WO1993009798A1 - Methodes therapeutiques et diagnostiques basees sur l'expression de nt-3 specifique de tissus et sur la liaison de recepteurs - Google Patents

Methodes therapeutiques et diagnostiques basees sur l'expression de nt-3 specifique de tissus et sur la liaison de recepteurs Download PDF

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WO1993009798A1
WO1993009798A1 PCT/US1992/009652 US9209652W WO9309798A1 WO 1993009798 A1 WO1993009798 A1 WO 1993009798A1 US 9209652 W US9209652 W US 9209652W WO 9309798 A1 WO9309798 A1 WO 9309798A1
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disease
nervous system
system cells
binding
disorder
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PCT/US1992/009652
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English (en)
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C. Anthony Altar
Peter Distefano
Stanley Wiegand
Nancy Ip
Carolyn Hyman
Roseann Ventimiglia
Ronald Lindsay
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Regeneron Pharmaceuticals, Inc.
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Publication of WO1993009798A1 publication Critical patent/WO1993009798A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/185—Nerve growth factor [NGF]; Brain derived neurotrophic factor [BDNF]; Ciliary neurotrophic factor [CNTF]; Glial derived neurotrophic factor [GDNF]; Neurotrophins, e.g. NT-3
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators

Definitions

  • the present invention relates to
  • neurotrophin-3 (NT-3), a member of the NGF/BDNF/NT-3/NT-4 gene family and to therapeutic and diagnostic methods utilizing neurotrophin-3 in the treatment of neurological disorders.
  • a family of neurotrophic factors has been identified that includes ⁇ -nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF),
  • Neurotrophin-3 neurotrophin-3
  • HDNF hippocampus-derived neurotrophic factor
  • NT-4 neurotrophin-4
  • Nerve growth factor (NGF) is by far the best characterized neurotrophic factor (Levi-Montalcini and Angeletti, 1968, Physiol. Rev. 48:534-569; Thoenen and Barde, 1980, Physiol. Rev. 60:1284-13335).
  • mice submaxillary gland is a rich source of NGF allowed for the purification and amino acid sequence analysis of mouse NGF (Angelitti, et al., 1973, Biochemistry 12:100-115) and DNA sequence analysis of mouse and human NGF (Scott, et al., 1983, Nature 302: 538-540; Ullrich, et al., 1983, Nature 303:821-825).
  • Comparison of mouse and human NGF showed that the protein is conserved within animals and in support of this, NGF-like activities have been isolated from several species (Harper and Thoenen, 1981, Ann. Rev. Pharmacol. Toxicol. 21:205- 229).
  • BDNF Brain-derived neurotrophic factor
  • NT-3 which bears structural similarity to both NGF and BDNF, has been isolated from mouse (Hohn, et al., 1990, Nature, 344:339-341), rat (Maisonpierre, et al., 1990, Science 247: 1446-1451; Ernfors, et al., 1990, Proc. Natl. Acad. Sci. USA 87: 5454-5458), and human (Rosenthal, et al., 1990, Neuron 4:767-773; PCT Application No.
  • hippocampus (Ayer-LeLievre, et al., 1988, Science 240: 1339-1341; Ernfors, et al., 1990, Proc. Natl. Acad. Sci. USA, 87: 5454-5458; Ernfors, et al., 1990, Neuron 5: 511-526; Whetmore, et al., 1991, Neurol. 109: 141-152; Hofer, et al., 1990 EMBO J., 9:2459-2464,
  • NGF, BDNF and NT-3 support the growth of both overlapping and unique sets of neuronal growth factor
  • NGF supports the development and maintenance of peripheral sympathetic and neural crest-derived sensory neurons (reviewed in Thoenen and Barde, 1980, Physiol. Rev., 60:1284-1325; Levi-Montalcini, 1987,
  • BDNF has been observed to support the survival of both placode and neural crest derived sensory neurons (Hofer and Barde, 1988,
  • NGF Basal forebrain cholinergic neurons
  • the effects of the three proteins are mediated by their interaction with specific receptors present on sensitive cells.
  • the trkA, trkB and trkC members of tyrosine protein kinases have been
  • the present invention relates to methods for the diagnosis and treatment of neurological disorders involving target cells for NT-3. Patterns of NT-3 expression in vivo and the distribution of tissue specific displaceable receptor binding sites for NT-3 in mammals are described.
  • the invention is based, in part, on the discovery that high NT-3 receptor binding densities exist in mammalian brain sections in the anterior nucleus of the olfactory bulb, layer 1 of neocortex, the nucleus of the lateral olfactory tract, the dentate gyrus, CA1, CA3 and CA4 of the hippocampus, and the caudate-putamen.
  • high NT-3 receptor binding densities exist in mammalian brain sections in the anterior nucleus of the olfactory bulb, layer 1 of neocortex, the nucleus of the lateral olfactory tract, the dentate gyrus, CA1, CA3 and CA4 of the hippocampus, and the caudate-putamen.
  • the invention is further based on the discovery that mRNA encoding the NT-3 receptor, trkC, is expressed in the striatum. Pathological conditions involving such regions which display high levels of NT-3 expression and/or moderate to high density NT-3 receptor may be diagnosed or treated in accordance with the invention.
  • the striatum is an area of the brain involved in a number of neurological disorders, including Huntington's chorea, in particular
  • the present invention provides for methods of diagnosing and treating disorders of the striatum that utilize NT-3, fragments of NT-3, NT-3 derivatives, or antibodies that bind to NT-3 or NT-3 receptors.
  • the invention is still further based on the discovery that NT-3 is retrogradely transported by certain sensory neurons as well as neurons of the hippocampus and neostriatum. Therefore, the present invention, in particular embodiments, provides for methods of diagnosing and treating disorders of sensory neurons, the hippocampus, and the neostriatum that utilize NT-3, fragments of NT-3, NT-3
  • NT- 3, fragments of NT-3, NT-3 derivatives, or antibodies that bind specifically to NT-3 receptors may be labeled and utilized in an imaging protocol to locate cells and tissues in the body that express the NT-3 receptor in vivo.
  • Anti-NT-3 antibodies may likewise be utilized to image native NT-3 bound to the surface of such target cells. Abberrant expression patterns imaged using such techniques may be analyzed to diagnose pathological conditions.
  • NT-3 compositions or anti-NT-3 antibodies may be used to potentiate or block the biological effect of NT3 on target cells and tissues in vivo.
  • Specific diseases that may be diagnosed and/or treated in accordance with the invention are described herein.
  • FIGURE 1 Amounts of [ 125 I]NT-3 bound to horizontal rat brain sections during association and dissociation binding assays. Each value is the mean ⁇ s.e.m. fmol of NT-3 bound per mg protein calculated from the dpm per section from each 4 animals.
  • n 4
  • FIGURE 4 Coronal and horizontal rat brain sections were incubated in 300 pM [ 125 I]NT-3 alone or with 300 nM NT-3 to define non-displaceable binding in adjacent sections. Note the high levels of displaceable binding in the dentate gyrus, CA pyramidal layers of hippocampus, caudate-putamen, and superficial neocortex.
