intracellular growth factor (i.e., insulin and NGF) stimulation of cells and represents a class of intracellular growth factor (i.e., insulin and NGF) stimulation of cells and represents a class of intracellular growth factor (i.e., insulin and NGF) stimulation of cells and represents a class of intracellular growth factor (i.e., insulin and NGF) stimulation of cells and represents a class of intracellular growth factor (i.e., insulin and NGF) stimulation of cells and represents a class of intracellular growth factor (i.e., insulin and NGF) stimulation of cells and represents a
  • the first method makes use of stably-transfected promoter-directed gene constructs designed to constitutively synthesize complementary antisense mRNA sequences. This technology generally results in hybridization arrest of protein translation for a given gene product. It has recently been suggested that RNase may play a role in this mechanism by cleaving RNA/DNA duplexes formed between antisense mRNA and DNA. Other possible mechanisms include impaired nuclear processing or the inability of the RNA/DNA duplex to be efficiently translated.
  • An alternative approach to vector-driven antisense translational arrest involves the synthesis of short 5' or 3' synthetic antisense oligodeoxyribonucleotides (antisense DNA).
  • RNAs when added to eukaryotic cells in vitro (reviewed in Van der Krol et al, Biotechniques.6:958-973 (1988).
  • oligonudeotides that are complementary to selected cellular or viral target nucleic acid sequences to modulate the expression of the target nucleic acid sequence.
  • There have been several reports on the use of specific nucleic acid sequences to inhibit viral replication [see for example Goodchild et al, Proc. Natl. Acad. Sci. USA. 85:5507-5511 (1988); Wickstrom et al, Proc. Natl. Acad. Sci. USA.85:1028-1032 (1988); and Kawasaki. Nucl. Acids Res.. 13:4991 (1985)].
  • Several laboratories have attempted to develop modified oligonudeotides that are relatively membrane permeable and nuclease resistant.
  • phosphorothioates may, in addition to binding to complementary target nudeic add sequences, also direct the inhibition of primer binding to HTV reverse transcriptase [Matsukura et al,
  • Another example includes an oligonudeotide joined through a linking arm to a group that imparts amphophilic character to the final product in order to increase the effidency of membrane transport [PCT Publication No. WO 88/09810, published December 15, 1988].
  • oligonudeotides i.e. antisense oligonudeotides linked to reactive agents that are able to modify the target nudeic acid.
  • reacting agents are intercalating agents which can bind to the duplex by internal insertion between adjacent base pairs or bind to external nudeoside and phosphate elements respectively.
  • intercalators that have been attached to oligonudeotides and oligonudeotide analogs indude acridine, anthridium, and photoactivatable psoralen [reviewed in Zon, Pharm. Res.. 5:539-549 (1988)].
  • Another sudi group of reactive groups coupled to oligomers include metal complexes such as EDTA-Fe(II), o-phenanthroline-Cu(I), or porphyrin- Fe(II) [reviewed in Krol et al, BioTechniques. 6:958-976 (1988)]. These compounds can generate hydroxyl radicals in the presence of molecular oxygen and a redudng agent. The resulting radicals can cleave the complementary strand following attack on the target nudeic add backbone. There have been many recent publications dealing with inhibition by antisense oligonudeotides.
  • RNA antisense oligonudeotides directed against basic fibroblast growth factor For example, proliferation of human malignant melanomas has been inhibited iri vitro by antisense oligonudeotides directed against basic fibroblast growth factor [Becker et al, EMBO ⁇ ., 8:3685 (1989)].
  • the generation of RNA antisense to part of the human N-myc gene via an episomally replicating expression vector has been observed to block transdifferentiation of neuroectodermal tumor cell lines [Whitesell et al, Mol.
  • An antisense oligonudeotide to transforming growth factor beta 3 inhibited epithelial-mesenchymal transformation of embryonic cardiac endothelial cells in explant cultures [Potts et al.Proc. Natl. Acad. Sci. USA, 88:1516-1520 (1991)] .
  • Autocrine loops have been observed for various growth factor molecules and tumor cell lines. Certain tumor cells are known to synthesize and respond to growth factors that are required for normal cellular growth and division. Via autocrine signaling, the cells respond to substances they themselves produce. Auto ⁇ ine loops might serve to accelerate or amplify a cellular response in tumor cells because that cell is less dependent on its environment for its existence.
  • auto ⁇ ine loops have been experimentally defined by the use of antisense approaches for the disruption of the autocrine loop.
  • the mere ability of an oligonudeotide that is antisense to a particular factor to adversely effect cellular growth is indicative that the cell is synthesizing that factor and the factor is required for growth or survival of the cell.
  • the existence of cellular receptors for the factor or even the release of the growth factor into the environment may not be detectable.
  • Antisense oligonudeotides have been used to demonstrate that transforming growth factor-beta serves as an autocrine cell differentiation fartor responsible for the transformation of epithelial cells to mesenchymal cells [Potts et al, Proc. Natl. Acad. Sd. USA, 88:1516 (1991)]. Antisense approadies have been used to demonstrate that basic fibroblast growth factor appears to be required for the autocrine-stimulated proliferation of both human melanomas [Becker et al, EMBO J span 8:3685 (1989)] and transformed human astrocytes [Morrison et al, J. Biol. Chem., 266:728 (1991)]. Antisense oligonudeotides against growth hormone inhibit lymphocyte proliferation
  • Specific protein phosphorylation inhibitors have been used for studying the effed of a number of kinases and their actions in the phosphorylation of key cellular proteins for the biological activity of nerve growth factor on its target cells.
  • K252a isolated from the culture broth of Nocardiosis sp. and its derivatives, are described in U..S. Patent Nos. 4,555,402; 4,877,776; and 4,923,986, which documents are incorporated herein by reference.
  • K252a and staurosporine were initially characterized as potent protein kinase C (PKC) and cydic nudeotide-dependent kinase inhibitors in vitro [Kase et al, Biochem. Biophys. Res. Comm.
  • Nanomolar concentrations of K252a and its derivatives in vitro have been found to inhibit, in a somewhat selective fashion, protein kinase C, cyclic AMP-and cydic GMP-dependent protein kinases, myosin light chain kinase, and calmodulin-dependent phosphodiesterase [Kase et al, Biochem.
  • EGF responses in PC12 cells they are able to block the earliest detertable signalling processes induced by NGF, including NGF induced tyrosine phosphorylation.
  • K252a also has been shown to inhibit the NGF induced outgrowth of neurites from primary cultures of embryonic dorsal root ganglion explants as well as to completely block the survival activity of NGF on primary cultures of embryonic chick sympathetic neurons [Matsuda and Fukuda, Neurosd. Lett..87:11-17 (1988); Borasio, Neurosci. Lett.. 108:207-12 (1990)].
  • the present invention is directed to a method of treating mammals bearing a tumor cell of a type characterized by expression of a BDNF.
  • the present invention relates to the identification of an autocrine survival loop in BDNF-expressing tumor cells and means for interrupting the autocrine loop in order to cause cell death.
  • the present invention is further direrted to recombinant cells that serve as a model system for cells, induding tumor cells, that are dependent on an autocrine loop for survival.
  • recombinant cells provide a means for screening compounds for therapeutic efficacy in the treatment of tumors that utilize autocrine survival loops.
