WO2004053071A9 - Method for discovering neurogenic agents - Google Patents
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- WO2004053071A9 WO2004053071A9 PCT/US2003/038670 US0338670W WO2004053071A9 WO 2004053071 A9 WO2004053071 A9 WO 2004053071A9 US 0338670 W US0338670 W US 0338670W WO 2004053071 A9 WO2004053071 A9 WO 2004053071A9
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- C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
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Definitions
- bromodeoxyuridine (BrdU) and by immunostaining of postmortem brains with antibo ; dies against BrdU and neuronal markers.
- An endogenous marker of dividing cells ki67 protein, has also been used instead of BrdU for this PCT PATENT APPLICATION OF KELLEHER- ANDERS SON ET AL . purpose.
- the location of neurogenic areas in the CNS is limited to the dentate gyrus of the hippocampus and the subependymal layer of the striatum.
- BrdU has revealed significant number of new cells and new neurons being born in the dentate gyrus and the subependymal layer of the striatum.
- the process of generating new neurons occurs in the mature, adult brain in significant quantities throughout rodents, primates, and human species.
- Hippocampus is the well-known center of learning, memory, and other cognitive functions, processes which new information are added, edited, stored, and recalled constantly throughout life.
- neurogenesis becomes more widespread and perhaps functionally diverse.
- the newly born neurons of the subependyma also referred to as subventricular zone
- the newly born neurons have short survival period.
- a compound that can stimulate the endogenous neurogenesis either in a disease state or in a healthy PCT PATENT APPLICATION OF KELLEHER -ANDERS SON ET AL . state may be an effective drug for a number of human nervous system diseases.
- the current limitation is the lack of an effective., predictive in vitro assay that can be used to select a neurogenic compound for clinical drug development.
- a novel, in vitro assay which is effective and predictive, to be useful for discovering a compound that promotes neurogenesis in vivo.
- classes of compound structures that are shown to be particularly effective in promoting the
- This invention relates to the method of discovering a neurogenic drug to treat neurologic, psychiatric, and aging-related disorders. It also relates to the use of Fused Imidazoles/, Aminopyrimidines , Nicotinamides,
- Phenoxypiperidines and Aryloxypiperidines for use as therapeutic agents and analytical reagents by means of promoting neurogenesis. More particularly this invention relates to these agents as therapeutics for prevention and treatment of neurological diseases in mammals and reagents for detecting neurogenesis and proliferation.
- SSRIs selective serotonin uptake inhibitors
- fluoxetine the mixed noradrenaline/serotonin transporter blockers like tricyclic agent imipramine and noradrenaline uptake inhibitors like desipramine.
- the antidepressant- induced increase in -intraneuronal biogenic amines occurs quite rapidly.
- hippocampal volume loss might be considered a consequence of glucocorticoid-induced hippocampal neuronal loss
- Neurogenesis can be characterized as three successive stages: proliferation of endogenous stem cells and
- depression could potentially be caused by 1) inhibition of the endogenous hippocampal stem cell proliferation in the dentate gyrus, 2) inhibition of differentiation and PCT PATENT APPLICATION OF KELLEHER-ANDERSSON ET AL . dendrite development and 3) by loss of neurons (apoptosis) and their dendritic structure.
- apoptosis is observed in depression, hippocampal apoptosis as measured by DNA fragmentation from depressed patients appears to play only a minor role in the volume loss (Lucassen et al., 2001).
- the stress did cause a reduction in synaptic plasticity in the hippocampus (Xu et al., 1998).
- imipramine partially reversed the loss in LTP in socially stressed, depressive-like animals (Von Frijtag et al.,. 2001) suggesting imipramine effects on the plasticity phase of neurogenesis.
- Another animal model of depression presenting neurogenesis loss and hippocampal volume loss, stressed animals that chronically received the antidepressant, tianeptine, showed similar numbers of dividing cells as control animals (no social stress) a measure of proliferation (Czeh et al . , 2001).
