Classifications

• • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia

Definitions

• the invention relates to methods for treatment of neurodegenerative disease and aging, and methods for delivery of therapeutic growth factor into the mammalian brain. Specifically, the invention pertains to the use of growth factors that activate the trkB nervous system growth factor receptor (including brain-derived neurotrophic factor (BDNF) and nervous system growth factor-4/5 (NT-4/5)) to stimulate neuronal activity in the entorhinal cortex (EC).
• growth factors that activate the trkB nervous system growth factor receptor including brain-derived neurotrophic factor (BDNF) and nervous system growth factor-4/5 (NT-4/5)
• EC entorhinal cortex
• BDNF and NT-4/5 are neuronal growth factors which play a role in brain function through a variety of mechanisms, including stimulation of glutamate-mediated communication between cerebrocortical neurons and cortical astrocytes (Pascual, et al, Neuroreport, 12:2673-2677, 2001), and induction of dopamine formation (Theofilopoulous, et al., Brain Res. Dev. Brain Res, 127:111-122, 2001). These growth factors share functionality with other growth factors such as NT-3 and NGF, in regulating neuronal connectivities between regions of the brain implicated in cognition.
• the researchers measured neuronal populations for loss in the hippocampus, dentate hilus, cortex and thalamic medial geniculate nucleus. Animals were also evaluated for performance in a Morris Water Maze (the same test used by the present inventors).
• the invention provides a clinically useful protocol for improving cognitive function in primates through delivery of nervous system growth factors, such as BDNF and NT-4/5, into the entorhinal cortex (EC) of the brain.
• nervous system growth factors such as BDNF and NT-4/5
• EC entorhinal cortex
• nervous system growth factors are delivered to normal, degenerated or injured tissue in the EC.
• use of the invention can also exert effects on the HC and is likely to exert effects on other cortical tissues which contain trkB receptors, such as the frontal cortex, parietal cortex temporal cortex and visual cortex.
• Delivery is by direct infusion of the nervous system growth factor protein, or by introduction of an expressible nervous system growth factor-encoding transgene into the targeted coritcal tissue(s).
• a nervous system growth factor is delivered to the EC in animals in whom spatial learning abilities and memory has been impaired by aging. Based on measures of cognitive function, including the Morris Water Maze, the impairments in spatial learning and memory are significantly ameliorated by treatment with the nervous system growth factor according to the invention.
• the nervous system growth factor is BDNF, delivered to cortical tissues, including one or more sites in the EC, by one time infusion.
• the nervous system growth factor is BDNF, delivered to cortical tissues, including one or more sites in the EC, by chronic infusion.
• measured cognitive function in treated animals improves to a level equivalent to function in unimpaired animals.
• Practice of the invention enables one to improve cognitive function lost to neurodegeneration in the EC.
• the effects of the inventive method can extend to trkB receptor containing tissues other than the EC, such as the HC and the frontal, parietal and visual cortices, thereby offering the opportunity to substantially reverse the effects of neurodegeneration associated with disease (such as Alzheimer's) or aging.
• BDNF native or recombinant BDNF, NT4/5 or other nervous system growth factors of equivalent activity into targeted cortical tissues, including the EC, may be made by infusion of the protein, or active fragments thereof, into the tissue at specified coordinates.
• Recombinant nervous system growth factor may also be delivered via an expressible transgene, carried in a recombinant expression vector (viral, non-viral or via a host cell, such as a fibroblast).
• Surgical delivery of a nervous system growth factor composition into the brain may be achieved by means familiar to those of skill in the art, including direct infusion or chronic infusion utilizing a micropump (e.g., the Alzet osmotic pumps commercially available from DURECT Corporation [10240 Bubb Road, Cupertino, Calif.
• a micropump e.g., the Alzet osmotic pumps commercially available from DURECT Corporation [10240 Bubb Road, Cupertino, Calif.
• microinjection through a surgical incision see, e.g., Capecchi, Cell, 22:479-488 (1980)
• electropotation see, e.g., Andreason and Evans, Biotechniques, 6:650-660 (1988)
• infusion chemical complexation with a targeting molecule or co-precipitant (e.g., liposome, calcium), and, for expressible transgenes, microparticle bombardment of the target tissue (Tang, et al., Nature, 356:152-154 (1992)).
• a targeting molecule or co-precipitant e.g., liposome, calcium
• Example I A description of a surgical technique used to introduce rBDNF into the EC of male Fischer rats using a micropump is provided in Example I, below. Coordinates for the EC, and for specific grafting sites within the EC, are selected so as to cluster in an area of EC neuronal loss and/or loss of BDNF expression and/or loss of BDNF sensitive gene expression in the EC, such as gaba-b receptor expression (Example IV). Such areas may be identified clinically using a number of known techniques, including magnetic resonance imaging (MRI) and biopsy. In humans, non-invasive, in vivo imaging methods such as MRI will be preferred.