  • recombinant cells that express both BDNF and the trkB receptor, and thus depend upon a BDNF autocrine loop for survival, are utilized to identify agents which interrupt the BDNF autocrine loop and which can be used to treat tumor cells that similarly depend upon such autocrine loops for survival and/or proliferation.
  • the invention is directed to nucleic acids of at least six nucleotides that are antisense to a gene or cDNA encoding BDNF or a portion thereof.
  • Antisense refers to a nucleic acid capable of hybridizing to a portion of a BDNF RNA (preferably mRNA) by virtue of some sequence complementarity.
  • the antisense nucleic acids of the invention which are used to interrupt a BDNF autocrine survival loop may be oligonudeotides that are double-stranded or single-stranded, RNA or DNA or a modification or derivative thereof, which can be directly administered to a cell, or which can be produced intracellularly by transcription of exogenous, introduced sequences.
  • this invention is directed to the use of staurosporine, K252a or its derivatives, or other protein kinase inhibitors to interrupt a BDNF autocrine survival loop.
  • BDNF antisense nudeic acids and the K252a or its derivatives provided by the instant invention can be used for the treatment of tumors, the cells of which tumor type can be demonstrated to express BDNF.
  • the invention is directed to methods for inhibiting the expression of a BDNF nucleic acid sequence in a eucaryotic cell comprising providing the cell with an effective amount of a composition comprising an antisense BDNF nucleic acid of the invention.
  • staurosporine, K252a or its derivatives or other protein kinase inhibitors may be used to interrupt the BDNF autocrine loop at the level of the cell surface receptor by inhibiting phosphorylation of the BDNF receptor.
  • the identification of cells expressing functional BDNF or other neurotrophin receptors can be carried out by observing the ability of a neurotrophin to "rescue" such cells from the cytotoxic effects of a BDNF antisense nudeic add.
  • Another aspect of the invention provides for the diagnosis of human neuroblastoma or small cell lung cardnoma by detecting BDNF expression in cells obtained from patients.
  • the invention further provides pharmaceutical compositions comprising an effective amount of the BDNF antisense nucleic adds of the invention in a pharmaceutically acceptable carrier.
  • Methods for treatment of various diseases and disorders comprising administering the pharmaceutical compositions of the invention are also provided.
  • a pharmaceutical composition which comprises as its active ingredient, K252a, staurosporine, or a related compound in a pharmaceutically acceptable carrier.
  • This composition may be used in the treatment of various diseases and disorders related to BDNF- expressing tumor cells.
  • a further aspect of this invention is a pharmaceutical composition which comprises as one active ingredient, K252a, staurosporine, or a related compound and as a second active ingredient, the BDNF antisense nudeic adds of the invention.
  • the staurosporine, K252a or other protein kinase inhibitor may be combined with any other conventional pharmaceutical agent useful in the treatment or prevention of disorders associated with BDNF-expressing tumor cells.
  • Still a further aspect of this invention is a method for treating patients having neuroblastoma or small cell lung cardnoma by administering an effective amount of the materials and compositions described above.
  • Yet another aspect of this invention is a method of stimulating neurite outgrowth by administering particularly low doses of a protein kinase inhibitor such as K252a.
  • BDNF mRNA was purified and then placed into a wheat germ lysate in vitro translation system (Promega) in the absence or presence of BDNF oligonudeotides.
  • the synthesis of BDNF protein was followed by using 35S- methione in the reaction.
  • Control oligonudeotide refers to the use of a random 18-mer unrelated to the sequence of human BDNF.
  • FIG. 1 Efferts of 3'-AS-BDNF upon cell viability in culture. The percentage of cell viability (y axis) is shown for different concentrations of 3'- AS-BDNF (x axis, micromolar) added to the cultured neuroblastoma cells.
  • LA-N-5 cells B: LA-N-1 cells; C: SK-ES cells; D: SH-SY5Y cells.
  • FIG. 3 Effect of the co-addition of various neurotrophins with 3'- AS- BDNF upon neuroblastoma cell lines.
  • the indicated neuroblastoma cell lines were simultaneously incubated with 50 ⁇ M 3'-AS-BDNF and either no neurotrophin (open squares with center dot), BDNF (closed diamonds), NT-3
  • FIG. 3A SH-SY5Y cells
  • Figure 3B LA-N-1 cells
  • Figure 3C LA-N-5 cells
  • Figure 3D CHP-134 cells
  • Figure 3E CHP-404 cells.
  • FIG. 1 Northern blot analysis of total cellular RNA (10 ⁇ g per lane) derived from small cell lung cardnoma cell lines or adult rat brain (lane 1).
  • the northern blot was hybridized to a human BDNF probe.
  • Small cell lung cardnoma cell lines are as follows: H82 (lane 2), H209 (lane 3), H345 (lane 4), H378 (lane 5), H510 (lane 6), and N417 (lane 7).
  • RNA Northern (RNA) blot comparisons of BDNF expression in both human and rodent tumor cells lines.
  • Total RNA (10 ⁇ g) from each cell line was fractionated, transferred to membranes and hybridized with 32p.
  • BDNF as previously described [Maisonpierre, et al. Science 247:1446 (1990).
  • Neuroblastoma cell lines in panels B and C are represented by LAN5, SY5Y and N18TG2.
  • Figure 6 Morphological effects of antisense and sense BDNF oligomers on LA-N-5 neuroblastoma.
  • LA-N-5 neuroblastoma cells were seeded into 6-well Costar plates at a density of 3 x 10 5 cells per well in RPMI 1640 (Irvine Sdentific) supplemented with 10% fetal bovine serum (FBS), 1% penicillin, 1% streptomycin (P/S) and 2 mM glutamine. Eighteen hours after seeding, the cells were transferred into serum-free defined media [Zhan et al, Mol. Cell. Biol. 6:3541 (1986)]and treated for 72 hours with the reagents described above. Engineered BDNF was produced in CHO cells and purified from CHO cell conditioned media to homogeneity as previously described [Squinto et al, Cell 65:885 (1991)].
  • FIG. 7 Dual-staining flow cytometric assay to quantitate both DNA and protein content of LA-N-5 neuroblastoma cells.
  • LA-N-5 neuroblastoma cells were seeded into 10 cm plates at a density of 1 x IO 6 cells per plate and were cultured as described in Figure 6.
  • Cells were either untreated (Panel A) or treated for 48 hours with 10 ⁇ M 3'-AS-BDNF alone (Panel B), 3'- AS-BDNF with 100 ng/ml of BDNF (Panel C) or with high concentrations (100 ⁇ M) of control 3 -S-BDNF (Panel D).
  • FIG. 1 Identification of constitutively autophosphorylated trk receptors in neuroblastoma cell lines.
  • Panel A Anti-phosphotyrosine immunoblot of autophosphorylated trkB receptors that were specifically immunopredpitated from total protein lysates prepared from approximately
  • Panel B N18TG2 neuroblastoma cells were untreated or pretreated with 200 nM K252a prior to the preparation of cell lysates and trk-specific immunopredpitation.
  • Panel C Anti- phosphotyrosine immunoblot of total protein lysates prepared from NIH3T3 cells (3T3), 3T3(trkB) cells treated with BDNF, or untreated 3T3(autocrine) cells.
  • FIG. 10 Identification of constitutively autophosphorylated trk receptors in neuroblastoma cell lines.
  • Panel A Anti-phosphotyrosine immunoblot of autophosphorylated trkB receptors that were specifically immunopredpitated from total protein lysates prepared from approximately 3 X 106 NTH3T3 cells expressing trkB and treated with BDNF or from 2-5 X 10 untreated neuroblastoma cells.