  • FIGURE 5 Coronal adult cat brain sections were incubated in 300 pM [ 125 I]NT-3 alone or with 300 nM
  • NT-3 to define non-displaceable binding in adjacent sections. Note the similarity of binding as observed in the rat shown in Figure 4.
  • FIGURE 6 Horizontal sections from two adult humans were collected through the basal ganglia and incubated in [ 125 I]NT-3 alone or with 300 nM NT-3 as in Figures 4 and 5. Intense and highly displaceable labeling was present in the caudate nucleus, putamen, neocortex, and claustrum.
  • FIGURE 7 Northern blot analysis of
  • trkA 2.6 Kb Xho1 Fragment
  • trk B 1.1 Kb Hpa/Sca
  • FIGURE 8 Northern blot analysis depicting the time-course effect of NGF, BDNF and NT-3 on the induction of C-FOS.
  • C-FOS 1.1 Kb Pst1 Fragment;
  • GAPD4 1.25 Kb Pst1 Fragment.
  • FIGURE 9 Northern blot analysis of trkB in rat brain.
  • the predominant band (arrow, approximately 7.0 to 9.0 Kb) was measured by densitometric scanning, as shown in Figure 11.
  • FIGURE 10 Northern blot analysis of trkC mRNA expression in rat brain. The predominant band (arrow, approximately 15 Kb) was measured by
  • FIGURE 11 Quantitation of the predominant trkB transcript in developing rat striatum.
  • FIGURE 12 Quantitation of the predominant trkC transcript in developing rat striatum.
  • FIGURE 15 Dose-dependent induction of C-FOS expression in dissociated cerebellum cultures in vitro by neurotrophin-3.
  • FIGURE 16 Dose-dependent induction of C-FOS expression in dissociated cerebellum cultures in vitro by brain-derived neurotrophic factor.
  • FIGURE 17 Dose-dependent induction of
  • FIGURE 18 Effect of NT-3 on dopaminergic neuron survival in vitro. Cultures of E14 ventral mesencephalon were prepared as described previously (Hyman, et al., 1991, Nature 350:230-233). Following a four hour attachment period, the culture medium was switched to a serum-free formulation at which time purified human recombinant NT-3 was added to the indicated concentrations. The cultures were maintained for 7 days in vitro, then they were
  • FIGURE 19 Effect of NT-3 on dopamine uptake in vitro. Cultures were prepared as in
  • FIG. 18 Purified human recombinant NT-3 was added at the time of the culture media switch as described in Figure 18. Each concentration of NT-3 was tested in sets of five replicate dishes. Cultures were maintained for 7 days in vitro, at which time they were processed for the measurement of high affinity dopamine uptake.
  • FIGURE 20 Effect of NT-3 on GABA uptake in vitro. Cultures were prepared as in Figure 18.
  • NT-3 Purified human recombinant NT-3 was added to the cultures at the time of the media switch as described. Each concentration of NT-3 was tested in sets of five replicate dishes. Cultures were maintained in vitro for 7 days, then they were processed for the
  • FIGURE 21 Effect of NT-3 on GAD activity in vitro. Cultures were prepared as in Figure 18.
  • NT-3 Purified human, recombinant NT-3 was added at the time of the media switch as described. Each concentration of NT-3 was tested in sets of five replicate dishes. Cultures were maintained for 7 days in vitro, and were then processed for measurement of GAD activity.
  • FIGURE 22 Effect of neurotrophin concentration on choline acetyl transferase (CAT) activity in E17 cultures of rat septal cholinergic neurons.
  • CAT choline acetyl transferase
  • FIGURE 23 Retrograde transport of [ 125 I]NT-3 from the right sciatic nerve of rats. NT-3 was
  • FIGURE 24 Retrograde transport of NT-3, BDNF and NGF from the right sciatic nerve of control and pyridoxine treated rats.
  • FIGURE 25 Transport of NT-3 and BDNF in the L4-L5 spinal cord segment of control and
  • the present invention provides for the use of biologically active NT-3 molecules in diagnostic and therapeutic regimens for detecting and treating neurological disorders involving target cells for NT-3.
  • the methods of the invention also provide for inhibition or neutralization of NT-3 activity using anti-NT-3 antibodies, antisense RNA or ribozymes.
  • NT-3 derivatives of NT-3, including biologically active fragments thereof, or antibodies that bind to NT-3 receptors, including anti-idiotypic antibodies that mimic NT-3, may be used to image neuronal cells that express the NT-3 receptor. Abberant patterns of receptor expression may be analyzed to diagnose pathological-conditions.
  • the NT-3 compositions or antibodies that bind to NT-3 receptors may be conjugated to a radiolabel, radiopaque label, fluor, enzyme, etc. Such conjugates may be used
  • Imaging protocols including but not limited to CAT scans, x-rays, fluorograms, MRI or PET scans, etc. to analyze the tissue distribution of NT-3 receptor in vivo.
  • Anti-NT-3 antibodies may similarly be used in such imaging protocols to image native NT-3 bound to the surface of such target cells.
  • conjugates may also be used to identify NT-3 receptors in biopsy or autopsy specimens.
  • NT-3, or NT-3 derivatives, including biologically active fragments thereof may be administered to potentiate its biological effect on a target tissue.
  • NT-3 gene products derived from the same species for therapeutic or diagnostic purposes, although cross-species NT-3 may be useful in certain specific embodiments of the invention.
  • Neurological disorders in which stimulation of NT-3 responsive target cells corrects the disorder may be treated in this manner.
  • neutralizing anti-NT-3 antibodies may be utilized to block the effects of native NT-3 on target cells.
  • antisense RNA or ribozymes may be used to inhibit or block the production of native NT-3 or peptide antagonists of NT-3 may be utilized.
  • Neurological disorders involving the over- proliferation of cells that express the NT-3 receptor and respond to NT-3 may be treated in this manner.
  • such protocols may be used to treat neurological tumors that express the NT-3 receptor and proliferate in response to NT-3.
  • a number of disorders of neurological tissue which express NT-3 and/or the NT-3 receptor may be treated in accordance with the invention. Where peripheral nerves are involved, a clinical test may be performed to determine whether the condition improves in response to the application of NT-3 or the
  • the efficacy of the treatment may be first assessed in an appropriate animal model system.
  • disorders involving neurological tissues that express NT-3 and/or the NT-3 receptor which may be treated in accordance with the invention include but are not limited to:
  • disorders of olfaction and taste such as anosmia and dysgeusia
  • disease affecting the neocortex including but not limited to Alzheimer's disease and clinical defects produced by stroke as well as predominantly cortical dementing illnesses, including but not limited to Pick's disease, cortical-basal ganglionic degeneration, diffuse cortical Lewy body disease and Jacob-Creuzfeldt disease and including disorders involving the superficial layer of the neocortex caused by lesions or processes which compress or invade the brain from its external surface, including but not limited to consequences of brain trauma as exemplified by a laceration or contusion, subdural hematoma, epidural hematoma and tumor metastases;
  • the NT-3 utilized in accordance with the invention may be prepared by recombinant DNA
  • NT-3 may be prepared from eukaryotic cells that express recombinant NT-3 as follows.
