WO2014107686A1 - Methods for treating neuron damage - Google Patents

Methods for treating neuron damage Download PDF

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Publication number
WO2014107686A1
WO2014107686A1 PCT/US2014/010385 US2014010385W WO2014107686A1 WO 2014107686 A1 WO2014107686 A1 WO 2014107686A1 US 2014010385 W US2014010385 W US 2014010385W WO 2014107686 A1 WO2014107686 A1 WO 2014107686A1
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Prior art keywords
gga
damaged
cns
mirna
trans
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PCT/US2014/010385
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French (fr)
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Hiroaki Serizawa
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Coyote Pharmaceuticals, Inc.
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Publication of WO2014107686A1 publication Critical patent/WO2014107686A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/12—Ketones
    • A61K31/121—Ketones acyclic
    • 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

  • This invention relates generally to methods for treating a disease in a subject mediated in part by mieroRRA (raiRNA)-induced regulation of mRNA translation (e.g., miRNA-378 or miRNA-71 1 increased activity) comprising administering to the subject a therapeutically effective amount of 5-irans-gemaylgeraiiyl acetone (GGA) or a derivative thereof.
  • mieroRRA mieroRRA
  • GGA 5-irans-gemaylgeraiiyl acetone
  • Geranylgeranyl acetone is an acyclic isoprenoid compound with a retinoid skeleton that has been shown to induce expression of heat shock proteins in various tissue types.
  • GGA is a known anti-ulcer drug used commercially and in clinical situations.
  • GGA has also been shown to exert cytoprotective effects on a variety of organs, such as the eye, brain, and heart (See for example Ishii Y., et al., Invest Ophthalmol Vis Set 2003; 44: 1982-92; Tanito M, et al, J Neurosci 2005; 25:2396-404; Fujiki M, et al, J Neurotrauma 2006; 23 : 1 164-78; Yasuda H, et al., Brain Res 2005; 1032: 176-82; Ooie ' ! ' . et al., Circulation 2001 ; 104: 1837-43; and Suzuki S. et al.
  • Kidney Int 2005; 67:2220-20 The effects and cytoprotective benefits of GGA in these settings is less understood as is the relationship of isomers of GGA to these cytoprotective benefits. Of particular interest, is the effect of GGA on extranuclear neurodegeneration both on an intracellular or extracellular basis.
  • Neurodegeneration is often the result of increased age, sporadic mutations, environmental exposures, disease, and/or protein aggregation in CNScelis: pathogenesis may also be related to a combination of the aforementioned factors.
  • Neurodegenerative diseases are often characterized by a progressive neurodegeneration of tissues of the nervous system and a change/loss of functionality of the neurons themselves as well as surrounding glial and immune cells. There is a need in the art for therapeutic compositions and methods that can treat these damaged cells without exerting toxic side effects.
  • This invention relates to pharmaceutical methods and uses of geranylgeranyl acetone (GGA) and GGA derivatives.
  • GGA geranylgeranyl acetone
  • One aspect relates to a method for treating a disease in a subject mediated in part by regulating mR A translation via modulation of miRNA including but not limited to miRNA-378 or miRNA-711 increased activity comprising administering to the subject a therapeutically effective amount of 5-trans-GGA or a derivative thereof,
  • this invention provides a method for treating damaged cells involved in newodegeneration exhibiting altered miRNA (e.g., increased miRNA-378 and/or miRNA-71 1 activity), said method comprising administering an effective amount of 5-trans- GGA or a derivative thereof as defined herein so as to decrease regulatory miRNA activity (such as miRNA-378 or miRNA-71 1 ) specifically in damaged neurons and/or neuron support ce!is proximate to damaged neurons, if! a preferred embodiment, the decrease miRNA correlates to an increase in HSP 70 protein,
  • this invention provides a method for treating damaged ceils in a subject in need thereof, wherein the damage to the cells arises from repression of mRNA translation (including but not limited to increased miRNA-378 and/or miR A-11 activity, said method comprising decreasing mi. NA-378 or miRNA-71 1 activity) specifically in damaged neurons and/or neuron support cells proximate to damaged neurons said method comprising administering a therapeutically effective amount of 5-trans-GGA or a derivative thereof to the subject, in a related embodiment, the decrease miRNA correlates to an increase in HSP 70 protein.
  • the effective amount of GGA is from about 1 mg/kg/day to about 12 mg/kg/day, or from about 1 mg/kg day to about 5 mg kg day, or from about 6 mg/kg/day to about 12 mg/kg/day, or preferably, about 3 mg/kg/day, about 6 mg kg day, or about 12 mg/kg/day.
  • a method of treadng a neural disease comprising administering GGA or a derivative thereof in an amount of about 1 - about 2 mg/kg sublingual! ⁇ ' to a patient, preferably a human patient, in need thereof.
  • the amount is administered as a daily amount, in another embodiment, the amount is administered once daily.
  • the neural disease is amyotrophic lateral sclerosis (ALS).
  • the patient has difficulty to swallow or is incapable of swallowing an oral dosage form, such as a solid oral dosage form.
  • the patient shows modulation of one or more of families of heat shock proteins such as heat shock protein 70 (HSP70), HSP1 10, HSP90, HSP60, HSP40 and HSP27.
  • GGA is administered in accordance with this invention.
  • the GGA is trans GGA, preferably trans GGA which is free of or substantially free of as GGA,
  • pro vided herein is a method of treating a damaged cell having altered miRNA such as increased miRNA-378 and/or miRNA-71 1 activity, the method comprising: contacting the damaged neuron with a therapeutically effective amount of 5-trans-GGA or a derivative thereof, and if miRNA-378 and/or miRNA-71 1 activity decreases in the contacted neuron, continuing to contact the damaged neurons with a therapeutically effective amount of 5-trans-GGA or a derivative thereof.
  • the damaged neuron is in a subject suffering from a neurodegenerative disease.
  • the neurodegenerative disease is ALS, Alzheimer's Disease (AD), or Parkinson's Disease (PD).
  • the 5-trans-GGA or a derivative thereof is administered sublingual! ⁇ ' to the subject, in one embodiments, the damaged neuron has reduced HSP activity.
  • a method of treating a damaged neuron having reduced HSP activity comprising: contacting the damaged neuron with a therapeutically effective amount of 5-trans-GGA or a derivative thereof, and if HSP activity increases in the contacted neuron, continuing to contact the damaged neurons with a therapeutically effective amount of 5-trans-GGA or a derivative thereof, in one embodiment, the damaged neuron is in a subject suffering from a neurodegenerative disease.
  • the neurodegenerative disease is ALS, Alzheimer's Disease (AD), or Parkinson's Disease (PD).
  • the 5-trans-GGA or a derivative thereof is administered sublmgually.
  • the amount is administered as a daily amount. In one embodiment, the amount is administered once daily. It; one embodiment, the GGA or the derivative thereof is GGA, In one embodiment, the GGA is irons GGA free of or substantially free of cis GGA.
  • a method of modulating miRNA-378 or miRNA- 71 1 activity in a damaged neuron comprising contacting the damaged neuron with an effective amount of 5-trans-GGA or a derivative thereof.
  • the damaged neron is damaged at least in pari by ALS, Alzheimer's Disease (AD), or Parkinson's Disease (PD).
  • a method of treating a damaged neuron comprising causing neuro-inflammation in the damaged neuron, wherein the neuroinflammation results at least in part from contacting the damaged cell with an effective amount of 5-trans-GGA or a derivative thereof.
  • a method of treating a damaged neuron comprising increasing HSP acti ity in the damaged neuron, wherein the increasing HSP activity results at least in part from contacting the damaged cell with an effective amount of 5-trans-GGA or a derivative thereof.
  • a method of treating a damaged neuron having reduced GRP78 activity comprising: contacting the damaged neuron with a therapeutically effective amount of 5-trans-GGA or a derivative thereof, and if GRP78 activity increases in the contacted neuron, continuing to contact the damaged neurons with a therapeutically effective amount of 5-trans-GGA or a derivative thereof.