  • Panel B N18TG2 neuroblastoma cells were untreated or pretreated with 200 nM K-252a prior to the preparation of cell lysates and trk-specific immunoprecipitation.
  • Panel C Anti-phosphotyrosine immunoblot of total protein lysate prepared from NIH3T3 cells (3T3), NIH3T3(trkB) cells treated with BDNF, or untreated 3T3-autocrine cells.
  • Panel B were seeded into 24-well plates as des ⁇ ibed in Figure 7. After seeding, all cells were transferred to growth factor-deficient media. Parental 3T3 cells were maintained in 50 pM FGF. The cells were treated for 48 hours with various concentrations of K252a (ranging from 0 to 250 nM). Cell viability for all cell lines was determined with the glucose utilization assay on duplicate samples. Panel A; Open squares-SH-SY5Y (without BDNF); Closed squares-LA-N-5 (with BDNF); Closed drdes-N18TG2 (with BDNF). Panel B; Open squares- 3T3 (with FGF); Closed squares-3T3-autocrine (with BDNF). Figure 12. Differential effect of K252a on small cell lung cardnoma (NCI-H69), lung adenocarcinoma (Calu-3), 3T3(autocrine) and neuroblastoma
  • N18TG2 (N18TG2) cells.
  • Cells were seeded into 24-well plates as described in Figure 7. The cells were treated for 48 hours with 0, 50 and 100 nM K252a. Cell viability for all cell lines was determined with the glucose utilization assay on duplicate samples. Histograms (left to right) are indicated as follows: Solid histogram; NCI-H69; Bold diagonally hatched histogram; N18TG2; dotted histogram; 3T3(autocrine); Fine diagonally hatched histogram; Calu-3.
  • the present invention provides methods and pharmaceutical compositions for therapeutically treating mammals bearing tumor cells which express neurotrophins to inhibit or interfere with the growth of the tumor cells and their progeny.
  • the compositions and methods of the present invention involve administering to the affected mammal an effective amount of a substance which interferes with the tumor cells' autocrine survival loop.
  • One mechanism for practicing the present invention involves the use of nudeic adds of at least six nucleotides that are antisense to a gene or cDNA encoding BDNF or a portion thereof.
  • Antisense refers to a nudeic acid capable of hybridizing to a portion of a BDNF RNA (preferably mRNA) by virtue of some sequence complementarty.
  • Another mechanism involves the use of the compound staurosporine,
  • the invention provides a recombinant autocrine loop cell model that embodies many features of tumor cells (such as neuroblastomas and SCLC's) that utilize autocrine survival loops.
  • a cell utilizing an autocrine survival loop refers to a cell which expresses a molecule that is necessary for its own survival. Interruption of Autocrine Loop
  • the antisense nucleic acids of the invention interrupt the BDNF- autocrine loop on an intercellular level, by preventing the synthesis of BDNF by the cell which depends on BDNF expression for survival.
  • the mechanism of K252a and related compounds to interrupt the BDNF-autocrine loop occurs at the level of the BDNF receptor, by preventing the activation and phosphorylation of the trk B receptor.
  • Another rouge of tyrosine protein kinase inhibitors, the thiazolidinedione rouge of inhibitors [Geissler et al, _
  • Biol. Chem., 265:22255-22261 (1990)] may act in a similar manner to K252a.
  • Other protein kinase inhibitors such as calphostin C, staurosporine, K252b, KT5720, KT5823, and KT5926 (Kamiya Biomedical Company, Thousand Oaks, California) may also be used.
  • Other mechanisms for interrupting the BDNF autocrine loop are also encompassed by this invention.
  • the antisense nucleic acids of the invention can be oligonudeotides that are double-stranded or single-stranded, RNA or DNA or a modification or derivative thereof, which can be directly administered to a cell, or which can be produced intracellularly by transcription of exogenous, introduced sequences.
  • the BDNF antisense nucleic acids provided by the instant invention can be used for the treatment of tumors, the cells of which tumor type can be demonstrated (in vitro or in vivo) to express the BDNF gene. Such demonstration can be by detection of BDNF RNA or of BDNF protein.
  • BDNF antisense oligomers not only prevent growth of such tumors, but can also result in death of tumor cells by an unusual mechanism involving programmed or "apoptotic" death, which is characterized by loss of DNA prior to loss of cellular protein. [Arends et al, Am. I. Pathol. 136:593 (1990)].
  • the invention further provides pharmaceutical compositions comprising an effective amount of the BDNF antisense nudeic adds of the invention in a pharmaceutically acceptable carrier. Methods for treatment of various diseases and disorders comprising administering the pharmaceutical compositions of the invention are also provided.
  • the invention is directed to methods for inhibiting the expression of a BDNF nucleic acid sequence in a eucaryotic cell comprising providing the cell with an effective amount of a composition comprising an antisense BDNF nucleic acid of the invention.
  • the identification of cells expressing functional BDNF or other neurotrophin receptors can be carried out by observing the ability of a neurotrophin to "rescue" such cells from the cytotoxic effects of a BDNF antisense nudeic acid.
  • Another aspect of the invention provides for the diagnosis of human neuroblastoma or small cell lung carcinoma by detecting BDNF expression in cells obtained from patients. Such detection can be carried out by detecting BDNF RNA or protein expression.
  • the antisense nucleic acids of the invention are of at least six nudeotides and are preferably oligonudeotides (ranging from 6 to about 50 nucleotides).
  • the oligonudeotides can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double- stranded.
  • the oligonudeotide can be modified at the base moiety, sugar moiety, or phosphate backbone.
  • the oligonudeotide may indude other appending groups such as peptides, or agents fadlitating transport across the cell membrane [see e.g. Letsinger et al, Proc. Natl. Acad. Sci. USA, 86:6553-6556
  • a BDNF antisense oligonudeotide is provided, preferably of single-stranded DNA.
  • such an oligonudeotide comprises a sequence antisense to the last 6 codons of human BDNF.
  • the oligonudeotide may be modified at any position on its structure with substituents generally known in the art.
  • the BDNF antisense oligonudeotide may comprise at least one modified base moiety which is selected from the group including but not limited to 5-fluorouradl, 5-bromouracil, 5-chlorouracil, 5-iodouradl, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5- carboxymethylaminomethyl-2-thiouridine, 5- carboxymethylaminomethyluradl, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2- dimethylguanine, 2methyladenine, 2-methylguanine, 3-methylcytosine, 5— methylcytosine, N6-adenine, 7-methylguanine, 5methylaminomethyluracil, 5- methoxyaminomethyl-2thiouracil, beta-D-mannosy
  • the oligonudeotide comprises at least one modified sugar moiety selected from the group induding but not limited to arabinose. 2- fluoroarabinose, xylulose, and hexose.
  • the oligonudeotide comprises at least one modified phosphate backbone selected from the group consisting of a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a 15 phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal or analog thereof.
  • the oligonudeotide is an -x-anomeric oligonudeotide.
  • An ot-anomeric oligonudeotide forms specific double- stranded hybrids with complementary RNA in which, contrary to the usual ⁇ - units, the strands run parallel to each other [Gautier et al, Nucl. Acids Res., 15:6625-6641 (1987)].
  • the oligonudeotide may be conjugated to another molecule, e.g., a peptide hybridization triggered cross-linking agent, transport agent hybridization-triggered deavage agent, etc.