  • Conditioned medium may be collected from a bioreactor (Charles River) seeded with eukaryotic cells expressing NT-3 and diluted with distilled water at about a 1.25:1 ratio and then may be continuously loaded at 4°C onto a 15 ml (1.6 cm i.d.) S-Sepharose
  • 10.6L (diluted) may be applied in a period of about five days.
  • the column may be washed with 50 ml of 20 mM MES buffer (a solution of 4-morpholinethane-sulfonic acid, the pH of which has been adjusted to about 5.8 with NaOH) followed by 50 ml of the same buffer containing 250 mM sodium chloride followed by the same MES buffer without sodium chloride until absorbance at 280 nm reaches baseline level.
  • the column may then be washed with about 25 ml of 20 mM bicine buffer at pH 8.5 followed by 15 ml of the same buffer containing 100 mM sodium chloride.
  • the column may subsequently be eluted with 150 ml, 100 mM to 750 mM sodium chloride gradient in 20 mM bicine buffer (pH 8.5).
  • the flow rate may be desireably about 2.5 ml/min.
  • Absorbance of the eluate may be monitored at 280 nm at a sensitivity of 0.2 absorbance unit full scale.
  • Fractions containing recombinant NT-3 may be identified by dorsal root ganglia (DRG) explant bioassay.
  • DRG dorsal root ganglia
  • the concentrated sample may then be applied onto a 120 ml (1.6 cm i.d.) Sephacryl S-100HR gel filtration column (Pharmacia) packed and equilibrated with 20 mM HEPES Buffer pH 7.6 containing 400 mM NaCl.
  • the column may be eluted at about 0.25 ml/min. 2 ml size fractions may be collected and assayed for NT-3 activity in the DRG explant bioassay.
  • the present invention also provides for fragments of NT-3 which are either antigenic or functionally active.
  • Functionally active fragments include fragments of NT-3 that are NT-3 agonists as well as fragments that are NT-3 antagonists. If it is desireable to supplement NT-3 activity, an agonist fragment may be used. If it is desireable to inhibit NT-3 activity, an antagonist fragment may be used.
  • Agonist fragments preferably comprise at least two of the three disulfide bridges found in the native NT-3 molecule and/or are capable of achieving at least about thirty percent, and preferably at least about fifty percent, of the activity of intact NT-3 measured under the same conditions, e.g. in a DRG assay.
  • NT-3 activity refers to an antibody that decreases the activity of native NT-3 in the DRG assay, described infra, by at least about 30 percent.
  • Any of the antibodies used in accordance with the invention include but are not limited to polyclonal, monoclonal, chimeric, single chain, and Fab fragments.
  • an animal host may be immunized using the appropriate immunogen formulated in an adjuvant to increase the immune response.
  • adjuvants include but are not limited to Freund's (complete and incomplete), mineral gels, such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhold limpit hemocyanin, dinitrophenol, BCG (bacille Calmette-Guerin) and
  • Monoclonal antibodies may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in
  • Antibody fragments may also be generated from whole antibody molecules using known techniques such as pepsin digestion and reduction of disulfide bonds.
  • the active ingredient which may comprise NT-3, a biologically active derivative or fragment thereof, or an antibody or antibody fragment prepared as described above should be formulated in a suitable pharmaceutical carrier for administration in vivo by any appropriate route including but not limited to injection (e.g., intravenous, intraperitoneal,
  • epithelial or mucocutaneous linings e .g. , oral mucosa, rectal and intestinal mucosa, etc.
  • sustained release implant including a cellular or tissue implant.
  • the active ingredient may be formulated in a liquid carrier such as saline, incorporated into liposomes, microcapsules, polymer or wax-based and controlled release preparations, or formulated into tablet, pill or capsule forms.
  • a liquid carrier such as saline
  • the concentration of the active ingredient used in the formulation will depend upon the effective dose required and the mode of administration used.
  • the dose used should be sufficient to achieve
  • circulating plasma concentrations of active ingredient that are efficacious.
  • a circulating serum concentration level ranging from about 1 ng/ml to 10 ⁇ g/ml may be used; preferably ranging from about 1 ng/ml to
  • Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
  • NT-3 receptors are distributed in a tissue- specific fashion. Neurological disorders involving pathologies of such tissues are candidates for diagnosis and/or treatment in accordance with the invention. Diseases or disorders involving nervous system cells (including neural cells as well as supporting cells, etc.) that are responsive to NT-3 are the targets of the methods of the invention. For example, high densities of NT-3 receptor occur in the anterior nucleus of the olfactory bulb, layer 1 of the neocortex, the nucleus of the lateral olfactory tract, the dentate gyrus, CA1, CA3, and CA4 of the
  • NT-3 receptor Moderate to low densities of the NT-3 receptor occur in the neocortex nucleus accumbens, spinal gray, basolateral amygdala, interpedunucular nucleus, superior colliculus, medial septum and cerebellum. Disorders involving such regions may be diagnosed or treated using the methods of the invention.
  • NT-3 an NT-3 derivative
  • NT-3 including a biologically active agonist NT-3 fragment, or an anti-idiotypic antibody which mimics the binding and biological effects of NT-3.
  • Such treatment will stimulate the growth or differentiation or survival of the target cells .
  • Neurological disorders involving the over-proliferation of cells that express the NT-3 receptor and which proliferate in response to NT-3, or which achieve an undesireable phenotype as a result of exposure to NT-3 may be treated by inhibiting the effect or production of native NT-3. This may be accomplished by administering an effective dose of neutralizing anti-NT-3 antibody to inhibit the
  • NT-3 biological effect of NT-3; or by delivery of anti-sense RNA or ribozymes to the cellular source of NT-3 to inhibit the production of native NT-3; or by administration of an NT-3 antagonist.
  • NT-3 responsive tissues have been damaged or are in the process of degeneration
  • treatment which augments NT-3 activity may be
  • NT-3 responsive tissue has been damaged may be made by any method known in the art. For example, if neuroimaging techniques, such as CAT scan, MRI, PET scan, etc.
  • damage to a structure which exhibits binding to NT-3 for example, as set forth in Table 2, infra, treatment to augment NT-3 activity may be initiated. But damage to such a structure may also be ascertained by other techniques, including, but not limited to, biopsy or on the basis of clinical examination or history, EEG, EMG, etc.
  • a diminuition in NT-3 activity may be desireable in conditions that involve either
  • Such conditions may include tumors, for example, in which NT-3 may be employed in an autocrine loop, or in the epilepsies or certain disorders of the striatum that are associated with production of excessive involuntary movement.
  • tumors it may be possible to identify an increase in NT-3 receptors, endogenous NT-3, or responsiveness to NT-3 using histologic and/or cell culture techniques; if such a condition is determined, diminuition of NT-3 activity may be desireable.
  • NT-3 molecules or possibly anti-NT-3 antibodies may assist in the treatment of such disorders as anosmia (the absence of smell) or dysgeusia (the distortion of normal taste).
  • anosmia the absence of smell
  • dysgeusia the distortion of normal taste
  • anosmia that may be treated by therapeutic applications of .NT-3 include, but are not limited to, head trauma (which may be caused by severing of the neurons crossing through the cribriform plate as well as complications associated with the normal aging process), multiple sclerosis, Parkinson's disease or a frontal lobe tumor.