  • a method of treating a damaged neuron comprising increasing G .P78 activity in the damaged neuron, wherein the increasing GRP78activity results at least in part from contacting the damaged cell with an effective amount of 5-trans- GGA or a derivative thereof.
  • a pharmaceutically acceptable composition preferably in a unit dosage form, comprising about 50 - about 200 mg of GGA or a derivative thereof and at least one pharmaceutically acceptable excipieut.
  • the pharmaceutically acceptable composition is for sublingual administration.
  • Pharmaceutically acceptable exciptents including those suitable for sublingual delivery, are known to the skilled artisan for use in accordance with this invention.
  • the pharmaceutically acceptable composition comprises GGA.
  • the GGA is trans GGA, preferably trans GGA which is free of or substantially free of as GGA.
  • a method of treating a neural disease comprising administering geranylgeranyl acetone (GGA) or a derivative thereof in an amount of about 1 - aboui 2 mg kg sublingual! ⁇ ' to a human patient in need thereof.
  • the amount is administered as a daily amount.
  • the amount is administered once daily, in one embodiment, the neural disease is amyotrophic lateral sclerosis (ALS).
  • the GGA or the derivative thereof is GGA.
  • the GGA is tram GGA free of or substantially free oicis GGA.
  • a pharmaceutically acceptable composition comprising about 50 - about 200 mg of GGA or a derivative thereof and at least one pharmaceutically acceptable excipient.
  • the pharmaceutically acceptable composition is for sublingual administration.
  • the GGA or the derivative thereof is GGA.
  • the GGA is trans: GG A, free of or substantially free of cis GGA.
  • FIG. 1 shows the time course of body weights of test animals.
  • FIG. 2 shows imputed clinical scores of in vivo tests
  • FIG. 3 shows imputed neuroscores of in vivo tests.
  • FIG. 4 shows a graph of phamiacokinetic data for CNS-102 administered to rats intravenously (see Example 26 for details).
  • FIG. 5 shows a graph of pharmacokinetic data for CNS-102 administered to rats orally (see Example 25 for details).
  • FIG. 6 shows a graph of total concentration of a first formulation of CNS-102 over time in brain and in plasma following administration to rats.
  • FIG. 7 shows a graph of total concentration of a second iorinuiation of CNS-102 over Lime in brain and in plasma following administration to rats (see Example 27 for details).
  • FIG. 8A illustrates the protection of cells by CNS-102 and CNS-101 in the presence ofGGTL
  • FIG. 8B illustrates the protection of cells by CNS- 102 and CNS- 103 in the presence of GGTL
  • FIG. 8C illustrates neurite outgrowth ratio for CMS- 102/CNS- 101 versus Log, 0 concentration.
  • FIG. 9 illustrates FISP expression in rat brain tissue after oral administration of CNS- 102.
  • FIG 10 illustrates concentration dependence of neuroprotection by CNS- 102.
  • PIG 11. illustrates survival plot, for drug treatment groups in SOD1 mice.
  • FIG. 12 illustrates comparative neurological motor function outcomes of SOD 1 mice.
  • FIG. 13 shows the HSP 70 expression (red) in CNSI 02/PBS Treated group with an objective lens of 4X.
  • FIG 14. Relative HSP70 mRNA expression levels in rat PBMCs Shrs post- dosing. Values are normalized to HSP70 mK A expression in PBMCs from vehicle treated rats.
  • B Dose-linearity plot for CNS-102 plasma exposure by AUG and C n)8x in connection with data from A.
  • FIG. 15 Shows a schematic timeline of KA model study.
  • FIG 18 Shows daily average weight for treatment groups.
  • FIG 19 Shows daily average body weighs from day 106 to end.
  • FIG 20 Shows the average neuroscore after treatment.
  • FIG 21 Shows average neuroscore vs. time.
  • FIG 22 Average 4-paw grip strength for surviving animals.
  • FIG 23 CMS 102 in the kainic acid rat experimental design.
  • CNS102 enhances hippocampal HSP70 and GRP78.
  • FIG 25 CNS102 Dose-response of HSP related proteins in 24 h treated 2A cells by Western Blot
  • FIG. 26 Shows dose-response in N2A Ceils Transfected with a 5'UTR. Luclferase Construct. ⁇ 0050]
  • FIG 27 CNS102 promotes prenylation and r.eurite outgrowth in the N2A model [005! ]
  • FIG 28 Expression of microglia marker in rat hippocampus by western blot.
  • FIG 29 Footprints and Quantitative Analysis of Stride Length from CatWalk Behavioral Test ai P127.
  • FIG 30 graphically shows run time on catwalk.
  • FIG 31 Shows mouse tail hang result.
  • FIG 32 Neuronal damage in the Hippocampus of Kainic Acid Treated Rats.
  • FIG 33 PK Parameters for C S 102 Administered IV and PO.
  • FIG. 34 Compares P K f o r sublingual versus oral gavage.
  • FIG. 35 Shows PK of CNS 102 repeat dose and brain penetration.
  • FIG. 36 Shows a CNS102 dosing paradigm.
  • compositions and methods are intended to mean that the compositions and methods include the recited elements, but not excluding others.
  • compositions and methods shall mean excluding other elements of any essential significance to the combination for the stated purpose, Thus, a composition consisting essentially of the elements as defined herein would not exclude other materials or steps that do not materially affect the basic and novel characteristic(s) of the claimed invention. "Consisting of shall mean excluding more than trace elements of other ingredients and substantial method steps. Embodiments defined by each of these transition terms are within the scope of this invention.
  • neuroprotective refers to reduced toxicity of neurons as measured in vitro in assays where neurons susceptible to degradation are protected against degradation as compared to control. Neuroprotective effects may also be evaluated in vivo by counting neurons in histology sections.
  • neuroneuron ' ' or “neurons” refers to all electrically excitable cells that make up the central and peripheral nervous system.
  • the neurons may be cells within the body of an animal or ceils cultured outside the body of an animal.
  • the term '"neuron 1" or “neurons” also refers to established or primary tissue culture cell lines that are derived from neural ceils from a mammal or tissue culture cell lines that are made to differentiate into neurons.
  • Neuroon or “neurons” also refers to any of the above types of cells that have also been ' modified to express a particular protein either extrachromosomally or intrachromosomally.
  • Neuron or “'neurons ' ' also refers to transformed neurons such as neuroblastoma ceils. Examples of specific types of neurons include Basket ceils, Betz cells. Medium spiny neurons, Purkinje cells, Pyramidal cells, Renshaw cells, Granule cells, and Anterior horn cells.
  • neuroneuron support cells refers to non-neuronal cells of the nervous system such as glial cells, astrocytes, and support cells within the brain such as glia.
  • glial cells include microglia, and microglia such as oligodendrocytes, ependymai cells, radial glia, Schwann cells, satellite cells, and enteric glial cells.
  • '"ALS refers to amyotrophic lateral sclerosis disease.
  • AD Alzheimer's disease
  • treatment means any treatment of a disease or condition in a patient, including one or more of:
  • neurodegenerative disease refers to diseases thai compromise the cell viability of neurons.
  • neurodegenerative diseases include but are not limited to ALS, AD, Parkinson ' s Disease, multiple sclerosis, and prion diseases such as Kuru, Creutzfe tdt-Jakob disease, Fatal familial insomnia, and Gerstmann-Straussler- Scheinker syndrome.
  • Neurodegenerative diseases can be recapitulated in vitro in tissue culture cells.
  • AD can be modeled in vitro by adding pre-aggregated p-amyloid peptide to the cells.
  • ALS can be modeled by depleting an ALS disease-related protein, TDP- 43.
  • Neurodegenerative disease can also be modeled in vitro by creating protein aggregates through providing toxic stress to the cell One way this can be achieved is by mixing dopamine with neurons such as neuroblastoma ceils. These Neurodegenerative diseases can also be recapitulated in vivo in mouse models.