  • Oligonudeotides of the invention may be synthesized by standard methods known in the art, e.g. by use of an automated DNA synthesizer (such as are commerdally available from Biosearch, Applied Biosystems, etc.).
  • an automated DNA synthesizer such as are commerdally available from Biosearch, Applied Biosystems, etc.
  • phosphorothioate oligos may be synthesized by the method of Stein et al, Nucl. Acids Res..16:3209 (1988)
  • methylphosphonate oligos can be prepared by use of controlled pore glass polymer supports [Sarin et al, Proc. Natl. Acad. Sci. USA.85:7448-7451 (1988)], etc.
  • the BDNF antisense oligonudeotide comprises catalytic RNA, or a ribozyme [see, e.g., PCT International Publication WO 90/11364, published October 4, 1990; Sarver et al. Science
  • the oligonudeotide is a 2'-0- methylribonudeotide [Inoue et al, Nucl. Acids Res.. 15:6131-6148 (1987)], or a chimeric RNA-DNA analogue [Inoue et al. FEBS Lett.. 215:327-330 (1987)].
  • the BDNF antisense nucleic add of the invention is produced intracellularly by transcription from an exogenous sequence.
  • a vector can be introduced in vivo such that it is taken up by a cell, within which cell the vector or a portion thereof is transcribed, produdng an antisense nucleic acid (RNA) of the invention.
  • RNA antisense nucleic acid
  • Such a vector would contain a sequence encoding the BDNF antisense nucleic acid.
  • Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA.
  • Vectors can be constructed by recombinant DNA technology methods standard in the art
  • Vectors can be plasmid, viral, or others known in the art, used for replication and expression in mammalian cells.
  • Expression of the sequence encoding the BDNF antisense RNA can be by any promoter known in the art to act in mammalian, preferably human, cells.
  • Such promoters can be indudble or constitutive.
  • Such promoters indude but are not limited to: the SV40 early promoter region [Bernoist and Chambon, Nature, 290:304-310 (1981)], the promoter contained in the 3' long terminal repeat of Rous sarcoma virus
  • the antisense nudeic adds of the invention comprise complementary to least a portion of a RNA transcript of a BDNF gene, preferably a human BDNF gene. However, absolute complementarily, although preferred, is not required. A sequence "complementary to at least a portion of an RNA", as .
  • referred to herein means a sequence having suffi ⁇ ent complementarily to be a ble to hybridize with the RNA, forming a stable duplex (or triplex, in the case of double-stranded BDNF antisense nudeic adds).
  • the ability to hybridize will depend on both the degree of complementarily and the length of the antisense nudeic add. Generally, the longer the hybridizing nudeic add, the more base mismatches with a BDNF RNA it may contain and still form a stable duplex (or triplex, as the case may be).
  • One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex.
  • the physiologically active substance K252a is a derivative of a substance K252 which was produced by culturing a mi ⁇ oorganism of the genus Nocardiosis [Matsuda et al, U. S. Patent No. 4,555,402].
  • K252 is definedin Murakata et al, U. S. Patent No. 4,877,776 as a compound represented by the
  • Rl and R2 are H or OH ;
  • X is COOH, COOR or CH20H ;
  • Y is H, R or COR; and
  • Z is OH, OR or SR, where R is a lower alkyL
  • K252 has been shown to inhibit the growth of human uterine cancer HeLa cells, human breast cancer cells MCF7, human colon adenocarcinoma cells COLO320DM, human lung cardnoma cells PC10 by means of protein kinase inhibitory activity.
  • Derivatives of K252 are shown in Murakata et al, U. S. Patent No,
  • Wi, W2, R l , R 2 , R 3 , R 4 , X and Y represent various substituents.
  • the trk B tyrosine kinase receptor is inactivated. Suppression of the phosphorylation of cellular proteins is believed to be due to the direct effect of K252a or staurosporine or their derivatives to specifically interfere with BDNF mediated cellular responses.
  • Other protein kinase inhibitors such as thiazolidine-diones, which inhibit EGF-induced receptor phosphorylation, may act similarly to interfere with BDNF-mediated cellular responses.
  • the materials of this invention may be used to treat tumors, of a type which has been shown to express BDNF.
  • Such tumors indude but are not limited to neuroblastoma, small cell lung carcinoma, and some neuroepithelial tumors.
  • a single stranded DNA antisense BDNF oligonudeotide is used in the treatment of neuroblastoma.
  • staurosporine or K252a is used in the treatment of neuroblastoma.
  • rumor types which express BDNF RNA can be identified by various methods known in the art. Such methods indude but are not limited to hybridization with a BDNF-spedfic nucleic acid, e.g. by Northern hybridization, dot blot hybridization, by observing the ability of RNA to be translated in vitro into BDNF, etc.
  • a BDNF-spedfic nucleic acid e.g. by Northern hybridization, dot blot hybridization, by observing the ability of RNA to be translated in vitro into BDNF, etc.
  • primary tumor tissue from a patient can be assayed for BDNF expression prior to treatment.
  • compositions of the invention comprising an effective amount of a substance which interferes with the BDNF autocrine survival loop in a pharmaceutically acceptable carrier, can be administered to a patient having a tumor which is of a type that expresses BDNF RNA.
  • compositions of the present invention for inhibiting the growth of BDNF-expressing tumor cells therefore comprise a therapeutically effective amount of a substance capable of interfering with a BDNF-autocrine loop in admixture with a pharmaceutically acceptable carrier.
  • the pharmaceutical compositions having tumori ⁇ dal activity may be utilized in conventional type formulations such as, e.g., solutions, syrups, emulsions, injectables, tablets, capsules, or suppositories.
  • Suitable carriers are well known to those of skill in the art of pharmacology [see, e.g., Remingtons Practice of Pharmacy, 9th, 10th and 11th Ed.] Exemplary carriers indude sterile saline, lactose, sucrose, caldum phosphate, gelatin, dextrin, agar, pectin, peanut oil, olive oil, sesame oil, squalene and water. Additionally, the carrier or diluent may include a time delay material, such as glycerol monostearate or glycerol distearate alone or with a wax.
  • suitable chemical stabilizers may be used to improve the stability of the pharmaceutical preparation. Suitable chemical stabilizers are well known to those of skill in the art and indude, for example, dtric acid and other agents to adjust pH, chelating or sequestering agents and antioxidants.
  • compositions of the pharmaceutical composition containing K252a, staurosporine, or a derivative thereof, or any other protein kinase inhibitor may conveniently be presented in unit dosage form and may be prepared by any of the conventional methods.
  • the composition may be in a form adapted for slow release in vivo, as is known in the art. All methods include the step of bringing into association the active ingredient with the carrier which may constitute one or more accessory ingredients.
  • the amount of the substance which will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. In one embodiment of this invention, it would be desirable to determine the antisense cytotoxidty of the tumor type to be treated in vitro, e.g. in the assay systems described in the examples infra, and then in useful animal model systems prior to testing and use in humans. Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, oral, and intranasal.
  • intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir.
  • intraventricular catheter for example, attached to a reservoir, such as an Ommaya reservoir.
  • the invention also provides for pharmaceutical compositions comprising substances which interfere with a BDNF-autocrine loop administered via liposomes, micropartides, or microcapsules.
  • compositions comprising substances which interfere with a BDNF-autocrine loop administered via liposomes, micropartides, or microcapsules.