  • dysgeusia that may be treated by therapeutic applications of NT-3 include, but are not limited to, Bell's palsy, familial dysautonomia and multiple sclerosis as well as distortions of taste that arise on the basis of olfactory dysfunction.
  • NT-3 expression and receptor binding within the lateral, intermediate and medial portions of the superficial entorhinal cortex, dentate gyrus, the granule cell layer of the dentate gyrus hilus as well as the CA1, CA3 and CA4 pyramidal layers of the hippocampus.
  • the expression and binding of NT-3 within these distinct tissues of the brain indicate therapeutic applications which include but are not limited to, memory deficits resulting from global cerebral ischemia which occurs in survivors of cardiac arrest.
  • therapeutic applications of biologically active NT-3 molecules may be utilized in the treatment of
  • Alzheimer's disease This region of the brain is involved early in the pathology of the disease and there is speculation (Saper and German 1987,
  • Alzheimer's disease may initiate in this region of the brain, spread through the cortex and hippocampus, and retrogradely affect the basal forebrain and other brainstem nuclei.
  • hippocampal tissue (a) expresses both trkB and trkC, (b) NT-3 induces FOS expression of both trkB and trkC, (c) NT-3 induces FOS expression and
  • neorofilament protein produces and increase in AChE - positive and calbindin - immunopositive cells. Since patients diagnosed with Parkinson's disease, Huntington's disease and Alzheimer's disease show a marked decrease in both mRNA transcripts and protein for calbindin (lacopino and Christakos, 1990, Proc. Natl. Acad. Sci. USA 87: 4078-4082) treatment with NT-3 may be used to increase expression of calbindin in such patients. 5.4.3. THE NEOCORTEX AND SUPERFICIAL
  • the examples described infra also demonstrate NT-3 expression and binding within the neocortex and superficial layers of the neocortex.
  • Disorders involving the neocortex which occur in a number of diseases may be treated including but not limited to Alzheimer's disease stroke victims and predominantly cortical dementing illnesses, including but not limited to, Pick's disease, cortical-basal ganglionic degeneration, diffuse cortical lewy body disease and Jacob-Creutzfeld disease.
  • the pathology of human victims, in contrast to other animal models, is that predominately all layers of the cortex are affected, with the regional distribution being determined by the territory of the blocked cerebral vessel. In the area surrounding the infarction there is a zone sometimes referred to as the "ischemic penumbra" in which the blood-brain barrier is
  • NT-3 or derivative or NT-3 peptide fragment may work to minimize the clinical deficits produced by a stroke.
  • superficial layers of neocortex might be lesions or processes which compress or invade the brain from its external surface, including, but not limited to:
  • brain trauma such as laceration or contusion, subdural hematoma, epidural hematoma and tumor metastases.
  • NT-3 expression and receptor binding within the caudate-putamen.
  • Therapeutic applications of NT-3 to the caudate-putamen may be utilized to treat, by way of example and not by limitation,
  • choreoathetic dystonia syndromes due to perinatal hypoxia and striatonigral degeneration and progressive supranuclear palsy.
  • Huntington's disease is an untreatable, progressive, dominantly inherited disease with 100% penetrance. Huntington's disease characteristically presents itself in early midlife, after the
  • the pathology of Huntington's disease consists principally of degeneration of the so-called "medium spiny" neurons of the caudate nucleus and putamen.
  • Degeneration is noted in other areas of the nervous system as well, including the frontal cortex, thalamus and the deep cerebellar nuclei. These cell losses result in decreases in the amounts of the
  • Symptoms include behavioral deterioration
  • striatum ⁇ an area demonstrated herein to express high levels of NT-3 transcripts and high density NT-3 receptors ⁇ treatment with NT-3 or a biologically active derivative or fragment thereof may be used in accordance with the invention to treat Huntington's disease.
  • Chorea-acanthocytosis is a hereditary choreatic syndrome of young adults who lack the severe intellectual deterioration or behavioral disturbances of Huntington's disease. Some patients have epileptic seizures. There is neuronal cell loss in the caudate nucleus and the putamen. The disease is distinguished by its autosomal recessive inheritance, muscle
  • the present invention also involves therapeutic applications for the treatment of
  • the basal ganglia is a principal target of perinatal "hypoxic-ischemic injury resulting in the neurological impairments of "cerebral palsy".
  • cerebral palsy There are three principal clinical variants of cerebral palsy: spastic hemiplegia, spastic diplegia and
  • Therapeutic applications of the invention also include treatment of striatonigral degeneration and progressive supranuclear palsy (Steele-Richardson- Olszewski Syndrome), which are so-called “Parkinsonplus syndromes” with clinical features of Parkinson's disease which do not respond to conventional
  • NT-3 promotes neurite survival and outgrowth in the sensory neurons of dorsal root ganglion cultures.
  • In vitro cultures enriched in lumbar or cervical ganglia are more responsive to NT-3 in relation to in vitro
  • JNT-3 may act preferentially on large diameter, large fiber proprioceptive neurons such as those found in the lumbar and cervical ganglia. Therefore, NT-3 may be useful in treating peripheral neuropathies affecting these larger sensory neurons which occur in patients with diabetes or patients undergoing chemotherapy for cancer or AIDS treatment (e.g., taxol, vincristine, cisplatin, di-deoxyinosine).
  • DIAGNOSTIC APPLICATIONS e.g., taxol, vincristine, cisplatin, di-deoxyinosine.
  • a number of in vitro or in vivo diagnostic tests which involve assaying NT-3 or the NT-3 receptor may be utilized to assist in diagnosis and the
  • NT-3 may be assayed in various biopsy or autopsy tissues by methods known in the art.
  • immunoassays may be used to detect expression of the NT-3 protein.
  • assays include but are not limited to ELISA,
  • nucleotide probes that are complementary to the NT-3 sequence may be used in RNA hybridization methods in situ to detect expression of the NT-3 mRNA
  • transcripts in the biopsy or autopsy sample may include the use of PCR (polymerase chain reaction). Tissue concentrations of NT-3 protein or mRNA transcripts may be correlated with disease.
  • the NT-3 receptor may be assayed in biopsy or autopsy samples or in vivo.
  • NT-3 or NT-3 derivatives may be modified, for detection, e.g., by labeling with a radioisotope, a radioopaque compound, a fluor, an enzyme etc.
  • conjugates may be used for imaging the NT-3 receptor in biopsy or autopsy samples in vitro or for imaging in vivo.
  • antibodies that mimic NT-3 and/or bind to the NT-3 receptor may similarly be used to image the NT-3 receptor distribution in biopsy or autopsy samples in vitro or in vivo. Imaging techniques well known in the art can be used to such ends, e.g., CAT SCAN, X-ray, etc. Aberrations in the distribution of receptor may be correlated with disease.
  • NT-3 preferentially accumulates in the lumbar and cervical ganglia thus providing a method for diagnosis of sensory neuron disorders.
  • a biopsy sample of such neurons of an afflicted patient may be utilized to determine the level of NT-3 protein and to compare to the level of NT-3 from an analogous sample from a normal individual.