  • a transgenic mouse that expresses a mutant Sodl protein has similar pathology to humans with ALS.
  • a transgenic mouse that overexpresses APP has similar pathology to humans with AD.
  • miR A-378 * refers to a miRNA with die following: an official symbol MIR378A (Gene ID 494327, GenBank Accession No: NC 000005.9) for human; and an official symbol Mir378 (Gene ID 723889, GenBank Accession No: NR .029879) for Mus musculus.
  • MIR378A Gene ID 494327, GenBank Accession No: NC 000005.9
  • Mir378 Gene ID 723889, GenBank Accession No: NR .029879
  • miRNA-71 H refers to a miRNA with the following: an official symbol IR7N (Gene ID 1003 13843, GenBank Accession No: NC_ 000003.1 1) for human; and an official symbol Mir71 1 (Gene ID 751536, GenBank Accession No: NC_000075.6) for Mus musculus.
  • IR7N Gene ID 1003 13843, GenBank Accession No: NC_ 000003.1 1
  • Mir71 1 Gene ID 751536, GenBank Accession No: NC_000075.6
  • HSP 70 refers to a family of ubiquitously expressed heat shock proteins. Proteins with simitar structure exist in virtually all living organisms. The Hsp70s are an important pan of the cell's machinery for protein folding, and help to protect ceils from stress.
  • GenBank Accession numbers CAA28382.1 , P08107.5, and NP_ 005337.2 represent the protein sequence for HSP 70 and GenBank Accession numbers NM_005346.4, N _005345.5, NM 006644.2 represent the nucleotide sequence of the protein.
  • HSP70.3 which has an official symble of Hspaia, Gene ID 1 3740, and GenBank Accession No.: NC_000083.6. Each of these GenBank sequences are herein incorporated by reference in their entirety, (00 5]
  • reporter refers to a gene or label that is attached to a regulatory sequence of another gene of interest and can be used as an indication of whether a certain gene has been taken up by or expressed in the cell or organism population.
  • Non-limiting examples of reports include green fluorescent protein and derivatives (i.e. yellow fluorescent protein and red fluorescent protein), luciferase, dsRed, GUS gene, and the like.
  • alky refers to substituted or unsubstituted, straight chain or branched aikyl groups with Cj-C
  • aryi refers to a 6 to 10 membered, preferably 6 membered ary! group. An aryi group may be substituted with 1 -5, preferably 1 -3, halo, alkyl, and/or -O-alkyS groups.
  • an effective amount of GGA is the amount of GGA required to produce a protective effect in vitro or in vivo.
  • the effective amount in vitro is about from 0, 1 nM to about i mlvl
  • the effective amount in vitro is from about 0.1 nM to about 0.5 nM or from about 0.5 nM to about 1.0 nM or from about 1.0 nM to about 5.0 nM or from about 5.0 nM to about 10 nM or from about 10 nM to about 50 nM or from about 50 nM to about i 00 nM or from about ] 00 nM to about 500 nM or from about 500 nM to about 1 mM
  • the effective amount for an effect in vivo is about 0.1 rng to about 100 mg, or preferably, from about 1 mg to about 50 mg, or more preferably, from about 1 mg to about 25 mg per kg/day.
  • the effective amount in vivo is from about 10 rag/kg/day to about 300 mg/kg/day, about 20 mg/kg/day to about 00 mg/kg/day, about 30 mg/kg/day to about 80 mg/kg/day, about 40 mg/kg/day to about 70 mg/kg/day, or about 50 mg/kg/day to about 60 mg/kg/day. In still some other embodiments, the effective amount in vivo is from about 100 mg kg/day to about 1000 mg/kg/day.
  • a patient suffering from acute CNS injuiry would be administered iv with a bolus concentration of 5-trans-GGA or its derivate wherein the bolus concentration provides for a Tmax of less than about 7 hours.
  • the bolus concentration provides for a Tmex of less than about 10 hours, less than about 9 hours, less than aboul 8 hours, less than about 7 hours, less than about 6 hours, less than about 5 hours, less than about 4 hours, less than about 3 hours, or less than about 2 hours.
  • Routes of administration refers to the method for administering GGA to a mammal. Administration can be achieved by a variety of methods. These include but are not limited to subcutaneous, intravenous, transdermal, sublingual, or intraperitoneal injection or oral administration,
  • halogenating is carried out as converting a hydroxy group to a halo group
  • halo or “halo group” refers to fluoro, chloro, bromo and iodo.
  • stereoselectively is defined as providing over 90% of the E isomer for the newly formed double bond.
  • Geometrical isomer “ or “geometrical, isomers” refer to compounds that differ in the geometry of one or more oiefime centers. or “(E)” refers to the tram orientation and "Z” or “(Z)” refers to the cis orientation.
  • Geranylgeranyl acetone refers to a compound or the formula:
  • compositions comprising the compound are mixtures of geometrical isomers of the compound.
  • the 5-trans isomer of geranylgeranyl acetone refers to a compound of the formula I :
  • the 5-cis isomer of geranylgeranyl acetone refers to a compound of the formula II:
  • C n C. mention such as Ci-Cio, CpC*, or Cj-Q when used before a group refers to that group containing m to n carbon atoms.
  • alkyl refers to monovalent saturated aliphatic hydrocarbyl groups having from 1 to 10 carbon atoms (i.e., CVC;o alky! or I to 6 carbon atoms (i.e., Cj-Ce alkyl), or i to 4 carbon atoms.
  • This term includes, by way of example, linear and branched hydrocarbyl groups such as methyl (CH 3 -), ethyl (CH 3 C3 ⁇ 4-), «-propyl (CH 3 CH 2 C3 ⁇ 4-), isopropyl ((CH ⁇ CH-), ⁇ -butyl (CH3CH2CH2CH2-), isobuty] ((CH 3 )2CHCH 2 -), m-butyl
  • aryl refers to a monovalent, aromatic mono- or bicyclic ring having 6-10 ring carbon atoms. Examples of aryl include phenyl and naphthyl. The condensed ring may or may not be aromatic provided that the point of attachment is at an aromatic carbon atom. For example, and without limitation, the following is an aryl group:
  • -CO2H ester refers to an ester formed between the - ⁇ CCbH group and an alcohol, preferably an aliphatic alcohol.
  • Co-crystal or as sometimes referred to herein "co-precipitate” refers to a solid, preferably a crystalline solid, comprising GGA or a GGA derivative, and urea or thiourea, more preferably, where, the GGA or the GGA derivative reside within the urea or thiourea lattice, such as in channels formed by urea or thiourea.
  • Complexed refers to GGA or a GGA derivative bound by certain quantifiable intermolecular forces, non-limiting examples of which include hydroge bonding and Van- Der Waais' interactions, and also by etitropic effects.
  • chiral moiety refers to a moiety that is chiral. Such a moiety can possess one or more asymmetric centers. Preferably, the chiral moiety is enantiomerically enriched, and more preferably a single enantiomer.
  • Non limiting examples of chiral moieties include chiral carboxylic acids, chiral amines, chiral amino acids, such as the naturally occurring amino acids, chiral alcohols including chiral steroids, and the likes.
  • cycloalkyP refers to a monovalent, preferably saturated, hydrocarbyl mono-, bi-, or tricyclic ring having 3-12 ring carbon atoms. While cycioalkyl.
  • cycioalkyl refers preferably to saturated hydrocarbyl rings, as used herein, it also includes rings containing 1 -2 carbon-carbon double bonds.
  • Nonlimiting examples of cycioalkyl include cyclopropyl. cyclobuiy h eyclopent l, cyciohexyl, cycloheptyl, adamentyl, and the like.