- hippocampal dentate gyrus (Gould et al., 2001; Eriksson et al., 1998), we can use multi-potential hippocampal stem cells to screen agents in vitro for neurogenic activity.
- phosphorylated CREB then binds to CRE binding site to promote the expression of BDNF and bcl—2, that 1 appear critical to cell survival and plasticity. Proof of
- neurotrophic factor BDNF' s involvement in depression comes from studies showing that antidepressants and
- electroconvulsive shock both caused an increase in BDNF levels (Nibuya et al., 1996) and that intrahippocampal injection of BDNF had antidepressant activity in two models of depression (Shirayama et al . , 2002) .
- AD therapeutics that address the neuronal loss, in the hopes of reducing the cognitive decline.
- One strategy enlists trophic agents, that regulate neuronal function and survival, as AD therapeutics (see Peterson and Gage, 1999) . Problems with systemic administration, side effects and locating trophic- sensitive neurons generated few clinical successes with these therapies.
- One AD therapeutic, AIT-082 promotes memory enhancement in AD individuals potentially by stimulating endogenous trophic factors (Ritzman and Glasky, 1999;
- Hippocampus is one of the main brain regions where neurogenesis in adult brain has been documented across several vertebrate species, including monkeys and humans (e.g., Gould et al., 2001; Eriksson et al., 1998). In fact, adult
- hippocampal neurogenesis contributes functionally to cognitive capacity.
- Shors et al. 2001
- inhibition of PCT PATENT APPLICATION OF KELLEHER-ANDERSSON ET AL neurogenesis in adult rat hippocampus, in the absence of the destruction of existing neurons, caused impaired memory function.
- a focal ischemic model reversible photothrombic ring stroke, caused increased neurogenesis in rat cortex by 3- 6% (Gu et al . , 2000). Seizures induced by electroconvulsive shock in adult rats increased neurogenesis in dentate gyrus of hippocampus (Scott et al, 2000; Madsen et al, 2000). Also, rats gamma-irradiated to kill mitotic cells in the CNS showed reduced numbers of nascent neurons and reduced LTP in slice cultures. These observations highlight the likelihood that a cellular mechanism for neurogenesis within adult human CNS, especially in hippocampus, does exist both as a normal
- a small molecule that induces hippocampal neurogenesis that is blood brain barrier penetrable would allow for a potentially novel oral therapeutic for Alzheimer's disease.
- AD therapeutics progressing in clinical trials, target neurodegeneration in the hopes of reducing the neuronal loss and cognitive decline.
- Apoptotic death involving caspase pathways and DNA fragmentation has been measured in in vitro and animal models of AD and in Alzheimer's diseased brain tissue.
- the extent of apoptosis leading to neuronal loss is of continual debate with most agreeing it has some effect, but that other neuronal death pathways definitely play a role PCT PATENT APPLICATION OF KELLEHE - NDERS SON ET AL .
- neuroprotective agents may improve or reverse the cognitive decline observed in MCI and AD.
- neurogenesis in a mammalian neuroepithelium proliferation and differentiation of an olfactory neuron precursor in vitro.
- Opioids intrinsically inhibit the genesis of mouse cerebellar granule neuron precursors in vitro: differential impact of mu and delta receptor activation on proliferation and neurite elongation. Eur J Neurosci. vol 12 ( ): 1281-93.
- VEGF Vascular endothelial growth factor
- Intracerebroventricular infusion of insulin-like growth factor-1 ameliorates the age-related decline in hippocampal neurogenesis. Neuroscience vol. 107 (4), pp603-613.
- Acetylcholine stimulates cortical precursor cell proliferation in vitro via
- CREB cAMP response element binding protein
- Fibroblast growth factor-2 activates a latent neurogenic program in neural stem cells from diverse regions of the adult CNS. J Neurosci. vol 19 (19) : 8487-97.
- Rathbone MP Middlemiss PJ, Gysbers J , Andrew C, Herman MA, Reed JK, Ciccarelli R, Di Iorio P, and Caciagli F.
- hippocampal progenitor cells in vitro temporal expression of intercellular coupling and voltage- and ligand-gated responses.