  • An aberrancy in the level of NT-3 may correlate with the presence of a sensory neuron disorder.
  • the actual determination of the level of NT-3 protein may be carried out by a method comprising contacting the sample with an anti-NT-3 antibody such that immunospecific binding can occur.
  • the present invention provides for a method of diagnosing NT-3 related peripheral nervous system disorders comprising
  • NT-3 responsiveness to NT-3 and indicates the presence of a peripheral nervous system disorder that is NT-3 related.
  • the present invention also provides for a method to diagnose a central nervous system disorder.
  • Evaluation of retrograde transport may be performed by any method known in the art, including but not limited to MRI, CAT, or scintillation scanning as discussed infra. Such methods may be used to identify the location of a nervous system lesion, as retrograde transport should substantially diminish upon reaching the lesion.
  • kits for such retrograde evaluation comprising in a container a detectably labeled NT-3 protein, derivative or
  • Such a label can be a radioactive isotope, or other label known in the art.
  • the present invention also provides for a method of treating sensory neuron disorders comprising administering, to a patient in need of such treatment, an effective amount of an NT-3 protein, derivative or peptide fragment capable of supporting the survival, growth and/or differentiation of motor neurons as demonstrated in an in vitro culture system.
  • effective amounts of neurotrophic factor may desirably be determined on a case by case basis, as sensory neurons from different tissue sources or from different species may exhibit different sensitivities to neurotrophic factor.
  • CAT choline acetyltransferase
  • NT-3 was produced by the preferred method described supra in Section 5.1.
  • the NT-3 aliquots were monitored for protein content by amino acid analysis and for biological activity using the dorsal root ganglia (DRG) explant culture system using muNGF as a standard.
  • DRG dorsal root ganglia
  • the NT-3 was iodinated by the
  • the [ 125 I]NT-3 was labeled to a specific activity of 2800-4400 cpm/fmol (1872-2876 Ci/mmole of NT-3) and stored at a concentration of 80-100 nM at 2-8°C. [ 125 I]NT-3 was used within 1-5 days in all studies to avoid the observed increase in non-displaceable [ 125 I]NT-3 binding that occurred after this time.
  • the brain of an adult male cat was sectioned coronally from the frontal cortex to the medulla and sections from 22 levels were collected as described for the rat. Twenty ⁇ m thick sections of a human brain were collected through the basal ganglia and included the caudate, putamen, neocortex, and adjacent fiber bundles.
  • Binding assays were conducted according to the procedure of Richardson, et al. (1986, Neurosci. 20:23-36) and with modifications for dry film
  • each section was preincubated for 1 hour at 22oC in 100 mM phosphate-buffered saline, pH 7.4, containing 0.5 mM MgCl 2 and 0.5 mM PMSF. Equivalent binding was observed for sections that were preincubated for 1, 3, or 24 hours.
  • Sections were then incubated in DMEM tissue culture medium containing high glucose, 10% heat-inactivated fetal calf serum (70° C for 0.5 hours), 25 mM HEPES buffer, 4 ⁇ g/ml leupeptin, 0.5 mM PMSF, (BRL,
  • adjacent brain sections were incubated in the same solutions with the addition of 300 nM NT-3 to define displaceable binding.
  • a range of concentrations of muNGF, hCNTF, and BDNF were each used to compete with [ 125 I]NT-3 binding in several experiments.
  • the sections were washed for 0.5 hr in the phosphate buffer. In the absence of excess unlabelled NT-3, equivalent amounts of total and non-displaceable binding were obtained with 3 minutes, 10 minutes and 2 hour washes with unlabeled buffer.
  • association experiment association experiment
  • dissociation experiment dissociation experiment
  • K d and B max values were calculated by equilibrium saturation analysis with each of four brains according to the best fit to a parabola by iterative, nonlinear regression analysis (Rodbard and Lewald, 1970, Acta Endocrinol. 147:79-103).
  • IC 50 values were calculated by the method of Bliss and
  • binding sites in horizontal and coronal sections of rat brain This binding was displaced by 70-90% by a 1000-fold greater concentration of NT-3 whereas 10-15% higher levels of non-specific binding were obtained at higher ligand concentrations. Because binding was particularly robust and displaceable in the caudate-putamen, neocortex, and hippocampus, horizontal sections that contained these areas were used for subsequent quantitative studies with
  • neocortex (anterior nucleus of the olfactory bulb, nucleus of the lateral olfactory tract), hippocampus (dentate gyrus, CA1, 3, and 4), caudate-putamen, and the neocortex (Table 2).
  • the first layer of the neocortex was prominently labeled in the frontal, parietal, and cingulate regions. Layer 1 was also heavily labeled throughout the entorhinal cortex.
  • nucleus accumbens Intermediate levels of binding were present in nucleus accumbens, basolateral amygdala, interpeduncular nucleus, ventral and dorsal horns of spinal cord, and superior colliculus. Lower amounts of specific binding were present in the lateral geniculate, medial septum, and cerebell ⁇ m. No displaceable binding was detected in white fiber buncles including the corpus callosum, anterior commissure, internal capsule, interbulbar internal capsule, or in the globus pallidus, most thalamic nuclei, hypothalamus, or in other regions of the pons, medulla, or other amygdaloid nuclei. No
  • [ 125 I]NT-3 binding sites were found in circumventricular organs such as the choroid plexus or ependymal cell layers. Specific binding was also absent in the liver, muscle, kidney, pancreas, and heart. TABLE 2
  • NT-3 binding were evaluated from visual evaluation of autoradiographs generated with 300 pM [125I]NT-3.
  • the non-specific binding defined with 300 nm NT-3 appeared uniform throughout the brain sections and accounted for only 10-20% of the total amount of bound ligand. This allowed the relative amounts of specific binding to be estimated from the total binding images. 6.3. DISCUSSION
  • heterogeneous brain binding site is nonetheless phylogenetically conserved.
  • These maps illustrate the brain regions in which endogenous NT-3 may function as a neurotrophic factor and also provide the basis for both in vitro and in vivo studies to assess the neuronal specificities and therapeutic targets of NT-3.
  • the displaceable binding of [ 125 I]NT-3 ranged from 60-90% of the total binding.
  • topography of the 10-40% non-displaceable binding probably represents NT-3 receptor-independent
  • the capacity of high and low affinity NT-3 binding sites were 26 and 170 fmol/mg protein
  • the high affinity site is at least twice as dense as that for rhNGF (Altar, et al., Proc. Natl. Acad. Sci. USA 88:281-285; Altar, et al., 1991 J. Neurosci. 11:828-836).
  • the high affinity binding site for [ 125 I]BDNF appears to be even more numerous and more ubiquitously localized than those for NGF or NT-3.
  • cholinergic areas include cranial nerve nuclei III and IV, the pedunculopontine or parabrachial cholinergic projections to thalamus, hypothalamus, and inferior colliculus, and the projection of preoptic
  • piriform cortex may contribute to the relatively intense NT-3 binding observed in these areas.
  • neurotransmitter systems in piriform and entorhinal cortex that project to hippocampus may contain these NT-3 binding sites.
  • NGF receptors are relatively low in the, adult rat cerebellum and other areas in which NGF binding sites and receptor immunostaining are quite dense.