  • the condensed rings may or may not be non-aromatic hydrocarbyl rings provided that the point of attachment is at a cycioalkyl carbon atom,
  • a cycioalkyl group [0098]
  • halo refers to F, CI, Br, and/or I,
  • heteroaryl refers to a monovalent, aromatic mono-, bi-, or tricyclic ring having 2-14 ring carbon atoms and 1-6 ring heteroatoms selected preferably from N, O, S, and P and oxidized forms of N, S, and P, provided that the ring contains at least 5 ring atoms.
  • Nonlimiting examples of heteroaryl include furan, imidazole, oxadtazole, oxazole, pyridine, quinoline, and the like.
  • the condensed rings may or may not he a heteroatom containing aromatic ring provided that the point of attachment is a heteroaryl atom.
  • heterocyclyl refers to a non-aromatic, mono-, bi-, or tricyclic ring containing 2-10 ring carbon atoms and 1 -6 ring heteroatoms selected preferably from N, O, S, and P and oxidized forms of N, S, and P, provided that the ring contains at least 3 ring atoms. While heterocyclyl preferably refers to saturated ring systems, it also includes ring systems containing 1 -3 double bonds, provided that they ring is non-aromatic.
  • heterocyclyl examples include, azalactones. oxazoline, piperidinyl, piperazinyl, pyrrolidinyl, tetrahydroruranyl, and tetrahydropyrany!.
  • the condensed rings may or may not contain a non-aromatic heteroaiorn containing ring provided that the point of attachment, is a heterocyclyl group.
  • hydrolyzing refers to breaking an R H -0-CO, R h -0-CS-. or an R -0
  • SO2- moiety to an R * -OH, preferably by adding water across the broken bond.
  • a hydrolyzing is performed using various methods well known to the skilled artisan, non limiting examples of which include acidic and basic hydrolysis.
  • oxo refers to a OO group, and to a substitution of 2 gemiria! hydrogen atoms with a C-0 group,
  • [1)103 j The term “pharmaceutically acceptable” refers to safe and non-toxic for in vivo, preferably, human administration.
  • pharmaceutically acceptable salt refers to a salt that is pharmaceutically acceptable.
  • the terra "salt" refers to an ionic compound formed between an acid and a base.
  • such salts include, without limitation, alkai metal, alkaline earth metal, and ammonium salts.
  • ammonium salts include, salts containing protonated nitrogen bases and alkylated nitrogen bases.
  • Exemplary, and non-limiting cations useful in pharmaceutically acceptable salts include Na. , Rb, Cs, H4, Ca, Ba, imidazoiium, and ammonium cations based on naturally occurring amino acids.
  • such sails include, without limitation, salts of organic acids, such as caroboxylic acids and sulfonic acids, and mineral acids, such as hydrogen halides, sulfuric acid, phosphoric acid, and the likes.
  • exemplary and non-limiting anions useful in pharmaceutically acceptable satis include oxalate, maleate, acetate, propionate, succinate, tartrate, chloride, sulfate, bisalfate, mono-, di-, and tribasic phosphate, mesylate, tosylate, and the likes.
  • substantially pure warn isomer refers to a trans isomer that is by molar amount 95%, preferably 96%, more preferably 99%, and still more preferably 99.5% or more a trans isomer with the rest being the corresponding cis isomer,
  • R'-R 5 is defined herein and q is 0-2.
  • each double bond is in a trans or E configuration.
  • a cls form of GGA or a GGA derivative will contain one or more of these bonds in a cis or Z configuration
  • This invention describes methods and compositions for treating diseases mediated, at least in pari, by miRNA-378 and/or miRNA-71 1 increased activity.
  • the increased activity may be due to an increase in the production of the raiRNA or an increase in the effectiveness of the udiRNA.
  • the increase in activity may be direct or indirect.
  • one aspect of the invention relates to a method for treating a disease in a subject mediated in part by niiRNA-378 or miRNA-7i l increased activity comprising administering to the subject a therapeutically effective amount of 5 -trans -GGA or a derivative thereof.
  • this invention provides a method for treating damaged neurons exhibiting increased miRNA-378 and/or miRNA-71 1 activity, said method comprising administering 5-trans-GGA or a derivative thereof in an amount effective to decrease miRNA-378 or miRNA-71 1 activity specifically in damaged neurons and/or neuron support cells proximate to damaged neurons.
  • this invention provides a method for treating damaged neurons in a subject in need thereof, wherein the damage to the neurons is associated with increased miRNA-378 and/or raiR A- 11 activity, said method comprising decreasing miRNA-378 or miRNA-7 i i activity specifically in damaged neurons and/or neuron support cells proximate to damaged neurons said method comprising administering a therapeutically effective amount of 5-trans-GGA or a derivative thereof to the subject.
  • the decrease in the mlRNA-378 and/or miRNA-71 1 activity correlates to an increase in HSP 70 expression.
  • the subject has a disease mediated, at least in part, by miRNA-378 and/or miRNA-71 1.
  • the damaged neurons have increased miRNA-378 and/or miRNA-71 1 activity.
  • the method comprises decreasing miRNA-378 and/or miRNA-71 1 activity in neurons or neuron support ceils.
  • the miRNA-378 and/or miRNA-711 activity is decreased in neuron support cells, in a related embodiment, the neuron support cells are glial cells.
  • the subject being treated according to the methods described herein is a subject with a neurodegenerative disease
  • neurodegenerative diseases include Alzheimer's disease, Parkinson's disease, multiple sclerosis, prion diseases such as Ktiru, Creutzfeltdt-Jakob disease. Fatal familial insomnia, and Gerstmann-Straussier-Scheinker syndrome, amyotrophic lateral sclerosis, or damage to the spina! cord.
  • GGA and the 5-trans isomer of GGA are also contemplated to prevent neural death during epileptic seizure.
  • the subject being treated is one that has experienced acute CNS injuries, including but not limited to, CNS injury associated with the occurrence of. for example, stroke, aneurism, surgery, arteriovenus malformation (AVM), radiation, vascular dementia, epileptic seizures, cerebral vasospasm, acute or traumatic brain injury, and hypoxia of the brain as a result of, for example, cardiopulmonary arrest or near drowning,
  • acute CNS injuries including but not limited to, CNS injury associated with the occurrence of. for example, stroke, aneurism, surgery, arteriovenus malformation (AVM), radiation, vascular dementia, epileptic seizures, cerebral vasospasm, acute or traumatic brain injury, and hypoxia of the brain as a result of, for example, cardiopulmonary arrest or near drowning,
  • CNS injury associated with the occurrence of. for example, stroke, aneurism, surgery, arteriovenus malformation (AVM), radiation, vascular dementia, epileptic seizures, cerebral vasospasm, acute or traumatic brain injury, and hypoxia
  • CNS- 102 or ail trans GGA 12 mg/kg/day prolonged survival in the superoxide dis nutase 1 (SODl) mouse model of amyotrophic lateral sclerosis (ALS). Furthermore, CNS- 102 improved the clinical and neurological scores, reaching statistical superiority to scores in vehicle treated mice at certain time points.
  • systemic CNS- 102 dosing has also provided neuroprotection in the hippocampus against locally injected kainic acid. The benefit is prolonged with peak protection at 72 hours following a single oral dose.
  • CNS- 102 protects neutite extension of the neuroblastoma cells (Neuro2A) from toxicity caused by an inhibitor of geranylgeranyl transferase.
  • thai CNS 102 the all-trans isomer, is substantially more potent than teprenone (by over an order of magnitude) as a neuroprotective agent. with efficacy noted using a daily oral dose of 12 mg/kg. Typical al!ometric scaling predicts a human equivalent dose of 1 -2 mg kg, making CNS102 a potential therapeutic,
  • HSPs heat shock proteins
  • CNS 102 likely targets multiple relevant pathways. It has the capacity to affect protein quality control and immune responses within the CNS, thereby slowing disease progression and possibly promoting neurite outgrowth and regeneration.