• MRI magnetic resonance imaging
• Materials useful in the methods of the invention include nervous system growth factor protein (BDNF, NT-4/5, NT-3 or a growth factor of equivalent effect on neuronal growth and activity in the EC), active protein fragments, in vivo compatible recombinant expression vectors, packaging cell lines, helper cell lines, synthetic in vivo gene therapy vectors, regulatable gene expression systems, encapsulation materials, pharmaceutically acceptable carriers and polynucleotides coding for growth factors of interest.
• BDNF nervous system growth factor protein
• NT-4/5, NT-3 or a growth factor of equivalent effect on neuronal growth and activity in the EC active protein fragments
• in vivo compatible recombinant expression vectors packaging cell lines, helper cell lines, synthetic in vivo gene therapy vectors, regulatable gene expression systems, encapsulation materials, pharmaceutically acceptable carriers and polynucleotides coding for growth factors of interest.
• Known nervous system growth factors include nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), nervous system growth factor-3 (NT-3), nervous system growth factor-4/5 (NT-4/5), nervous system growth factor-6 (NT-6), ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), the fibroblast growth factor family (FGF's 1-15), leukemia inhibitory factor (LIF), certain members of the insulin-like growth factor family (e.g., IGF-1), the neurturins, persephin, artemin, the bone morphogenic proteins (BMPs), the immunophilins, the transforming growth factor (TGF) family of growth factors, the neuregulins, epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and others.
• NGF nerve growth factor
• BDNF brain-derived neurotrophic factor
• NT-3 nervous system growth factor-3
• NT-4/5 nervous system growth factor-6
• CNTF ciliary neurotrophic factor
• BDNF is a 27 kDa homodimer originally derived from human brain which shares high sequence homology (and some functionality) with NGF, NT-3 and NT-4/5, and influences many neuron types in the CNS.
• BDNF was first shown to promote the outgrowth of spinal sensory neurons, but has since been shown to support the survival and outgrowth of sensory neurons, ganglion neurons, dopaminergic neurons, cholinergic neurons, GABAergic neurons and motor neurons.
• BDNF can signal the differentiation of pluripotent neural crest cells into sensory neurons. Its effects are cell selective—BDNF exerts no supportive effect on NGF-sensitive sympathetic neurons.
• BDNF is produced primarily in the brain and spinal cord by glial cells, but is also produced by Schwann cells associated with peripheral motor neurons. It activates signal transduction by the dimerization and autophosphorylation of the TrkB receptor.
• Recombinant and native BDNF protein from different species, including humans, as well as NT-4/5 and immunoassays therefor, are commercially available from several sources, including, for rNT-4/5, Promega Corporation (2800 Woods Hollow Road Madison, Wis. 53711-5399); and, for rBNDF, Regeneron Pharmaceuticals, Inc. (777 Old Saw Mill River Road, Tarrytown, N.Y. 10591).
• coding polynucleotides, precursors and promoters for a number of human nervous system growth factors are known, as are coding sequences for nervous system growth factors of other mammalian species.
• GenBank M61176 sets forth the coding sequence (mRNA) for BDNF (see also, XM — 006027); BDNF precursor is set forth at BF439589; and a BDNF specific promoter is set forth at E05933.
• mRNA coding sequence for BDNF
• BDNF precursor is set forth at BF439589
• a BDNF specific promoter is set forth at E05933.
• a similar range of coding sequences for other nervous system growth factors, including NT-4/5 and NT-3, are also available through GenBank and other publicly accessible nucleotide sequence databases.
• Human growth factors are preferred for use in therapy of human disease according to the invention due to their relatively low immunogenicity as compared to allogenic growth factors.
• growth factors of other species e.g., non-human primates
• growth factors of other species e.g., non-human primates
• the strategy for transferring transgenes into target cells in vivo includes the following basic steps: (1) selection of an appropriate transgene; (2) selection and development of suitable and efficient vectors for gene transfer; (3) demonstration that in vivo transduction of target cells and transgene expression occurs stably and efficiently; (4) demonstration that the in vivo gene therapy procedure causes no serious deleterious effects; and (5) demonstration of a desired phenotypic effect in the host animal.
• the expression vector selected should meet the following criteria: 1) the vector must be able to infect targeted cells and thus viral vectors having an appropriate host range must be selected; 2) the transferred gene should be capable of persisting and being expressed in a cell for an extended period of time (without causing cell death) for stable maintenance and expression in the cell; and 3) the vector should do little, if any, damage to target cells.