  • the granule cell layer of cerebellum is also the most densely labeled brain area following in situ
  • trkC antisense probe hybridization with trkC antisense probe.
  • the strong, residual trkC signal generated with trkC sense cRNA probe (Lamballe, et al., 1991, Cell 66:967-979) or a relatively diminished translation of trkC message into functional receptor are two possible explanations for the presence of trkC antisense message but not NT-3 binding in the cerebellar granule cell layer.
  • trkC expression is also very pronounced in hippocampus, neocortex, and neostriatum (Lamballe et al., 1991, Cell 66: 967-979) and these areas were most intensely labeled with [ 125 I]NT-3.
  • trkC mRNA and [ 125 I]NT-3 binding will ascertain the resemblance of these two markers.
  • Hippocampi were dissected from E16-E18, or
  • E20 rat embryos of Sprague-Dawley rats and collected in F10 medium.
  • the tissues were minced, rinsed twice with F10 medium (Gibco) and trypsinized with 0.25% trypsin (Gibco) for 20 minutes at 37°C. Trypsin was inactivated by the addition of a serum-containing medium composed of minimum essential medium (MEM) supplemented with fetal calf serum (FCS, 10%), glutamine (2 mM), penicillin (25 U/ml) and
  • MEM minimum essential medium
  • FCS fetal calf serum
  • FCS fetal calf serum
  • penicillin 25 U/ml
  • polyornithine-laminin (10 ⁇ g/ml) in DME plus 10% fetal calf serum. After 4 hours of culture, the medium was changed to DME plus 1 mg/ml BSA and N2 media
  • BDNF and NT-3 Approximately 40% of the hippocampal cells showed a fos response to BDNF and NT-3. The effects of BDNF and NT-3 on fos induction were not additive. All of the cells that showed the fos response to BDNF and NT-3 were neurons. Double staining with fos and calbindin showed that calbindin-immunopositive cells were among the cell population that responded with fos induction.
  • BDNF and NT-3 were found to induce an increase (50%) in the amount of neurofilament protein. They also produced an increase in the number of AChE-positive and calbindin-immunopositive cells. In contrast, NGF had no apparent effect. Dose response studies of BDNF and NT-3 on the number of calbindin-immunopositive cells showed that the response
  • NT-3 produced a 20-fold increase in the number of calbindin-positive cells which was accompanied by an increase in calbindin-mRNA levels. Delaying the addition of NT-3 to the cultures for 4 days did not appear to affect the increase in the number of calbindin-positive cells, suggesting that NT-3 acts to induce the calbindin-phenotype instead of acting as a survival factor. Developmental profiles of the increase in calbindin-positive cells produced by BDNF and NT-3 were compared. The effect of NT-3 was much more striking for hippocampal neurons earlier in development (E16-E18) and declined later in development (E20), while the reciprocal effect was observed for BDNF.
  • the only cell populations that appeared to respond to both BDNF and NT-3 thus include AChE positive and calbindin positive cells.
  • Other cell populations examined which did not appear to respond to BDNF and NT-3 in terms of survival include GABAergic cells, glutamatergic cells and somatostatin-positive cells. Decreases of up to 60-80% in the levels of both the message and protein for calbindin were observed in the hippocampus of patients who were diagnosed as having Parkinson's, Huntington's or Alzheimer's disease (lacopino and Christakos, 1990, Proc. Natl. Acad. Sci. USA 87:4078-4082).
  • GABAergic cells GABAergic cells
  • glutamatergic cells glutamatergic cells
  • somatostatin-positive cells Decreases of up to 60-80% in the levels of both the message and protein for calbindin were observed in the hippocampus of patients who were diagnosed as having Parkinson's, Huntington's or Alzheimer's disease (lacopino and Christa
  • calbindin protein in the hippocampal neurons may have significant physiological implication in that it may be important in preventing cell death in select populations of hippocampal neurons.
  • Striatal neuronal cultures were prepared from E17 rat brains as follows: striatal tissue was minced in calcium- and magnesium-free Hank's balanced salt solution and dissociated by enzymatic treatment with 0.25% trypsin and DNAase (0.2 mg/ml) followed by mechanical trituration. Dissociated cells were seeded at a density of 8 ⁇ 10 6 cells on 100 mm dishes which had been previously coated with polylysine and
  • Striatal and hippocampal astrocyte cultures were prepared from P1 rat brains as follows: tissues were minced and enzymatically dissociated by treatment 0.25% trypsin and DNAase 0.2 mg/ml). Following a 5 minute centrifugation at low speed, and an
  • the cell suspension was dissociated mechanically by trituration.
  • Cells were then passed through a Nitex filter cartridge and seeded at a density of 71,000 cells/cm 2 in T75 tissue culture flasks in DME-FCS.
  • Immunocytochemical staining was used to determine the purity of the astrocyte and neuronal cultures.
  • Antibodies to glial fibrillary acidic protein (specific to astrocytes) and neurofilament protein (specific to neurons) were used in combination with other cell-type-specific markers to delineate the cellular composition of the cultures.
  • Total RNA was prepared from striatal RNA
  • neuronal cultures at 4 DIV
  • striatal astrocyte cultures at 28 DIV
  • RNA samples were prepared from striatal or whole brain tissue samples by extraction in 0.3M LiCl/6M urea followed by phenol/chloroform extraction (Auffray and Rougeon, 1980, Eur. J. Biochem 107:303-314).
  • RNAs (10 ug/lane) were fractionated by electrophoresis through 1% agarose-formaldehyde gels (Bothwell et al., 1990, in: Methods for Cloning and Analysis of Eukaryotic Genes, pp. 42-43, ed., Jones and Bartlett, Boston) followed by capillary transfer to nylon membranes. Probes to trkB (1.1 kb, spanning the intracytoplasmic tyrosine kinase domain) and trkC (800 bp, spanning the
  • intracytoplasmic tyrosine kinase domain were labeled by random hexamer labeling with 32 P dCTP (Stratagene Prime It).
  • Membranes were hybridized overnight in 0.5 sodium phosphate buffer, pH 7.9 , containing 1 percent bovine serum albumin, 7 percent SDS and 100 ⁇ g/ml sonicated salmon sperm at 65°C. Filers were rinsed briefly in 2 ⁇ SSC and 0.1 percent SDS and then washed twice in 1 ⁇ SSC and 0.1 percent SDS and exposed to
  • TrkC mRNA expression was detected in rat striatum as early as E17 ( Figure 10). Expression of trkC mRNA reaches a peak between P7 and P20, where a 1.5-1.9 fold increase in transcript level is detected relative to whole adult rat brain ( Figure 12).
  • trkC mRNA is expressed in cultured striatal neurons (4 DIV) but not in striatal or hippocampal astrocytes (28 DIV).
  • BDNF and NT-3 may act as trophic factors in the striatum.