  • CNS-102 up-regulates expression of HSP70 in addition to other selected HSPs including HSP40, and HSP90. This has been demonstrated in vitro, in the murine neuroblastoma ceil neuroZA system, and in vivo in the rat cerebral cortex and hippocampus. The expression of HSP70 was detectable through 96 hours in the cortex and hippocampus of rats receiving a single dose of 12 mg/kg of CNS- 102. Comparative in- vitro studies demonstrate that CNS- 102 is a more potent inducer of HSPs than the cis-isomer CNS- 103.
  • CNS- 102 increases the survival in the SODl mouse model, which in part may be due to a molecular chaperone effect.
  • HSPs are recognized to function as ATP-dependent chaperones, assisting in the folding of newly synthesized proteins and the sequestering of misfoided proteins. Protein misfolding leading to secondary cellular damages is postulated to be a significant component of the pathophysiology of ALS.
  • a high threshold for induction of the stress response is thought to contribute to increased vulnerability to the toxicity of protein aggrega tes in motor neurons.
  • HSPs gene induction may improve protein refolding in ALS. consistent with the reported protein refolding and survival benefits in a spinal and bulbar muscular atrophy (SMBA) mouse model dosed with teprenone.
  • SMBA spinal and bulbar muscular atrophy
  • HSP70 has been reported to play an important role in stabilizing axons and dendrites and supporting the growth cone. Therefore it may prevent the loss of motor neurons by its chaperonal activity on the cytoskeleton, supporting the development of a new therapeutic based on CNS-102 to treat neurodegenerative diseases.
  • peripheral, blood based biomarkers are preferable to matrices that are more difficult to obtain such as cerebrospinal fluid.
  • the major source of HSPs in biood are peripheral blood monocytes (PBMCs), but HSPs can also be detected in serum.
  • PBMCs peripheral blood monocytes
  • HSPs can also be detected in serum.
  • PBMC derived HSPs were focused on, because the neuroprotective function is attributed to intracellular HSP70, while extracellular HSP70 is considered a proinflammatory mediator.
  • PBMCs peripheral blood monocytes
  • ELiSA kits for intracellular HSP70 and optimized assay conditions were identified.
  • a yield of approximately 7 million PBMCs from a rat terminal bleed provides sufficient protein for an HSP70 ELISA assay.
  • HSP Heat Shock Protein
  • HSP70 acts in concert with a number of other HSP chaperones to mediate its beneficial effects on proteostasis, it is desirable to monitor the protein levels of multiple HSP family members. Due to the associated cost and the amount of protein required from a sample, it is challenging to accomplish this with multiple ELISA assays.
  • proteomics platforms offer technology to multiplex the measurement of candidate biomarker proteins, even from multiple organisms, in one 'panel ' by a method termed Multiple Reaction Monitoring or MRM. This obviates the need to develop expensive individual assays for the multiple proteins, as well as going from rats into human subjects.
  • Target biomarker proteins may be combined in a single assay without the need to design immune-affinity agents, or concerns about cross reactivity between reagents or interference from autoantibodies, This unbiased inclusion of markers based solely on their differentiating capacity, the wide dynamic range arid reproducibility and sensitivity are ail advantages in the MRM platform. Additional target biomarker proteins from existing biomarker panels can easily be included in the assay.
  • MRM assay For the MRM assay, at least 5 peptides per protein marker unique to rat and human respectively, are chosen based on available databases of observed peptides. These are synthesized and their transitions characterized. A multiplexed assay with the ability to measure detectable markers are developed using these synthetic peptides and can be applied to both species/sample types. Assay development is followed by assay verification in up to 10 representative samples. The accuracy, precision and reproducibility of the MRM assays are determined from a pooled sample of rat PBMC lysates that are aiiquoted and spiked with I ISP standards. Three of these aliquots are analyzed in 3 separate runs. Accuracy is expressed as % deviation for the difference of the average HSP concentrations from the nominal HSP concentrations. Precision is expressed as %CV within an anaiylicai run and reproducibility is expressed as %CY between analytical runs,
  • HSP70 levels are defined as parameters that need to be maintained constant in the sample handling protocol.
  • Sample handling experiments may be performed first by qPCR. before selecting the most critical sample handling conditions to be analyzed by MR.M. [0130]
  • HSP stability is considered acceptable under conditions that lead to a loss of less than 15% of the nominal HSP concentration. The stability data are documented in the sample handling protocol,
  • Method The inter-individual variability is assessed for the MRM analysis of HSP proteins and the qPCR analysis of HSP famil .members in 3 male and 3 female rats as well as in human PBMC samples from 3 males and 3 females.
  • the infra-individual variability in humans is assessed from the same healthy volunteers, who provided samples for inter-individual assessment. Samples are collected two weeks after the first set of samples. This is the same duration as the planned Multiple Ascending Dose (MAD) Phase 1 study.
  • the infra-individual variability assessment for HSP protein by MRM may not be feasible in rats, because a whole terminal blood sample may be required to obtain sufficient PBMC lysate for an MRM assay.
  • the infra-individual variability in rats is determined at a minimum HSP rn NA with sample collection 1 week apart to reflect the duration of the kainic acid study described in Section 3.5.3.
  • the intra- and inter- individual variability is expressed as %CV.
  • PBMC samples from approximately 3 ALS patients.
  • Patient samples with medical history can be obtained from commercial tissue sources (e.g. Folio) or academic sample repositories such as the one maintained by the North East ALS (NEALS) Consortium.
  • tissue sources e.g. Folio
  • NEALS North East ALS
  • KA kainic acid
  • Rats are randomly assigned to treatment groups. Group 1 acts as a negative, sham control, Group 2 will show lesion size and biomarker response to KA insult without drug treatment, Group 3 will characterize the biomarker response elicited by the drug in combination with sham surgery and the remaining groups will characterize the dose response to CNS- 102. Dosing staff, the surgeon and the staff analyzing lesion areas are blinded to treatment assignments. Rats are dosed sublingually once daily for 6 days.
  • PBMCs are isolated from whole blood. Brains are collected and one hemisphere is immersion fixed in 4% paraformaldehyde and dehydrated in sucrose in preparation for cryo-sectionmg. From the second hemisphere the hippocampus is dissected and stored in RNA later for extraction of mRNA. Following the last dose, blood samples are collected from satellite PK animals for a 24 hour plasma concentration time profile. In addition terminal bleeds are collected for biomarker analysis from Group 7 animals on Day -3 to establish baseline HSP expression. A schematic timeline of the study is shown in Figure
  • Frozen sections are stained with cresyl violet and the relative size of the lesion area in the CA3 region of the hippocampus is measured in a blinded fashion on digital microscope images in ImageJ according to an established protocol.
  • the group size may change depending on power calculations, * Satellite P animals; M: males
  • Terminal blood samples are analyzed for MRM target protein content in PB Cs and relative expression levels of HSP mRNA with established primers.
  • mR A will also be extracted from the hippocampus of the second brain hemisphere and the relative expression pattern of HSPs analyzed with the same primer set.
  • Plasma concentration of CNS-102 in the PK satellite animal samples are analyzed by Pharmaout laboratories. We plan to obtain the following key results from this study: (i) CNS- 102 plasma exposure at maximum and half maximum efficacy, i.e.
  • kits and methods for identifying compounds that regulate HSP 70 comprising: expressing, in a pool of cells, a nucleotide construct comprising the 3'UTR of HSP 70 and a reporter; adding a test compound to a first portion of the pool of cells; and determining the output level of the reporter; wherein an increase in the output level of the reporter in the first portion of cells indicates a compound that increases HSP 70 protein levels and a decrease in the output level of the reporter in the first portion of cells indicates a compound that decreases HSP 70 protein levels.
  • a further aspect provides for an assay or method for identifying compounds thai regulate HSP 70, aid assay or method comprising: expressing, in a pool of ceils, a nucleotide construct comprising the 3'UTR of HSP 70 ana a reporter; adding a test compound to a first portion of the pool of cells; adding a control to a second portion of the pool of ceils; and determining the output level of the reporter; wherein a higher output level of the reporter in the first portion of ceils compared to the second portion of ceils indicates a compound thai increases HSP 70 protein levels and a lower output level of the reporter in the first portion of cells compared to the second portion of cells indicates a compound that decreases HSP 70 protein levels.