- FGF-2-responsive neural stem cell proliferation requires CCg, a novel autocrine/paracrine cofactor. Neuron, vol 28 (2 ): 385-97.
- Glucocorticoid receptor and protein/RNA synthesis-dependent mechanisms underlie the control of synaptic plasticity by stress. PNAS USA. vol 95:3204-3208.
- a neurogenic drug is an agent that enhances the process of generating new neurons (neurogenesis) . Recent studies indicate that neurogenesis occurs in the adult human brains under normal as well as under degenerative conditions and that such adult-generated neurons do
- Neurodegenerative conditions including Alzheimer's disease, mild cognitive impairment, dementia, age-related cognitive decline, stroke, traumatic brain injury, spinal cord injury and the like are neurodegenerative conditions.
- Neuropsychiatric diseases including depression, anxiety, schizophrenia and the like also show nerve cell
- a neurogenic drug would be beneficial for countering and treating these diseases.
- the present invention discloses a method of
- Such drug will serve to prevent or treat neurodegenerative and neuropsychiatric disorders by promoting the birth of new neuron endogenously within the nervous system by administering the compounds of the present invention into the patient. This may involve delivery of the agents alone or together with transplanted stem cells or progenitor cells.
- compounds of the type Fused Imidazoles, Aminopyrimidines , Nicotinamides, Aminomethyl Phenoxypiperidines and Aryloxypiperidines are evaluated for their ability to promote neurogenesis by proliferation/ differentiation of human hippocampal multipotent stem/ progenitor cells and neuronal progenitors.
- Figure 1 Schematic description of neurogenesis processes captured in the assay and different potential sites of a neurogenic drug action.
- Alamar Blue a fluorescent dye, is used as an indicator of metabolic respiration to determine optimum plating density. Results at an initial plating density of 30,000 cells/well suggest a large difference in cell number on removal of mitogen from the N2b media
- Figure 3A Influence of known growth factors on proliferation and neurogenesis relative to control.
- Hippocampal progenitor cells were treated for seven days with differentiation media (without mitogen) in the
- Hippocampal progenitor cells were treated for seven days with differentiation media (without mitogen) in the
- the percentage of neurons calculated for each treatment are as follows: 48.5 + 6.3% for controls and 53.6+1.15 for LIF.
- the non-TUJl positive cells are mainly astrocytic (GFAP+) .
- Figure 4 Examples of proliferation profile of compounds selected from primary screening. Proliferation was measured after compound treatment for 7 days by Alamar Blue staining of live cells per well. Shown are relative values over the vehicle control.
- Typical ratio for vehicle control is 40-50% neurons. The ratio can change by either increased
- a screening of a large number of unknown agents e.g., protein factors, peptides, nucleic acids, natural
- a neural progenitor population derived as a stable cell line from partial differentiation of embryonic stem cells can also be used.
- a cell line is defined as a population of cells having been expanded for at least 10 cell-doublings.
- Cell lines that are genetically engineered to enhance the cells' mitotic capacity can also be used.
- the genetic modification consists of over-expression of functional c-myc protein intracellularly under a conditional activation system such as c-myc protein fused to a ligand-binding domain of an estrogen receptor.
- a conditional activation system such as c-myc protein fused to a ligand-binding domain of an estrogen receptor.
- a progenitor population that upon differentiation generates both neurons and glia in a single culture has been used. Presence of glia, either astrocytes and/or oligodendrocytes or their
- differentiation of the progenitors is initiated by withdrawing the mitogen from the culture. Serum as well as other growth-promoting factors should be avoided from the differentiating culture since they will significantly affect the reproducibility and interfere with the neurogenesis assay.
- Undifferentiated mitotic cells are harvested by enzyme treatment to remove residual mitogen, in the preferred embodiment, basic fibroblast growth factor (bFGF) .
- bFGF basic fibroblast growth factor
- the initial seeding density can be within the range of about 2,000-125,000 cells per well of a 96-well plate.