  • Chick dorsal root ganglia When cultured either as explants or dissociated neuron-enriched cultures, sensory neurons of the chick dorsal root ganglia (DRG) are responsive to a greater or lesser degree to NGF, BDNF, NT-3, NT-4 and CNTF. Chick embryo DRG neurons die within 24 hours of being placed in culture in the absence of any neurotrophic factor. Each of the above factors can support the survival and outgrowth of neurites of some of these neurons, ranging from 10-60%, depending on the factor, stage of neuronal development etc. It appears that each of the above neurotrophic factors has both distinct and overlapping specificities towards sup-populations of DRG neurons, although there is no clear evidence as to which sub-types of sensory neurons are supported by which neurotrophic factor. This example defines the comparative specificity of NT-3, NGF and BDNF.
  • DRG were collected from chick embryos at developmental stages ranging from E6 - E10. Starting in the sacral region ganglia from right and left sides were collected in pairs along the entire neural axis. 5-6 ganglia at, each level or pools of sacral, lumbar, thoracic or cervical ganglia were explanted in
  • Ganglia were cultured in F14 medium + 5% horse serum in the presence or absence of 5 ng/ml, NGF, BDNF or NT-3. After 24 hours the. extent of fiber outgrowth was measured on an arbitrary scale of 0 to +5, 0 being virtually no fibers,. +5 being a profuse halo of fibers (the saturating level seen with NGF, the most potent of the factors in this assay). 9.1.2. DISSOCIATED NEURON-ENRICHED CULTURES
  • Lumbar and thoracic ganglia from E8 embryos were collected separately, dissociated with 0.25% trypsin and freed of non-neuronal cells (Lindsay et al, 1985, Develop. Biol. 112, 319-328).
  • Purified neurons were seeded on a substrate of polyornithine- laminin at 8,000 neurons per 35 mm dish. Cells were cultured in the presence of F14 medium containing 5% horse serum. A dose response ranging from 1 pg to 10 ng of either NGF, BDNF or NT-3 was carried out. After 48 hours the number of process bearing neurons was determined in triplicate cultures (Lindsay et al., 1985, Develop. Biol, 12: 319-328). 9.2. RESULTS AND DISCUSSION
  • NGF supported the survival of a similar percentage of either lumbar or thoracic DRG neurons (40-50%). Although BDNF was less effective than NGF, the effects of BNDF were essentially the same towards lumbar or thoracic neurons - supporting survival of around 30% of the neurons. In contrast, NT-3
  • the DRG of the lumbar and cervical enlargements contain more large-diameter, large fiber proprioceptive sensory neurons than either sacral or thoracic ganglia.
  • NT-3 has selective activity on large-diameter, large fiber DRG neurons.
  • FOS IMMUNOHISTOCHEMISTRY Cells were treated for 30-300 minutes with BDNF, NT-3, or NGF at a concentration of 0.1-1 ng/ml at the end of the culture period. Cells were then rinsed once in BME and prefixed for 10 min in 2% paraformaldehyde in BME before fixation at room temperature in 4% paraformaldehyde in PBS for 30 min. After permeabilization with 0.1% triton for 10 min and blocking in 10% normal goat serum (NGS) and 1% BSA for 90 min, the cells were incubated for 2 days at 4°C with anti-FOS antibody (Oncogene) diluted 1/2000 in PBS with 5% NGS.
  • NGS normal goat serum
  • DAB hydrochloride
  • This example discloses the efficacy of neurotrophin 3 in supporting the survival or
  • E14 rat brain The preparation of cultures from E14 rat brain was carried out as described by Hyman, et al., 1991, Nature 350: 230-233. Briefly, all cultures were prepared from the ventral mesencephalon dissected from 14-day-old embryonic rats (E14). Typically, pooled tissue from two or three litters of rat embryos from timed-mated Sprague Dawley rats was trypsinized
  • the single-cell suspension was seeded onto 35-mm dishes (precoated with polyornithine and laminin) containing growth medium to give a density of 5 ⁇ 10 4 cells cm -2 .
  • MEM medium supplemented with glutamine (2 mM), glucose (6 mg/ml -1 ), penicillin G (0.5 U/ml -1 ), streptomycin (5 ⁇ g/ml -1 ) and FCS (7.5%) to allow cell attachment
  • cells were cultured in the presence or absence of BDNF in a serum-free, defined medium (Hyman et al., 1991, Nature 350:230-232) except that insulin was included at 20 ng/ml -1 .
  • To visualize dopaminergic cells cultures were fixed with 4% paraformaldehyde, washed extensively, permeabilized with 0.02% saponin in Sorensen's buffer with 1.5% horse serum and stained with a mouse monoclonal
  • GAD enzyme activity was determined according to the method of Kimura and Kuriyama (1975, Jpn J. Pharm. 25:189-195) by measuring the release of 14 CO 2 from L-[1- 14 C] glutamic acid. Cells on 35 mm dishes were lysed with 30 ⁇ l of a solution containing 50 mM KH 2 PO 4 (pH 7.2) and 0.25% Triton X-100, scraped and collected. Five microliters of the cell lysate was assayed for GAD enzyme activity.
  • the reaction mixture contained 0.57 mM of L-[1- 14 C] glutamic acid (NEN, NEC-715, 52.6 mCi/mmol), glutamic acid (3 mM), pyridoxal phosphate (0.2 mM) and AET (1 mM), in a KH 2 PO 4 buffer (50 mM, pH 7.2). Under these reaction conditions, the enzyme reaction was found to be linear for up to 2.5 hours.
  • Each treatment group had five 35 mm dishes, 3 of which were marked with a (-) and two with a (+). All culture dishes were washed once with incubation buffer and once with warm buffer containing +/- BZT and DABA as appropriate. To each culture dish was added 0.8 ml of +/- BZT and DABA buffer prior to incubation at 37°C for 5 minutes; followed by the addition of 0.2 ml of 3 H-DA (final concentration of 50 nM) and 14 C-GABA (final concentration of 500nM). Uptake of 3 H-DA + 14 C-GABA was allowed to proceed at 37°C for 15 minutes. The culture dishes were then placed on ice and the incubation solution was
  • the cultures were washed three times with an incubation buffer having the following composition: 100 ⁇ M pargyline, 1 mM ascorbate, 10 ⁇ M aminooxyacetic acid and 2 mM-beta-alanine. Cultures were then preincubated for 5 minutes at 37oC in incubation buffer; replicate cultures were preincubated in buffer additionally containing 5 ⁇ M benztropine (BZT) and 1 mM 2,4 diamino-n-butyric acid (DABA). 50 nM 3 H-DA and 500 nM 14 C-GABA were then added for 15 minutes at 37°C. The uptake of label was stopped by placing the dishes on a bed of ice and rinsing with ice coId buffer. The samples were solubilized with 0.5 N NaOH and 3 H and 14 C was measured by liquid scintillation counting. Specific uptake was defined as the following composition: 100 ⁇ M pargyline, 1 mM ascorbate, 10 ⁇ M aminooxyace
  • Dopaminerigic markers were also analyzed via immunocytochemical staining for tyrosine hydroxylase (Hyman, et al., 1991, Nature 350: 230-233).
  • a dose response test of NT-3 was carried out to determine if cells cultured in the presence of this factor would show an increased survival of
  • NT-3 induced a maximal effect of a 2.8 fold increase in 3 H-GABA uptake activity when included in the culture medium at a concentration of 20 ng/ml for a period of 7 days in vitro (Figure 20).