  • the nucleotide further comprises a promoter region for directing expression, of the 3'UTR.
  • promoter refers to a nucleic acid sequence sufficient to direct transcription of a gene
  • promoter elements which are sufficient to render promoter dependent gene expression controllable for cell type specific, tissue specific or inducible by external signals or agents. Suitable promoters include, for example, cytomegalovirus, EFl-l ct, SV40, and PGK.
  • the 3'UTR of HSP 70 refers to the untranslated region on the 3 " end of the HSP 70 gene.
  • the 3' UTR comprises about 2000, about 1800, about 1600, about 1400, about i 300, or about 1253 bp from the end of the protein coding sequence.
  • control can refer to a non-active compound, a mock solution (a solution with no compound), and the like,
  • determining the output level refers to quantitatively or qualitatively measuring the output of the reporter used in the assay.
  • one suitable reporter system is luciferase. Firefly luciferase catalyzes the oxidative carboxylation of luciferin, a reaction with the highest efficiency of any known bioluminescence reaction. At the optimal reaction pl l of 7.8. light emission peaks at 562 nm. This form of light emission yields a very sensitive non-radioactive assay. For measurement of expressed luciferase activity in vitro, luciferase can be extracted from transfected cells through eel! lysis.
  • a typical firefly luciferase assay can then be carried out in an assay cuvette, ATP, Mg2+ and buffer are added to the iysate either separately or as a preform uiated solution.
  • the luminescent reaction is then triggered by an injection of luciferin, and die emitted light is recorded.
  • reporter is luciferase.
  • the cells are exposed to damage prior to addition of the test compound and/or control. Exposure to damage, especially neurons, may increase the sensitivity of the assay and/or allow for evaluation of the specificity of the assay.
  • Suitable vectors for expressing the 3'UT and reporter are well-known in the art, and include vectors capable of expressing a polynucleotide operative] linked to a regulatory element, such as a promoter region and/or an enhancer thai is capable of regulating expression of such DNA.
  • an expression vector refers to a recombinant DNA or UNA construct, such as a p!asmtd, a phage, recombinant virus or other vector that, upon introduction into an appropriate host cell, results in expression of the inserted DNA
  • Appropriate expression vectors include those that repHcable in eukaryotic cells and/or prokaryotic cells and those that remain episomai or those which integrate into the host ce!l genome.
  • vector refers to a non-chromosonia! nucleic acid comprising an intact rep] icon such that the vector may be replicated when placed within a cell, for example by a process of transformation.
  • Vectors may be viral or non-viral.
  • Viral vectors include retroviruses, adenoviruses, herpesvirus, baecuJoviruses. modified bacculoviruses, papovirus, or otherwise modified naturally occurring viruses.
  • Exemplary non- iral vectors for delivering nucleic acid include naked DNA; DNA complexed with cationic lipids, alone or in combination with cationic polymers; anionic and cationic liposomes; DKA-protein complexes and particles comprising DNA condensed with cationic polymers such as heterogeneous polylysine, defmed-iength oligopeptides, and polyethylene imine, in some cases contained in liposomes: and the use of ternary complexes comprising a virus and polylysine-DNA.
  • Non-viral vectors may include a plasmid that comprises a heterologous polynucleotide capable of being delivered to a target ceil, either in vitro, in vivo or ex-vivo.
  • the heterologous pol ynucleotide can comprise a sequence of interest and can be operably linked to one or more regulatory elements and may control the transcription of the nucleic acid sequence of interest,
  • a vector need not be capable of replication in the ultimate target cell or subject.
  • the terra vector may include expression vector and cloning vector.
  • a "viral vector *" is defined as a recombinant ly produced virus or viral particle that comprises a polynucleotide to be delivered into a host cell, either in vivo, ex vivo or in vitro.
  • viral vectors include retroviral vectors, adenovirus vectors, adeno-associated virus vectors, alphavirus vectors and the like.
  • Alphavirus vectors such as Semliki Forest virus-based vectors and Sindbis virus-based vectors, have also been developed for use in gene therapy and immunotherapy.
  • a vector construct refers to the polynucleotide comprising the retroviral genome or pari thereof, and a therapeutic gene.
  • retroviral mediated gene transfer or “retroviral transduction” carries the same meaning and refers to the process by which a gene or nucleic acid sequences arc stably transferred into the host ceil by virtue of the virus entering the ceil and integrating its genome into the host celt genome.
  • the virus can enter the host cell via its normal mechanism of infection or be modi ied such thai it binds to a different host cell surface receptor or ligand to enter the cell.
  • retroviral vector refers to a viral particle capable of introducing exogenous nucleic acid into a cell through a viral or viral-like entry mechanism.
  • a further aspect of the invention relates to a compound identified by an assay as described herein,
  • kits comprising a poo! of cells expressing a nucleotide construct comprising the 3 * UTR of HSP 70 and a reporter; a means for detecting the output of the reporter; and instructions for use.
  • the ceils are neurons.
  • die reporter is selected from the group consisting of fluorescent protein, red fluorescent protein, dsRed, yellow fluorescent protein, luciferin, and aequorin and luciferase. Compo srids
  • This invention relates to compounds and pharmaceutical compositions of isomers of geranylgeranyl acetone. In certain aspects, this invention relates to a synthetic 5-trans isomer compound of forrnula I:
  • I is at least 80% in the 5E, 9E, 1 3E configuration.
  • the in vention provides for a compound of formula I wherein I is at least 85%, or at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 99,5%, or at least 99.9% in the 5E. 9E » 13E configuration.
  • the invention for the compound of formula 1 does not contain any of the cis-isomer of GGA.
  • Another aspect of this invention relates to a synthetic 5-cis isomer compound of formula SI:
  • the invention provides for a compound of formula II wherein ⁇ is at least 80% in the 5E, 9E, 13E configuration, or alternatively, at least 85%, or at least 90%, or at least 95%, or at least 96% r or at least 97%, or at least 98%, or at least 99%, or at least 99.5%, or at least 99.9% in the 5E, 9E, ⁇ 3 ⁇ configuration.
  • the compound of formula II does not contain any of the trans-isomer of GGA.
  • the configuration of compounds can be determined by methods known to those skilled in the art such as chiroptical spectroscopy and nuclear magnetic resonance spectroscopy.
  • GGA derivatives useful in this invention include those described in PC f publication no. WO 2012/031028 and PCT application no. PCT/US2012/027147, each of which are incorporated herein by reference in its entirety. These arid other GGA derivatives utilized herein are structurally shown below.
  • the GGA derivative utilized herein is of Formula I:
  • n 1 or 2;
  • each R 1 and R" are independently CVQ alkyi. or R' and together with the carbon atom they are attached to ibrrn a Cs-C 6 cvcloalkvi ring optionally substituted with 1 -3 C i -Q, alkyi groups; each of R , R 4 , and R 5 independently are hydrogen or Ci-C 6 alkyi;
  • Q 2 is hydrogen, R 6 , -0-R c s -NR'R 8 . or is a chiral moiety
  • R 6 is:
  • Cj-C f alkyi, optionally substituted with -C0 H or an ester thereof, C
  • -Q, alkoxy, oxo, -CR €R2, -OCR, CS-CB cycloalkyl.
  • each cycloalkyl. heterocyclyl. aryl, or heteroaryl is optionally substituted with 1 -3 alkyi groups, optionally substituted with 1-3 halo, preferably, fluoro, groups, or R ! g and R
  • each R .' and R 8 are independently hydrogen or defined as R"; refers to a mixture of cis and trans isomers at the corresponding position wherein at least 80% and, preferably, no more than 95% of the compound of Formula (I) is present as a trans isomer;
  • the GGA derivative utilized is of Formula (I-A):
  • a is 1. In another embodiment, n is 2.