• vectors known to have this capability include DNA viruses such as adenoviruses, adeno-associated virus (AAV), and certain RNA viruses such as HIV-based lentiviruses, feline immunodeficiency virus (FIV) and equine immunodeficiency virus (EIV.
• DNA viruses such as adenoviruses, adeno-associated virus (AAV), and certain RNA viruses such as HIV-based lentiviruses, feline immunodeficiency virus (FIV) and equine immunodeficiency virus (EIV.
• Other vectors with this capability include herpes simplex virus (HSV).
• HSV herpes simplex virus
• HIV-based lentiviral vector system which, like other retroviruses, can insert a transgene into the nucleus of host cells (enhancing the stability of expression) but, unlike other retroviruses, can make the insertion into the nucleus of non-dividing cells.
• Lentiviral vectors have been shown to stably transfect brain cells after direct injection, and stably express a foreign transgene without detectable pathogenesis from viral proteins (see, Naldini, et al., Science, 272:263-267 (1996), the disclosure of which is incorporated by reference; and Example V).
• the selected growth factor (protein or expressible transgene) will be delivered in a pharmaceutically acceptable carrier, to form a growth factor composition.
• a growth factor composition for use in the invention may be prepared by placing the growth factor protein or growth factor-encoding transgene (including, without limitation, those expressible in viral and non-viral vectors) into a pharmaceutically acceptable suspension, solution or emulsion. Suitable mediums include saline and liposomal preparations.
• pharmaceutically acceptable carriers may include sterile aqueous of non-aqueous solutions, suspensions, and emulsions.
• non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
• Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
• Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils.
• Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like.
• Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like.
• a composition of growth factor transgenes may be lyophilized using means well known in the art, for subsequent reconstitution and use according to the invention.
• unit dosage refers generally to the concentration of growth factor/ml of growth factor composition.
• the growth factor concentration is defined by the number of viral particles/ml of growth factor composition.
• each ml of growth factor composition will contain a concentration of protein or active peptide fragments between 1 and 25 ng/ml of carrier.
• each unit dosage of growth factor will comprise 2.5 to 25 ⁇ l of a growth factor composition, wherein the composition includes a viral expression vector in a pharmaceutically acceptable fluid and provides from 10 10 up to 10 15 growth factor expressing viral particles per ml of growth factor composition.
• viral vectors with an operable growth factor encoding transgene have been shown to express human growth factor after delivery to the brain and to the CNS for up to 12 months (Example V).
• human growth factor protein the exogenous growth factor can be expected to remain in the target tissue for periods somewhat shorter than may be achieved using growth factor expressible transgenes.
• the invention provides a chronically available source for growth factor in the brain.
• the process of aging simulates the neurological changes in the brain experienced in aging humans, including the loss of BDNF activity, EC neuronal cell populations, and loss of BDNF sensitive receptors (e.g., gaba-b).
• Data demonstrating the use and efficacy of the methods of the invention in aged animals are provided in the Examples.
• a non-aged animal model that models Alzheimer's Disease with a high degree of integrity are rats and primates in whom transection of the fornix pathway connecting the septum from the hippocampus has been performed.
• Clinical evaluation and monitoring of treatment can be performed using the in vivo imaging techniques described in the Examples, as well as through biopsy and histological analysis of treated tissue.
• neuronal numbers can be quantified in a tissue sample using, for example, anti-growth factor antibody (for immunoassay of secreted growth factor) (Example III), or by tracking growth factor sensitive gene expression, as demonstrated in Example IV.
• anti-growth factor antibody for immunoassay of secreted growth factor
• Example IV for tracking growth factor sensitive gene expression
• improved cognitive function is a clearly desirable end goal in aged, diseased or injured animals in whom such function has been impaired, and this goal may be achieved through use of the invention (Example II).
• Test animals underwent pre-operative water maze training, as described in Example II. Data presented in this pilot study were generated from analyzing: 10 BDNF-infused aged, 8 aged-intact, 9 vehicle-infused aged, 9 middle aged, and 20 young intact, and 2 vehicle-infused young rats. Aged (24 month-old), middle aged (11 month-old) and 10 young (5 month-old) male Fischer 344 rats were obtained from the Harlan/NIA rodent colony.
• Rats were anesthetized with a mixture of ketamine (50 mg/kg), acepromazine (0.5 mg/kg), and xylazine (2.6 mg/kg). After verifying that all reflex responses to cutaneous stimulation were absent, rats were implanted with following coordinates for entorhinal cortex in aged animals (relative to Bregma): ⁇ 9.3 mm anterior/posterior, ⁇ 5.6 mm medial lateral, 6 mm cannula length ventral to the skull surface.