  • it may be useful to use such compositions to achieve sustained release of the substances.
  • liposomes targeted via antibodies to specific identifiable tumor antigens e.g., cell surface antigens selective for neuroblastoma or SCLC
  • K252a or staurosporine or their derivatives may also be employed in accordance with the methods and compositions of this invention, alone or in combination with other therapeutic or diagnostic agents useful in the direct or adjunctive treatment of certain cancers. It is contemplated that K252a or a derivative thereof may be used in combination with the BDNF anti-sense nucleic acids of this invention.
  • agents e.g., antimetabolites, alkylating agents, vinca alkaloids, antineoplastic antibiotics, platinum derivatives, substituted ureas, adrenocortico steroids, cytokines, interleukins, interferons or antibodies, may also be employed in conjunrtion with such kinase inhibitors to treat a variety of cancers characterized by BDNF-expressing cells and related diseases.
  • the dosage regimen involved in administering an effective amount of, for example, K252a in a method for treating the below-described conditions will be determined by the attending physician considering various factors which modify the action of drugs, e.g. the condition, body weight, sex and diet of the patient, the severity of the tumor, time of administration and other clinical factors.
  • the dosage of the compositions of the invention used to treat the specific disease condition des ⁇ ibed herein may be varied depending on the particular disease and the stage of the disease.
  • compositions containing K252a or a derivative thereof, staurosporine, or other kinase inhibitors also contain another conventional therapeutic agent, such as cyclophosphamide, cytokines, interleukins, interferons or antibodies, as mentioned above.
  • another conventional therapeutic agent such as cyclophosphamide, cytokines, interleukins, interferons or antibodies, as mentioned above.
  • the antisense oligonudeotides of this invention may be combined with pharmaceutical compositions containing protein kinase inhibitors. When these agents are combined in a pharmaceutical composition, it is antidpated that each active ingredient will be present in the combined composition in the same concentration or slightly lower concentration than if this agent was administered alone.
  • compositions and methods of the present invention differs in prindple from that of the large majority of drugs in use for treatment of tumors assodated with BDNF-expression in use at the present time.
  • substances which interfere with the BDNF-autocrine survival loop display highly spedfic activity so that patients do not suffer the many disadvantages of conventional cancer therapy.
  • BDNF-expressing tumors susceptible to treatment by the present method and compositions include, but are not limited to, neuroblastoma, small cell lung carcinoma, and some neuroepithelial tumors.
  • Other tumor types which express BDNF can be identified by various methods known in the art. According to the method of the present invention, where desired, primary tumor tissue from a patient can be assayed for BDNF expression prior to treatment.
  • the methods and compositions of this invention may be utilized for veterinary purposes in the treatment of BDNF-expressing tumors that afflict horses, swine, cattle and fowl, for example. These disorders may be treated using quantities of the compound that may be used in treating the mammalian disorders described herein above.
  • BDNF mRNA expression appears to be unique to neuroblastoma with only a few exceptions (such as small cell lung cardnoma and a few neuroepithelial tumor cells). These results suggest that BDNF mRNA expression may serve as a useful marker clinically for human neuroblastoma, as well as SCLC and some neuroepithelial tumors.
  • BDNF mRNA expression may be a more useful and broad ranging clinical marker for both early and late stage neuroblastoma.
  • BDNF, NGF and NT-3 see Section 6, infra.
  • some neuroblastomas express functional receptors for BDNF, NGF or NT-3 based on the ability of these individual ligands to rescue such a cell type from 5 antisense cytotoxidty.
  • our data (Example 1., section 1.1) suggest that LA-N-5 neuroblastoma cells express functional receptors for NGF, BDNF, and NT-3, while LA-N-1 cells express only functional BDNF receptors, and that CHP-134 and CHP-404 neuroblastoma cells express both NGF and BDNF receptors but lack NT-3 receptors.
  • BDNF-dependent autocrine survival loop in neuroblastomas, the properties of such cells reflecting such a survival loop such as detertable levels of mRNA for trkB, or detectable levels of a constituitively phosphorylated trk receptor, were not readily detected.
  • a recombinant cell system was created.
  • This system utilized a BDNF/trkB mediated autocrine loop.
  • This system is based on a variant NTH 3T3 fibroblast cell line whose growth and survival in defined media normally requires either fibroblast growth factor (FGF) or platelet-derived growth fartor (PDGF) [Lee, and Donoghue, J.
  • FGF fibroblast growth factor
  • PDGF platelet-derived growth fartor
  • these autocrine NIH3T3 cells are in many ways similar to neuroblastomas dependent upon a BDNF autocrine survival loop. For example, they display a similar sequence-spedfic susceptibility to BDNF antisense oligomer, which can be overcome by exogenous BDNF. Furthermore, these cells do not secrete detectable levels of BDNF into the media, nor do they display detectable levels of a constitutively phosphorylated trkB receptor.
  • K252a and staurosporine act on BDNF/trkB-transfected NIH3T3 cells grown in defined media in a manner which is very similar to their effect on neuroblastomas, thus confirming that such cells provide a useful assay system for identifying agents that can be used to destroy autocrine loop dependent tumor cells through disruption of the autocrine loop.
  • Other autocrine loop model systems which are engineered to encode and express a particular fartor, as well as the receptor for that factor, may also be created and used, as contemplated herein, to identify agents that interrupt such autocrine loops.
  • Such factors include, but are not limited to, nerve growth factor, neurotrophin-3, neurotrophin-4, and ciliary neurotrophic factor.
  • antisense oligonudeotides directed against BDNF are cytotoxic in vitro to neuroblastoma cell lines, thus demonstrating that human neuroblastoma cells require BDNF as an autocrine survival molecule and that these neuroblastoma cells can be rescued from the cytotoxic effects of antisense BDNF by administering exogenous BDNF. Since most of these neuroblastoma cells do not express detectable levels of trk B mRNA, our data imply that additional receptors for
  • BDNF might exist. 1.1 Human and Rodent Neuroblastoma Cell Lines Express BDNF mRNA
  • the single neuroblastoma cell line that did not express BDNF mRNA was SH-SY5Y which is unique in that it is of thoradc - as opposed to neural crest-derived origin.
  • Dr. Mark Israel (UCSD) provided RNA blots of some of these neuroblastoma cell lines. Table I
  • K562 MCF7 *Data are a summary of Northern blotting results of human cell line RNAs probed with human BDNF. (+) indicates positive expression of hBDNF mRNA and (-) indicates a lack of BDNF mRNA. (+/-) indicates a very low level of expression. (Actual Northern blotting data is shown in the BDNF patent).
  • oligonudeotide Three antisense oligonudeotides were synthesized complementary to various regions of the human 5 BDNF gene [see PCT International Publication No. WO91/03568, published March 21, 1991] as set forth in Table II below. Each oligonudeotide (“oligo”) was made as an 18-mer and the complementary sense oligos served as controls in all experiments.
  • the 5' antisense oligo (5'-AS-BDNF; SEQ ID NO: 1) consisted of human BDNF DNA sequence beginning 3 nudeotides upstream of the presumptive ATG start codon and extending 4 codons downstream from this initiating methionine.
  • the second antisense oligo corresponded to
  • BDNF DNA sequence around the dibasic residue processing site (PS- AS- BDNF; SEQ ID NO: 3) and the third antisense oligo (the 3"-AS-BDNF; SEQ ID NO: 5) corresponded to the last 6 codons of human BDNF.