  • the GGA derivative utilized is of Formula (i-B):
  • the GG A derivative utilized is of Formula ( ⁇ - ⁇ ), (II-B), or (11-Cj:
  • the GGA derivative utilized is of Formula i ' il-D), fll-E), (II-F):
  • R 6 is Q-Cioary), such as naphthyl.
  • R" is a heieroaryl, such as quinolinyl.
  • GGA derivative utilized in this invention is of Formula (II):
  • n 0 or I :
  • n 0, I , or 2;
  • each R l and R 2 are independently Ci-C 6 aiky!, or R 1 and R 2 together with the carbon atom they are attached to form a C 5 -C7 eycioalkyl ring optionally substituted with 1-3 C
  • each of R J , R 4 , and 5 independently are hydrogen or Cj-Cg alkyi
  • Q is -X-CO-NR ,e R ,y , ⁇ X-CS-NR I8 R", or -X-S0 2 -NR'3 ⁇ 4 iy :
  • X is -0-, -S-, -MR 7 -, or -CR 8 R 9 ;
  • R 7 is hydrogen or together with R ! * or R ⁇ and the intervening atoms form a 5-7 membered heterocyclic ring optionally substituted with i-3 C 1-C alkyl groups; each R* and R 9 independently are hydrogen, C r C 6 alkyl, -COR 8i or -C0 2 R Si . or R 8 together with R sS or '" and the intervening atoms form a 5-7 membered heterocyciy! ring optionally substituted with 1 -3 -Cs alkyi groups; and
  • R 18 and R 19 independently is hydrogen: CJ-CA alkyl, optionally substituted with -CO 2 H or an ester thereof, Ci-Q alkoxy, oxo, -CR-CR 2 , -CCR, C 3 -C 8 cycloalkyl, C C 8 heteroeydyi, Ce-Cioaiyl, or Ca-Cio heteroaryl, wherein each R. independently is hydrogen or Ci- j alkyl; C3-CJ0 cycloalkyl; C3-Q heterocyclvl; C5-C10 aryl; or C2-C10 beteroaryl; wherein each cycloalkyl, heterocyclvl.
  • aryl, or heteroaryl is optionally substituted with 1-3 alky! groups, optionally substituted with 1-3 halo, preferably, fluoro, groups, or R ! 8 and 1 together with the nitrogen atom they arc attached to form a 5-7 membered heterocyele.
  • the compound of Formula (II) includes optical isomers such as enantiomers and diastereomers.
  • an ester refers preferably to a phenyl or a CpC;, alkyl ester, ich phenyl or alkyl group is optionally substituted with a amino group.
  • the compound of Formula (II) is of formula:
  • R ⁇ , R-, R , R ⁇ and Q are defined as in any aspect or embodiment herein.
  • the GGA derivative utilized is of formula:
  • R ⁇ R 2 , R 4 , R 5 , and Q are defined as in any aspect and embodiment here.
  • the compound of Formula (II) is of ibrmul
  • R 1 , R " R J , R 4 . R 5 , and Q ate defined as in any aspect or embodiment herein.
  • the GGA derivative utilized is of formula: wherein R', RA R " , R ⁇ ra, n, X, R' and R '" are defined as in any aspect and embodiment here.
  • the GGA derivati ve uiilized is of formula :
  • R 1 , R ⁇ R ⁇ R% m, n, and R' s are defined as in any aspect and embodiment here.
  • the GGA derivative utilized is of formula:
  • R is defined as in any aspect and embodiment here.
  • the GGA derivative utilized is of formula:
  • R l 8 is defined as in any aspect and embodiment here.
  • the GGA derivative utilized is of formula:
  • R ! S is defined as in any aspect and embodiment here.
  • the GGA derivative utilized is of formula:
  • the GGA derivative utilized is of formula: wherein R 18 and R 19 are defined as in any aspect and embodiment here.
  • ihe GGA derivative utilized is of formula:
  • R is defined as in any aspect and embodiment here.
  • the GGA derivative utilized is of formula:
  • R I S and R w are defined as in any aspect and embodiment here.
  • n is Q. in another embodiment,, m is 1.
  • n is 0. in another embodiment, n is 1. In another embodiment ; n is 2.
  • m+n is !. In another embodiment, m+rt is 2. In another embodiment, m+n is 3.
  • R 1 and R 2 are independently ⁇ -C(, aikyi. In another embodiment, R 1 and R 2 independently are methyl, ethyl, or isopropyl.
  • R 1 and together with the carbon atom the are attachec to form a C5-C7 cycioalkyl ring optionally substituted with 1 -3 CrC(, aikyi groups, in another embodiment, R ! and R 2 together with the carbon atom they are attached to form a ring that is:
  • R ⁇ R 4 and R * are independently CrC i; aikyi.
  • one of R 1 , R 4 , and R 3 are aikyi, and the rest are hydrogen, in another embodiment, two of R 3 , R 4 , and R 5 are aikyi, and the rest are hydrogen, in another embodiment, R-', R 4 , and * are hydrogen.
  • R 3 , R 4 , and R 5 are fOJ88j In another embodiment.
  • Q is -X-CO-NR R ' .
  • Q is -X-CS-N ' 3 ⁇ 4 ' g .
  • Q is -X-S02-NR%' 9 .
  • Q is -OCONHR 18 ---OCONR 18 R i9 , -NHCONHR 18 , -NHCONR R ! *, -OCSNHR ,s -
  • OCSNR ,8 R 19 OCSNR ,8 R 19 .
  • -NHCS HR 18 OCSNR ,8 R 19 .
  • -NHCSNR 18 R 19 OCSNR ,8 R 19 .
  • X is -0-. In another embodiment, X is -NR.'-. In anoiher embodiment, X is or -CR'PJ',
  • one of R" 3 and ,? is hydrogen.
  • one or both of R 1& and R 19 are Ci-C 6 aikyi.
  • one or both of R 1 * and R 19 are C
  • one or both of R n and K' v are Cj-Qo cycioalkyl,
  • one or both ofR' 8 and R i9 are C3-C10 cycioalkyl substituted with 1-3 aikyi groups.
  • one or both of R i8 and R ! are Cj-Cg heterocyeiyl.
  • R lS and ' ' * are C f i-Cto r l
  • one or both of R i S and R' 9 are Ca-Cioheteroaryl.
  • R; and R1 together with the nitrogen atom they are attached to form a 5-7 membered heterocycle.
  • R 0 is -C0 2 H or an ester thereof
  • R 20 is Cj-Cg.
  • R i0 is cycioalkyl.
  • R 20 is Cj-Cg heterocyeiyl.
  • R 20 is Ce-Cio aryl.
  • R*° is or Cj-Cio heleroaiyl.
  • examples of compounds utilized by this invention include certain com ounds tabulated below,
  • the GGA derivative utilized is of formula
  • m is 0 or 1 ;
  • n 0, 1 , or 2;
  • each R' and R " are independently Ci-Q alkyi, or R 1 and R 2 together with the carbon atom they are attached to form a C5-C7 cycloalkyi ring optionally substituted with 1 -3 C1-C alkyi groups;
  • each of R J , R ⁇ and R: x independently are hydrogen or Ci-C-6 alkyi;
  • Q is -X-CO-NR l8 R" or -X-S0 2 -N R l g 19 ;
  • X is -0-, -NR 7 -, or -CR 8 R 9 ;
  • R' is hydrogen or together with R or R ' and the intervening atoms form a 5- 7 membered ring optionally substituted with 1 -3 C Cs alkyi groups:
  • each R s and R 9 independently are hydrogen, Q-Ce alkyi, -COR 81 or -C0 R 8 ', or R* together with R' 8 or R 19 and the intervening atoms form a 5-7 membered cycloalkyi or heterocyclyl ring optionally substituted with 1 -3 Ci-Q, alky! groups; each R 18 and R' v independently is hydrogen, C C* alkyi.