• Water maze apparatus The first run of water maze testing was conducted in a black circular tank (diameter: 1.40 m; height: 0.60 m) filled with water (19-21° C.). A black escape platform was submerged 3 cm below the surface of the water in a specific location during training/acquisition trials. The escape platform was removed during probe testing. To provide a clear visible cue, four wooden posts were attached to the platform during cued trials. Black curtains were hung around the tank and four unique wall cues were hung to serve as environmental landmarks. For data analysis, the tank was divided into four quadrants: north, south, east, and west. Both collection and analysis of the data were performed using a San Diego Instruments (San Diego, Calif.) computer tracking system.
• San Diego Instruments San Diego, Calif.
• Pre-operative testing For all runs of water maze testing, the task consisted of 8 days of training, conducted in 4 training blocks of 6 trials (3 per day). Each training block included 5 acquisition trials (90 sec/trial max; 1 min inter-trial interval) followed by 1 probe trial (30 sec free swim). During all non-probe trials, the submerged escape platform was placed in the center of the “goal” quadrant of the pool. To begin each trial, rats were placed in the water, facing the maze wall, from one of four start positions evenly spaced around the pool (N, S, W, E). Start positions were chosen randomly at the beginning of each test day for all rats. Rats swam until they located the platform or for a maximum of 90-sec, after which the rat was guided to the platform. At the conclusion of each trial, rats remained on the platform for 30 seconds and were then removed by the experimenter and placed in a holding cage for 1 min.
• Cumulative search error (SE), time (latency), and distance (path length) to find the escape platform were used as measures of learning during the training trials. Every 6 th trial, rats were probed for learning of the platform location by removing (1 st run) or lowering (subsequent runs) the platform and recording the proximity average to the platform location and annulus crossings during a 30 sec free swim. Each of the learning measures from aged animals were examined and compared to young animals. Aged animals were considered aged impaired if their performance fell outside of the range of young animal performance. Acquisition data was used to form groups of aged-BDNF and aged-vehicle animals with equivalent levels of water maze performance before surgeries and post-operative testing.
• Post-operative testing After a three-week delay (during which time rats were receiving either BDNF or vehicle infusions), rats were re-tested on the identical multiple-trial place learning task (8 days, 4 blocks) that was used during pre-operative testing.
• Behavioral data analysis Data were compiled and analyzed in Stat View 5.0 for the Macintosh (Abacus Concepts, Berkeley, Calif.). Comparisons between groups were made using repeated-measures ANOVA for the training data, whereas, factorial ANOVAs were used for probe trial data. Behavioral data are presented as mean ⁇ standard error of the mean. Criteria for significant differences were set at the 95% probability level.
• hypothalamus, hippocampus, entorhinal cortex, prefrontal cortex, and the remainder of neocortex were sectioned from anesthesized animals, then immediately dissected and frozen in liquid nitrogen. Tissues were stored at ⁇ 80° C.
• Immunohistochemistry for BDNF was performed using a rabbit anti-BDNF antibody at a concentration of 1:6000 and sections prepared from the treated animals. Specificity of the antibodies was verified by omitting the primary antibody with a resultant loss of cellular labeling.
• BDNF levels of BDNF were determined using two-site enzyme-linked immunosorbent assays (ELISA) developed according to standard procedures (Conner et al., J. Neurosci, 17:2295, 1997). The assay was specific for BDNF and was relatively linear over the range for which it was used (1-100 pg/sample). Assays developed for BDNF showed no detectable cross-reactivity with other nervous system growth factor family members, even when these proteins were added to the assay at concentrations 20-fold in excess the upper assay limit (2000 pg/sample).
• ELISA enzyme-linked immunosorbent assays
• Detection of bound antigens was made by sequentially adding the appropriate detection (anti-BDNF (Promega G1641; 1:2500 dilution)) and HRP-conjugated (peroxidase conjugated anti-chicken IgY (Promega G1351; 1:1000 dilution) or peroxidase conjugated anti-mouse IgG (Dako p-260; 1:1000 dilution) antibodies (each incubated overnight at 4° C.). A soluble calorimetric reaction product was then generated, and optical density measurements were made on a microplate reader at an absorbance of 490 nm. In all cases, results were corrected for nonspecific interactions by subtracting values determined in IgG coated wells from those made in anti-nervous system growth factor coated wells.
• BDNF immunolabeling confirmed the accurate location of the cannulas within entorhinal cortex (FIG. 1).
• BDNF infusion significantly alters the expression of dozens of genes, mostly within the infusion site in EC, but also remotely in the hippocampus for a smaller number of genes.
• expression of the gaba-b receptor was increased by greater than 50-fold after BDNF infusion.
• Gaba-b receptor expression is reduced in both the EC and HC of aged-impaired animals.
• Such an alteration of responsiveness to putatively inhibitory neuronal signaling may mediate the behavioral effect of BDNF infusion.

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