  • the results show similar fold effects in assays conducted to examine independent phenotypic marker activities, indicating that the NT-3 acts as a survival-promoting activity for these neurons.
  • the results show similar fold effects in assays conducted to examine independent phenotypic marker activities, indicating that the NT-3 acts as a survival-promoting activity for these neurons.
  • NT-3 may stimulate the GABA uptake activity of the cells (a measure of their metabolic activity) independently of exerting its effect on the GAD activity.
  • the septal region from Sprague-Dawley rats after 17 days of gestation was dissected free from the surrounding tissue. Tissue fragments were pooled, washed three times with Hams F-10, and then
  • tissue culture dish transferred to a 35mm tissue culture dish and minced.
  • a single cell suspension was made by incubating the tissue with 0.25% trypsin for 20 minutes at 37°C.
  • the cells were dissociated by passing the fragments repeatedly through the constricted tip of a Pasteur pipet. The dissociated cells were then centrifuged at 500 ⁇ g for 45 seconds.
  • the cells were plated into 6 mm wells which had been coated with polyornithine (10 ⁇ g/ml) and laminin (10 ⁇ g/ml). The cell viability was checked, after 24 hours in culture, by the ability of the cells to exclude trypan blue.
  • the normal growth medium, 5HS/N3, for cultures composed of neurons and glia contained:
  • the growth medium was removed from the cultures by rinsing the cells twice with 100 ⁇ l of PBS.
  • the cells were lysed via one freeze-thaw cycle and a 15 minute incubation at 37°C in 50 mM KH 2 PO 4 pH 6.7, containing 200 mM NaCl and 0.25% (v/v) Triton X-100.
  • Two microliters of the cell lysate was removed and assayed for CAT activity according to the microFonnum procedure (Fonnum, 1975, J. Neurochem. 24:407- 409).
  • the final substrate composition consisted of 0.2 mM [ 14 C] Acetyl-CoA (NEN, 54.4, mCi/mmol), 300 mM NaCl, 8 mM choline bromide, 20 mM
  • acetyltransferase was tested by the addition of a specific inhibitor of CAT activity, N-hydroxyethyl-4- (1-napthylvinyl)pyridium (HNP), during the assay
  • Neurotrophin-3 slightly increased the level of CAT activity after a 7-day treatment period at a concentration of 100 ng/ml (Figure 22).
  • NGF nerve growth factor
  • Quantitative data are expressed as cpm in L4 plus L5 DRG and compared to L4, L5 cpm in the non-injected side.
  • [ 125 I]NT-3 was shown to be retrogradely transported from the crushed adult rat sciatic nerve to the L4, L5 DRG ( Figure 23).
  • the transport was specific in that a 100-fold excess of unlabeled NT-3 was able to block 80-90% of the transport.
  • rats were anesthetized with chloral hydrate (170 mg/kg) mixed with pentobarbital (35.2 mg/kg).
  • the right sciatic nerve was exposed and 2 ⁇ l of 125 I-labeled NT-3 or BDNF or NGF was injected into the nerve at the level of the obturator internus tendon. Wounds were sutured and the rats allowed to recover for 18 hr. At this time rats were killed by decapitation and the lumbar 4th (L4) and 5th dorsal root ganglia (DRG) removed and placed in 4%
  • L4-L5 spinal cord segment was also dissected and counted in fixative. Samples were counted in a gamma counter for 1 minute and the counts per minute for L4 plus L5 DRGs were assessed.
  • Figure 24 shows that all three labeled neurotrophins were transported to the ipsilateral (right) but not contralateral (left) DRG when injected into the right sciatic nerves of control rats.
  • Figure 24 also shows that [ 125 -I]NT-3 and [ 125 I]BDNF transport in the DRG were reduced 71% and 60%, respectively, in pyridoxine treated rats (p ⁇ 0.01). No significant change was observed for 125 I-NGF in pyridoxine treated rats. Interestingly, the transport of NT-3 and BDNF was not significantly altered in the spinal cords of pyridoxine treated rats compared to controls
  • Neurotrophin-3 was radioiodinated as described supra in Example Section 14.1.2.
  • NT-3 is retrogradely transported from the hippocampus to the neurons of the medial septum/diagonal band, although proportionately many fewer counts were transported for NT-3 than NGF.
  • Film and emulsion autoradiographic experiments (Table 5) showed that labeling associated with both neurotrophic factors was well localized to magnocellular neurons of the medial septum and diagonal band, cells which are known to provide the cholinergic input to hippocampus.
  • Magnocellular neurons were very densely labeled and quite numerous in NGF-injected animals compared to animals injected with NT-3. However, NT-3 was found also to be transported by a population of smaller neurons within the medial septum/diagonal band.
  • NT-3 was also transported by large
  • NT-3 were immunoreactive for tyrosine hydroxylase, a marker for the dopaminergic neurons of the pars compacta.
  • NT-3 was transported to several additional sites in the rat forebrain. As is the case for NGF, we have demonstrated transport of radiolabelled NT-3 to the supramammillary nucleus, but here the pattern of labeling is more intense than is apparent for NGF. Additionally, NT-3 was transported to a number of brain regions which do not appear to transport NGF at all. After injection into the dentate gyrus of the hippocampus, a few labeled cells were present bilaterally within the CA4/hilus
  • HPC dorsal hippocampus
  • MS/OB medial septum/diagonal band of Broca
  • V Mes
  • ventral mesencephalon r
  • right side l
  • left side All injections (striatum and hippocampus) were made on the right side of the brain.
  • Numbers with T prefix in the trophic factor column are animal code numbers. All other numbers represent cpm in the brain area indicated.

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Abstract

La présente invention se rapporte à une méthode thérapeutique et diagnostique basée sur l'expression de neurotrophine-3 (NT-3) humaine, et pouvant être spécifiquement utilisée comme traitement potentiel de la maladie d'Alzheimer, de la maladie d'Huntington ainsi que des troubles ou affections du système nerveux périphérique. La présente invention se rapporte également à l'activité de la NT-3 favorisant la survie sur des populations neuronales dopaminergiques. L'invention est en outre destinée à des applications thérapeutiques et diagnostiques basées sur l'aptitude de la NT-3 à être transportée de manière rétrograde à la fois dans le sytème nerveux central et dans le système nerveux périphérique.
PCT/US1992/009652 1991-11-12 1992-11-12 Methodes therapeutiques et diagnostiques basees sur l'expression de nt-3 specifique de tissus et sur la liaison de recepteurs WO1993009798A1 (fr)

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WO2000067032A1 (fr) * 1999-05-03 2000-11-09 Evotec Biosystems Ag Procedes permettant de diagnostiquer ou traiter la depression
WO2001064247A2 (fr) * 2000-02-29 2001-09-07 Cephalon, Inc. Methode de traitement du cancer a l'aide d'agents anti-neurotrophiques
WO2001064247A3 (fr) * 2000-02-29 2002-02-14 Cephalon Inc Methode de traitement du cancer a l'aide d'agents anti-neurotrophiques
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PT101051A (pt) 1994-02-28
CN1073264A (zh) 1993-06-16

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