- the preferred density is 40,000 cells per well of a 96-well plate, which has been optimized for best signal-to-noise ratio. Too low cell density retards the initiation of differentiation and results in poor plating efficiency, PCT PATENT APPLICATION OF KELLEHER-ANDERSSON ET AL . which interferes with the assay. Too high cell density leads to inhibition of neurogenesis due to cell-cell contact and paracrine factors, which also interferes with the assay.
- the actual cell number can be proportionally decreased or increased depending upon the surface area of the culture substrate used. For example, for a 384-well plate, which has approximately 1/4 of the surface area of a 96-well plate, the initial seeding density should be decreased accordingly (1/4).
- a neurogenic drug is to increase the number of neurons generated from their precursors.
- a molecule can bring about such increase in the neurogenesis by a number of different mechanisms. It can act as a mitogen for the neural stem/progenitor cells and increase the progenitor's cell number, which in turn results in increased number of neurons in the culture when
- PCT PATENT APPLICATION OF KELLEHER-ANDERSSON ET AL it can act as a mitogen for committed neuronal progenitors that differentiate only into neurons. Increasing this subpopulation would also increase the final number of neurons in the culture. Or, it can act as a survival factor to rescue immature neurons from undergoing cell death during differentiation, which will result in
- the assay method here captures all of these
- the assay is continued for seven days. A minimum of three days from the onset of differentiation should be allowed for stable expression of definitive neuronal markers to appear. A sufficient time is also required for a compound action on differentiation and/or proliferation to take place to a sufficient degree to be reliably detectable. Manifestation of drug-induced changes in neuron number takes a minimum of three days for the human cells to be detectable.
- the final neuron number is detected by immunostaining of the culture with antibodies against neurons and
- the collected cells were seeded at 40,000 cells per well of 96-well plates pre-coated with extracellular matrix proteins (e.g., Biocoat PDL, Fisher).
- the seeding media is a standard serum-free, growth factor- free, basal media that supports healthy neuronal/glial survival, such as N2 without phenol red.
- the fluorescence level is proportional to the number of respiring cells in the culture and is a measure of a proliferative activity of a test agent ( Figure 2) .
- the cells were fixed and stained with antibodies against neuron-specific antigens according to standard procedures.
- Typical antigens effective were Typelll-beta tubulin and
- the total cell number in each well was quantified by staining the cultures with a nuclear dye such as DAPI or Hoechst according to standard procedures.
- Example 1 Selection of a positive control.
- Several neurotrophic factors--including brain-derived neurotrophic factor, glia-derived neurotrophic factor, neurotrophic PCT PATENT APPLICATION OF KELLEHER- ANDERS SON ET AL . factor-3, and leukemia inhibitory factor--suggested to have neurogenic properties were tested in the assay described above. Only one (leukemia inhibitory factor) was effective ( Figure 3A and 3B) . Thus, the assay can discriminate test agents for selectively having a neurogenic activity.
- the positive control utilized is leukemia inhibitory factor (LIF) , a cytokine growth factor, at 20 ng/ml.
- LIF leukemia inhibitory factor
- LIF as the positive control is based on its properties to increase by 2-3 fold the number of neurons and glia. This effect validates both the neural stem cell system, in which, should a compound be effective in neurogenesis, the cells respond appropriately by enhanced differentiation and/or mitosis, and the assay method in which such cellular responses can be measured reproducibly and quantifiably .
- Example 2. Primary screening of unknown compounds.
- Imidazoles, Aminopyrimidines , Nicotinamides, Aminomethyl Phenoxypiperidines and Aryloxypiperidines are evaluated for their effect on neurogenesis according the assay method described above. From the preliminary analysis using the fluorescent plate reader, over 300 compounds to date showed initial positive activity. Those were re-analyzed by quantitative neuron counting. Among them, 30 compounds PCT PATENT APPLICATION OF KELLEHER-ANDERSSON ET AL . significantly increased cell number '( "proliferation" ,
- Table II contains the summary of EC50 of each compound tested. On the other hand, at high
- an agent would be administered to treat a neurodegenerative disease.