  • AS - refers to antisense sequence
  • S - refers to sense sequence
  • PS - refers to dibasic amino acid processing site.
  • the presumptive ATG start codon is highlighted in the 5'-S-BDNF sequence.
  • the Arg-Lys dibasic residue codons are indicated in the PS-S-BDNF sequence. All oligonudeotides were synthesized on an Applied Biosystems nucleic add synthesizer.
  • Eadi antisense BDNF oligonudeotide was first tested for its ability to inhibit the synthesis of BDNF using a wheat germ lysate in vitro translation system ( Figure 1).
  • the synthesis of BDNF (+/- antisense or sense oligonudeotide) was followed in this assay system by metabolic labeling with 35S-methionine, polyacrylamide gel electrophoresis, and fluorography ( Figure 1). It was observed that a random 18-mer (control oligo) had no inhibitory effect on BDNF synthesis in vitro, at both 1 and 6 ⁇ M concentrations.
  • the 5'-AS-BDNF (SEQ ID NO: 1) oligo had a slight inhibitory effect on BDNF synthesis in vitro at a concentration of l ⁇ M and profound inhibitory effects at 6 ⁇ M.
  • the 3'- AS-BDNF (SEQ ID NO: 5) and the PS-AS-BDNF (SEQ ID NO: 3) both effectively inhibited BDNF synthesis at concentrations of l ⁇ M and 6 ⁇ M.
  • the complementary sense oligos had little to no effect on the in vitro synthesis of BDNF at l ⁇ M but did have some inhibitory activity at very high concentrations (6>tM) (representative data shown for the 5'-S-BDNF oligo (SEQ ID NO: 2); Figure 1).
  • BDNF BDNF, PS-AS-BDNF, and 3'- AS-BDNF
  • Human neuroblastoma cells were cultured in Eagle's modified essential medium (EMEM) with 10% fetal bovine serum (FBS), 2mM glutamine, and 1% each of penidllin and streptomycin (complete media).
  • EMEM Eagle's modified essential medium
  • FBS fetal bovine serum
  • penidllin and streptomycin complete media
  • the cells were plated into 24-well Costar plates at a seeding density of 2 x 104 cells/well.
  • Antisense oligonudeotide uptake was carried out by adding antisense oligos directly to the cells in EMEM without serum.
  • the concentration of antisense or sense oligonudeotide added to the cell culture media ranged from 0.1 to 5Q*M ( Figure 2).
  • ITS supplement insulin, transferrin and selenium
  • LA-N-1 neuroblastoma cells were somewhat less sensitive than LAN-5 cells, as only 40% of these cells were killed with 1 responsible-M 3 -AS-BDNF at 96 hours.
  • LA-N-1 cells express less BDNF mRNA than LA-N- 5 cells.
  • SK-ES and SH-SY5Y cells (which do not express BDNF mRNA) were resistant to the cytotoxic effects of the 3'- AS-BDNF even at concentrations of 50 ⁇ M.
  • BDNF (SEQ ID NO: 5).
  • BDNF antisense oligomers but not the control oligomers, had profound effects on cellular morphology when added to neuroblastoma cultures at low concentrations ( Figure 6A-C). As would be expected if these effects were derived from the disruption of a BDNF autocrine loop, antisens-mediated changes in cell morphology could be prevented by the addition of exogenous BDNF ( Figure 6D).
  • Figure 3 (A - E) shows the results of coaddition experiments where either BDNF, NGF, or neurotrophin 3 (NT-3) (100 ng/ml of each purified recombinant factor, obtained from CHO cells transfected with the respective neurotrophin gene [see PCT International Publication No. WO 2011/001100 ng/ml of each purified recombinant factor, obtained from CHO cells transfected with the respective neurotrophin gene [see PCT International Publication No. WO
  • oligonudeotide 50 uM was added simultaneously with the 3'-AS-BDNF (SEQ ID NO: 5) oligonudeotide (50 uM) to various neuroblastoma cells (i.e., SH-SY5Y (A), LA-N-1 (B), LA-N-5 (C), CHP-134 (D), CHP-404 (E)).
  • Cell viability was determined on duplicate wells of a 24-well plate by trypan blue staining at 24 hour intervals after the addition of 3'-AS-BDNF +/- neurotrophic factor. Cell number was also recorded with a hemocytometer.
  • the oligonudeotide uptake was carried out by adding the oligo directly to the cell culture system under serum-free conditions for 4 hours.
  • BDNF antisense oligomers not only prevented neuroblastoma growth, but also resulted in the death of neuroblastoma tumor cells ( Figure 6B). If BDNF is indeed functioning in these cells as a neuronal survival molecule, it would be expected that the death due to disruption of a BDNF autocrine loop might occur by mechanisms similar to those described previously for neuronal cell death following neurotrophic factor deprivation [Martin, et al. L. Cell Biol. 106:829 (1988); Scott, et al. Neurobiol.
  • neuronal death may generally be marked by some of the same biochemical changes that characterize programmed cell death in other systems such as the thymus and the prostate (Batistatou, supra: Rukenstein. supra, Wylie, et al. Int. Rev. Cytol. 68: 251(1980)].
  • the activation of the endogenous caldum-dependent endonuclease that results in the loss of DNA prior to the loss of cellular protein may be a general feature of programmed or
  • apoptotic death [Arends, et al. Am T. Pathol. 136: 593 (1990)].
  • the DNA profile of LA-N-5 human neuroblastoma cells is typical of many normally cycling cell lines, with a large percentage of the cell population in the Gl phase of the cell cyde, and the remainder of the population in either S phase or in G2+M (Figure 7A).
  • Treatment with antisense BDNF oligomers results in the appearance of an apoptotic population of LA-N-5 cells, characterized by a significantly reduced DNA content in the absence of protein loss (Figure 7B); these changes are accompanied by a decrease in the percentage of cells in S-phase, as usually seen in apoptotic populations (Del Bino, supra).
  • BDNF rescue of antisense- treated autocrine cells prevented the appearance of the apoptotic population ( Figure 7C).
  • neuroblastoma cells were not susceptible to low concentrations of BDNF sense or random sequence oligomers, these oligomers resulted in neuroblastoma cell death, as well as death of cells not dependent on BDNF autocrine loops, when present at high concentrations (see below).
  • exogenous BDNF did not alter the effects of either sense or antisense oligomers on SY5Y cells ( Figure 8B), exogenous BDNF markedly shifted the antisense oligomer kill curve on LA-N-5 cells so that it matched that of the control sense oligomer ( Figure 8A).
  • BDNF-negative Ewing's sarcoma cell line was similar to SY5Y in its insensitivity to antisense BDNF oligomers
  • examination of four additional BDNF-positive cell lines revealed an extraordinarily susceptibility to antisense BDNF oligomers, as well as an ability to be rescued by BDNF, that was essentially indistinguishable from that of LA-N-5 cells (data not shown).
  • the antisense BDNF oligomer acts in a sequence-specific manner and only on BDNF-expressing neuroblastomas.
  • BDNF antisense oligomers selectively activate apoptotic cell death in neuroblastomas by disrupting an autocrine survival loop dependent on the continued synthesis of BDNF.
  • BDNF mRNA expression may serve as a useful marker dinically for human neuroblastoma.
  • the best clinical marker for neuroblastoma to date is N-myc amplification and that N-myc is only amplified in approximately 30% of all late-stage neuroblastoma (Stages IH and IV)
  • BDNF mRNA expression may be a more useful and broad ranging clinical marker for both early and late stage neuroblastoma.