- the agent would be administered to treat a neurodegenerative disease.
- neurodegenerative disease would be Alzheimer's disease, dementia, mild cognitive impairment, aged-related cognitive decline, Parkinson's disease, amyotrophic lateral
- sclerosis multiple sclerosis, demyelination, stroke, spinal injuries, traumatic injuries, neuropathic pain, and the like.
- this invention would be administered to treat a psychiatric disease.
- the psychiatric disease is depression, post-traumatic stress syndrome, stress, anxiety, schizophrenia, sleep deprivation, cogntive dysfunction, amnesia, and the like.
- an agent would be administered by any number of routes and multipotent stem cells or differentiated multipotent stem cells would be transplanted into brain.
Abstract
Description
Claims
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AU2003293409A AU2003293409A1 (en) | 2002-12-09 | 2003-12-05 | Method for discovering neurogenic agents |
EP03790356A EP1576134B1 (en) | 2002-12-09 | 2003-12-05 | Method for discovering neurogenic agents |
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US43235902P | 2002-12-09 | 2002-12-09 | |
US60/432,359 | 2002-12-09 | ||
US49367403P | 2003-08-08 | 2003-08-08 | |
US60/493,674 | 2003-08-08 |
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WO2004053071A2 WO2004053071A2 (en) | 2004-06-24 |
WO2004053071A3 WO2004053071A3 (en) | 2006-03-30 |
WO2004053071A9 true WO2004053071A9 (en) | 2012-08-16 |
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PCT/US2003/038670 WO2004053071A2 (en) | 2002-12-09 | 2003-12-05 | Method for discovering neurogenic agents |
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US (8) | US20040185429A1 (en) |
EP (1) | EP1576134B1 (en) |
AU (1) | AU2003293409A1 (en) |
WO (1) | WO2004053071A2 (en) |
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-
2003
- 2003-12-05 AU AU2003293409A patent/AU2003293409A1/en not_active Abandoned
- 2003-12-05 US US10/728,652 patent/US20040185429A1/en not_active Abandoned
- 2003-12-05 WO PCT/US2003/038670 patent/WO2004053071A2/en not_active Application Discontinuation
- 2003-12-05 EP EP03790356A patent/EP1576134B1/en not_active Expired - Lifetime
-
2008
- 2008-03-17 US US12/049,922 patent/US7560553B1/en active Active
-
2009
- 2009-07-09 US US12/500,073 patent/US7858628B2/en active Active
-
2010
- 2010-11-04 US US12/939,914 patent/US8030492B2/en active Active
- 2010-11-04 US US12/939,897 patent/US8058434B2/en active Active
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- 2011-10-07 US US13/269,507 patent/US8362262B2/en not_active Expired - Fee Related
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2013
- 2013-01-17 US US13/744,220 patent/US8674098B2/en active Active
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- 2014-01-31 US US14/170,344 patent/US8846914B2/en active Active
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US20100034784A1 (en) | 2010-02-11 |
US20120027733A1 (en) | 2012-02-02 |
EP1576134A2 (en) | 2005-09-21 |
AU2003293409A8 (en) | 2004-06-30 |
US20110135612A1 (en) | 2011-06-09 |
AU2003293409A1 (en) | 2004-06-30 |
EP1576134B1 (en) | 2013-03-06 |
WO2004053071A3 (en) | 2006-03-30 |
US8030492B2 (en) | 2011-10-04 |
US20110052552A1 (en) | 2011-03-03 |
US20140147424A1 (en) | 2014-05-29 |
US8058434B2 (en) | 2011-11-15 |
US20040185429A1 (en) | 2004-09-23 |
US7560553B1 (en) | 2009-07-14 |
US20130195816A1 (en) | 2013-08-01 |
US8674098B2 (en) | 2014-03-18 |
US8362262B2 (en) | 2013-01-29 |
EP1576134A4 (en) | 2007-05-30 |
US8846914B2 (en) | 2014-09-30 |
WO2004053071A2 (en) | 2004-06-24 |
US7858628B2 (en) | 2010-12-28 |
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