  • BDNF mRNA-positive neuroblastomas require a BDNF autocrine loop for their own proliferation and survival.
  • Our results suggest that at least some neuroblastomas may be effectively and specifically killed by treatment with antisense BDNF oligonudeotides.
  • Some neuroblastoma cell lines can be effectively rescued from the cytotoxic effects of antisense BDNF oligonudeotides by the addition of either BDNF, NGF or NT-3.
  • BDNF autocrine survival factor for small cell lung carcinoma tumors
  • RNA samples prepared from six different small cell lung carcinoma cell lines were obtained from Dr. Jim Battey's laboratory at the NIH.
  • the cell lines shown in Figure 4 are as follows: H82 (lane 2), H209 (lane 3), H345 (lane 4), H378 (lane 5), H510 (lane 6), and N417 (lane 7).
  • 10 ug of each of the cell line RNAs were used for the Northern blot, and the level of BDNF mRNA was compared directly with adult rat brain (lane 1).
  • Oligonudeotides are Cytotoxic For BDNF-Expressing Small Cell Lung Carcinoma Cells
  • Antisense BDNF nudeotides prepared as set forth in Example 1.2 were added directly to cultures of SCLC cells (H345 and H378) in vitro. Assays were conducted as set forth in Example 1.3.
  • the concentration of antisense or sense oligonudeotide added to the cell culture media ranged from 0.1 to 50 uM ( Figure 8).
  • ITS supplement insulin, transferrin and selenium was added to the cell culture wells and cell viability was assessed at 96 hours after oligonudeotide addition. Duplicate wells were assayed by trypan blue staining.
  • both the H345(8A) and H378(8B) cells were extremely sensitive to the cytotoxic effects of the 3 aannttiisseennssee BBDDNNFF((SEQ. ID NO:5) oligonudeotide, but not to 5 or sense oligonudeotide.
  • Figure 9(A & B) shows the result of co-addition experiments where BDNF (lOOng/ml, obtained from CHO cells transfected with the respective neurotrophin gene) was added simultaneously with the 3'-AS-BDNF (SEQ ID NO: 1
  • BDNF autocrine loop for their own proliferation and survival and that such cells may be effectively killed by treatment with antisense BDNF oligonudeotides.
  • K252a can art to spedfically block neurotrophin-mediated cellular responses.
  • PC-12 cells were grown to approximately 70% confluency in 100 mm tissue culture dishes with serum-containing medium (DME supplemented with 6% equine serum, 6% calf serum, 1% glutamine, 1% penidllin, 1% streptomycin). Growth factors and inhibitors were diluted into the same medium and administered to the cells in 200 microliter aliquots.
  • DME serum-containing medium
  • NGF and FGF stimulate early cellular responses in PC12 cdls via independent signal transduction pathways differentiated by the spedficity of staurosporine and K252a NGF- mediated responses.
  • Lysates from untreated cells and those treated with BDNF were compared with cells which had been pretreated with inhibitors alone [25 microM H-7, 100 nM K252a, and 100 nM staurosporine] and cells which had been pretreated with inhibitors and then administered 100 ng/ml BDNF [25 microM H-7, 100 nM K252a, and 100 nM staurosporine],
  • the 3T3 cell line chosen for these studies is dependent on FGF for survival in serum-free defined media.
  • 3T3 cells expressing both trkB and BDNF survive in defined media alone and serve as a useful model cell system for autocrine survival, resembling human neuroblastoma tumor cell lines.
  • both LA ⁇ N-5 and SK-N-LO cells express BDNF mRNA while SH-SY5Y and SK-ES cells do not express BDNF mRNA.
  • trkB expression at the mRNA level was only detectable in the LA-N-5 cell line.
  • Cells were seeded into 24-well plates at an approximate density of 2 x 10* cells per well.
  • Human neuroblastoma cells SCLC cells and lung adenocar ⁇ noma cells were cultured in RPMI 1640 with fetal bovine serum, while 3T3 cells were cultured in serum-free defined media with the appropriate neurotrophin (i.e., FGF, BDNF or NGF at 500 pM) [Glass et al, Cell, supral. TrkB/BDNF autocrine 3T3 cells (MBx) were cultured in defined media alone. All cell culture media contained 450 mg/dl of glucose at the time of cell seeding.
  • neurotrophin i.e., FGF, BDNF or NGF at 500 pM
  • K252a was added (solubilized in DMSO - see Example 1) at concentrations of 0, 10, 50, 100, 250, 500, 1000, and 2000 nM. All assays were performed in triplicate. Cell morphology was monitored visually and cell viability was assessed by the glucose utilization assay after 6 days in culture. Glucose concentration (mg/dl) was determined by transferring a 50 ul aliquot of the growth media to blood glucose strips and then reading these strips 2 minutes later in a blood glucose monitor. Glucose readings were averaged and then plotted versus the concentration of K252a to estimate the LD5 0 for each drug on each cell line. Table 3 shows the LD5 0 values for K252a for each cell line.
  • SK-N-LO cells apparently do not express trkB, our data also suggest that these cells may express a unique K252a-sensitive trk-like BDNF receptor or, alternatively, that the level of trkB expression in these cells is below the detectable limits. Further, our data demonstrates that protein kinase inhibitors such as K252a are cytocidal for small cell lung cardnoma cells but not cytoddal for lung adenocarcinoma cells. A comparison of the effect of K252a on 3T3-autocrine, neuroblastoma cells ( N18TG2), small cell lung carcinoma (NCI-H69) and lung adenocar ⁇ noma cells (Calu-3) is shown in Figure 12.
  • MG87 cells are 3T3 cells which require FGF for survival in defined medium.
  • MG87 trk-A cells require NGF.
  • MG87 trk-B cells require BDNF.
  • K252a and staurosporine are more efficadous (approximately 6-fold) for 3T3 fibroblasts expressing trk receptor kinases (MBx, MG87 trkA, and MG87 trkB) which require neurotrophins for survival in defined media relative to the parental 3T3 cells (MG87), which survive in defined media supplemented with FGF.
  • MG87 parental 3T3 cells
  • K252a may be an important selective antagonist of the neurotrophin/trk receptor signal transduction pathway and, therefore, might be potentially useful therapeutically for the killing of neurally-derived tumor cells dependent on a BDNF autocrine survival loop.
  • K252a cancer cell lines which express BDNF would be similarly affected by K252a.
  • some neuroepithelial tumors would be expected to be adversely affected by K252a and other protein kinase inhibitors.

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Abstract

Procédé de traitement de tumeurs exprimant la neurotrophine, consistant à interrompre la boucle de survie autocrine par l'administration d'une quantité pharmaceutiquement efficace d'une substance capable d'interrompre ladite boucle autocrine. Des acides nucléiques non codants et du K252a ou ses dérivés, par exemple, peuvent être utilisés dans des compositions pharmaceutiquement acceptables pour interrompre la boucle autocrine d'une cellule tumorale qui dépend de la neurotrophine qu'elle exprime pour sa survie. Un système modèle destiné à identifier d'autres moyens d'interrompre les boucles autocrines est également décrit.
PCT/US1992/003392 1991-07-03 1992-04-23 Procede et systeme de determination de l'activite de la neurotrophine WO1993000909A1 (fr)

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EP92915861A EP0593663A4 (en) 1991-07-03 1992-04-23 Method and assay system for neurotrophin activity
JP5502196A JPH06509333A (ja) 1991-07-03 1992-04-23 ニューロトロフィン活性に対する方法およびアッセイ系
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WO1993025683A1 (fr) * 1992-06-12 1993-12-23 Massachusetts Institute Of Technology Gene empechant la mort cellulaire programmee
US5468872A (en) * 1993-09-16 1995-11-21 Cephalon, Inc. K-252a functional derivatives potentiate neurotrophin-3 for the treatment of neurological disorders
US5516771A (en) * 1993-05-28 1996-05-14 Cephalon, Inc. Use of indolocarbazole derivatives to treat a pathological condition of the prostate
US5599808A (en) * 1994-02-18 1997-02-04 Cephalon, Inc. Aqueous indolocarbazole solutions
US5602309A (en) * 1993-10-04 1997-02-11 University Of Kentucky Research Foundation Transgenic mice which overexpress nerve growth factor
WO1997001344A3 (fr) * 1995-06-27 1997-03-27 Jackson H M Found Military Med Procede de ralentissement dynamique de la cinetique du cycle cellulaire, destine a potentialiser les lesions cellulaires
US5859311A (en) * 1995-11-27 1999-01-12 University Of Kentucky Research Foundation Transgenic mice which overexpress neurotrophin-3 (NT-3) and methods of use
EP1002534A1 (fr) * 1992-07-24 2000-05-24 Cephalon, Inc. Dérivés de K-252a et de bis-staurosporine
US6200968B1 (en) 1998-08-06 2001-03-13 Cephalon, Inc. Particle-forming compositions containing fused pyrrolocarbazoles
US6274576B1 (en) 1995-06-27 2001-08-14 The Henry Jackson Foundation For The Advancement Of Military Medicine Method of dynamic retardation of cell cycle kinetics to potentiate cell damage
WO2004101072A1 (fr) * 2003-05-16 2004-11-25 Universite Laval Modulation de chlorure du snc et ses utilisations
US6902732B2 (en) 1992-06-12 2005-06-07 Massachusetts Institute Of Technology Identification and characterization of a gene which protects cells from programmed cell death and uses therefor
WO2005051958A3 (fr) * 2003-11-20 2006-01-12 Cephalon Inc Compositions de formation de particules contenant des pyrrolocarbazoles fusionnes
US7718382B2 (en) 2004-05-14 2010-05-18 Universite Laval Method for identifying compounds for treatment of pain
US20150133529A1 (en) * 2012-05-16 2015-05-14 Rana Therapeutics, Inc. Compositions and methods for modulating bdnf expression
EP3029061A1 (fr) 2008-06-18 2016-06-08 The Texas A&M University System Cellules souches mésenchymateuses, compositions et procédés pour le traitement de lésions du tissu cardiaque
CN110438125A (zh) * 2012-03-15 2019-11-12 科纳公司 通过抑制脑源神经营养因子(bdnf)的天然反义转录物治疗bdnf相关疾病

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Cited By (27)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6312947B1 (en) 1992-06-12 2001-11-06 Massachusetts Institute Of Technology Identification and characterization of a gene which protects cells from programmed cell death and uses therefor
WO1993025683A1 (fr) * 1992-06-12 1993-12-23 Massachusetts Institute Of Technology Gene empechant la mort cellulaire programmee
US6902732B2 (en) 1992-06-12 2005-06-07 Massachusetts Institute Of Technology Identification and characterization of a gene which protects cells from programmed cell death and uses therefor
US6465617B1 (en) 1992-06-12 2002-10-15 Massachusetts Institute Of Technology Identification and characterization of a gene which protects cells from programmed cell death and uses therefor
EP1002534A1 (fr) * 1992-07-24 2000-05-24 Cephalon, Inc. Dérivés de K-252a et de bis-staurosporine
US5516771A (en) * 1993-05-28 1996-05-14 Cephalon, Inc. Use of indolocarbazole derivatives to treat a pathological condition of the prostate
US5654427A (en) * 1993-05-28 1997-08-05 Kyowa Hakko Kogyo Co., Ltd. Indolocarbazole derivatives to treat a pathological condition of the prostate
US5468872A (en) * 1993-09-16 1995-11-21 Cephalon, Inc. K-252a functional derivatives potentiate neurotrophin-3 for the treatment of neurological disorders
US5516772A (en) * 1993-09-16 1996-05-14 Cephalon, Inc. K-252 derivatives which enhance neurotrophin-induced activity
US5602309A (en) * 1993-10-04 1997-02-11 University Of Kentucky Research Foundation Transgenic mice which overexpress nerve growth factor
US5599808A (en) * 1994-02-18 1997-02-04 Cephalon, Inc. Aqueous indolocarbazole solutions
WO1997001344A3 (fr) * 1995-06-27 1997-03-27 Jackson H M Found Military Med Procede de ralentissement dynamique de la cinetique du cycle cellulaire, destine a potentialiser les lesions cellulaires
US6274576B1 (en) 1995-06-27 2001-08-14 The Henry Jackson Foundation For The Advancement Of Military Medicine Method of dynamic retardation of cell cycle kinetics to potentiate cell damage
US6455593B1 (en) 1995-06-27 2002-09-24 The Henry Jackson Foundation For The Advancement Of Military Medicine Method of dynamic retardation of cell cycle kinetics to potentiate cell damage
US5859311A (en) * 1995-11-27 1999-01-12 University Of Kentucky Research Foundation Transgenic mice which overexpress neurotrophin-3 (NT-3) and methods of use
US6200968B1 (en) 1998-08-06 2001-03-13 Cephalon, Inc. Particle-forming compositions containing fused pyrrolocarbazoles
US7795246B2 (en) 1998-08-06 2010-09-14 Cephalon, Inc. Particle-forming compositions containing fused pyrrolocarbazoles
EP2198922A1 (fr) * 2003-05-16 2010-06-23 Universite Laval Modulation de chlorure du SNC et ses utilisations
US7709207B2 (en) 2003-05-16 2010-05-04 Universite Laval Method for identifying compounds for treatment of pain
WO2004101072A1 (fr) * 2003-05-16 2004-11-25 Universite Laval Modulation de chlorure du snc et ses utilisations
EA010293B1 (ru) * 2003-11-20 2008-08-29 Сефалон, Инк. Частицеобразующие композиции, содержащие конденсированные пирролокарбазолы
WO2005051958A3 (fr) * 2003-11-20 2006-01-12 Cephalon Inc Compositions de formation de particules contenant des pyrrolocarbazoles fusionnes
EA014713B1 (ru) * 2003-11-20 2011-02-28 Сефалон, Инк. Способ получения частицеобразующих композиций, содержащих конденсированные пирролокарбазолы
US7718382B2 (en) 2004-05-14 2010-05-18 Universite Laval Method for identifying compounds for treatment of pain
EP3029061A1 (fr) 2008-06-18 2016-06-08 The Texas A&M University System Cellules souches mésenchymateuses, compositions et procédés pour le traitement de lésions du tissu cardiaque
CN110438125A (zh) * 2012-03-15 2019-11-12 科纳公司 通过抑制脑源神经营养因子(bdnf)的天然反义转录物治疗bdnf相关疾病
US20150133529A1 (en) * 2012-05-16 2015-05-14 Rana Therapeutics, Inc. Compositions and methods for modulating bdnf expression

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