  • Ci-Cg cycloalkyi, C3-C3 heterocyclyl, - Cie aryi, or is rCio heteroaryl or is C3-C1 0 cycloalkyi, Cj-Cg heterocyclyl, €(,- € ⁇ aryl, or C 2 -Cio heteroaryl wherein each cycloalkyi, heterocyclyl, aryl, or heteroaryl is optionally substituted with 1-3 alkyi groups, or- R » and R !9 together with the nitrogen atom they are attached to form a 5-7 membered heterocycle.
  • the GGA derivative utilized herein is of Formula H I: nil
  • n 0, 1 . or 2;
  • each R' and R 2 are independently C)-C 6 aikyl, or 1 and R 2 together with the carbon atom tiiey are attached to form a C5-C7 cycloalkyl ring optionally substituted with i -3 Ci-Ci, a!kyl groups;
  • each of R 3 , R 4 , and R 5 independently are hydrogen or Cj-Cs alkyi;
  • Q is selected from the group consisting of:
  • X when X is bonded via a single bond, X is -0-, -NR 7 -, or -CR 8 R 9 -, and when X is bonded via a double bond, X is -CR 8 -;
  • Y 1 is hydrogen or -O-R 10
  • R" is C
  • R 7 is hydrogen or together with R" and the intervening atoms form a 5-7 membered ring optionally substituted with i-3 C i -Ce aikyl groups; each R* and R 9 independently are hydrogen, Cj-C 6 alkyi, -COR 8 ' or -C0 2 R* ⁇ or R together with R 6 and the intervening atoms fonn a 5-7 membered cycloalkyl or heterocyclyl ring optionally substituted with i-3 Ci-Q aikyl groups;
  • R 10 is d-C 6 aikyl
  • R" and R are independently C r C 6 aikyl, C C w cycloalkyl, -C0 2 R !5 , or - CON( L S ) , or R Kl and R" together with the intervening carbon atom and oxygen atoms fonn a heterocycle optionally substituted with 1-3 Cj-C* alky! groups;
  • R u is C
  • R' 4 is hydrogen, Cj-Cs alkyl optionally substituted with a -CC1 ⁇ 2H or an ester thereof or a C 6 -Cio aryi, Cj- s alkenyl, Cr , alkynyl C3-C10 cycloaikyl or a C 3 -C 8 heierocydyi, wherein each cycloaikyl, hetcrocyclyl, or aryi, is optionally substituted with 3 -3 aikyl groups; each R independently are hydrogen, GVC IQ cycloaikyl, LVCV.
  • alkyl optionally substituted with 1-3 substituents selected frora the group consisting of -COiH or an ester thereof, aryi, or Cj-Ct heterocyclyl, or two groups together with the nitrogen atom they are bonded to form a 5-7 membered heterocycle:
  • R 16 is hydrogen or C r C ( , alkyl
  • R r is hydrogen, C C S alkyl substituted with 1-3 hydroxy groups, -CHO, or is CO2H or an ester thereof;
  • each R !8 and R l 9 independently is hydrogen, Ci-C « aikyl apiiorially substituted with - CG 2 H or an ester thereof, CVC ie cycloaikyl, C 3 -C : 8 heterocyclyl, Cs-Cjo aryi, or C 2 ⁇ C Vi heteroaryl, or is C3-C10 cycloaikyl, Cj-Cg heterocyclyl, C 6 -Cio aiyl, or C2-C10 heteroaryl wherein each cycloaikyl, heterocyclyl, aryi or heteroaryl is optionally substituted with 1-3 alkyl groups, or R ! 3 ⁇ 4 and R i9 together with the nitrogen atom they are attached to form a 5-7 membered heterocycle; and
  • each R 8 ! independently is (h-Q, alkyl
  • m is 0.
  • rn is 1. in another
  • n is 0. in another embodiment, n is 1. In another embodiment, n is 2.
  • the compound of Formula (01) is of formula:
  • X, ⁇ ', and Y" are defined as in any aspect or embodiment herein.
  • the GGA derivative utilized is of formula: wherein R 1 , R 2 , R ' ⁇ R", R 5 , R 6 , X, Y ⁇ and Y" are defined as in any aspect and embodiment here.
  • the GGA derivative utilized is of formula:
  • R', R 2 , R', R s , R 6 , X, and Y 2 are defined as in any aspect and embodiment here. ⁇ 0199)
  • the GGA derivative utilized is of formula:
  • R 2 R 3 R' 1 R 5 R 6 wherein R ! , R 2 , R. " ⁇ R 4 , R 3 , R 6 and X are defined as in any aspect and embodiment here. (0200]
  • the GGA derivative utilized is of formula:
  • R ⁇ R 2 , R 4 . R 3 , and Q are defined as in any aspect and embodiment here.
  • the GGA derivative utilized is of formula:
  • R 1 .. R 3 R 4 , R 5 , m, n, and R !S are defined as in any aspect and embodiment here.
  • the GGA derivati ve utilized is of formula: wherein R ', R 2 , R 4 , R 5 , R*. m, n, and R" are implanted as in any aspect and embodkneni here.
  • each R ! and W " are Cj- s a!kyi.
  • each R 1 arid R ⁇ are methyl, ethyl, or isopropyl.
  • R ' ⁇ R 4 , and R 5 are C
  • R 3 , R 4 , and R i are hydrogen.
  • R R A t and R 5 are methyl.
  • X is G.
  • X is -NR' .
  • R' is hydrogen.
  • R' together with R 6 and the intervening atoms form a 5-7 membered ring optionally substituted with 1-3 Cj-Ce alkyl groups.
  • X is -CR ' R -.
  • X is -CR -.
  • each R* and R 9 independently are hydrogen, C
  • R 8 is hydrogen
  • R y is hydrogen, Ci-Ce alkyl, -COR g! , or -C0 2 R 81 .
  • R y is hydrogen.
  • R 9 is methyl.
  • R 9 is -COaR*'.
  • R 9 is -COR 81 .
  • R ' together with R and the intervening atoms form a 5-7 membered ring.
  • R 9 is hydrogen or C ; -C 6 alkyl. In one embodiment, R 9 is hydrogen. in another embodiment, R ' ' is CrCe alkyl.
  • R h is Ci-Cg alkyl.
  • R° is methyl, ethyl, butyl, isopropyi, or tertiary butyl
  • R 6 is d-C 6 alkyl substituted with 1-3 a!koxy or 1-5 halo group.
  • R 6 is alkyl substituted with an aikoxy group, in another cmbodiraenr, R 6 is alky! substituted with 1-5, preferably, 1 -3, halo, preferably fluoro, groups.
  • R* is WLV.
  • R 19 is H
  • R' 8 is Ci-Q alkyl, optionally substituted with a group selected from the group consisting of -CO forcingH or an ester thereof, C3-C10 cycloalkyl, C'3-C's heterocyclyl, i-V Cio aryl, or C Qo heteroaryl.
  • R 18 is C3-C10 cycloalkyl, C3- Cg heterocyclyl, C & -C )o aryi. or Cj-Cio heteroaryl.
  • R IS is C3-C10 cycloalkyl.
  • R 6 is C 2 -C 6 alkenyl or C2- alkynyi.
  • R 6 is C Cio cycloalkyl
  • R° is C3-C10 cycloalkyl substituted with 1-3 Cj-Cg alkyl groups.
  • R 6 is cyclopropyl, cyclobutyi, cycloperjlyl, cyclohexyl, or adamentyi.
  • R 5 is aryl or C 2 -C jo heteroaryl.
  • R 6 is a 5-7 membered heteroaryl containing at least 1 oxygen atom
  • R 6 is C ( ,-C:o ary!, Cj-Cg heterocyclyl. or C Cm heteroaryl, wherein each aryl. heterocyclyl, or heteroaryl is optionally substituted with 1 -3 C1-C & alky I groups,
  • Y" is -O-R 1 1 .
  • Y 1 and Y ⁇ arc joined to form NR 13 .
  • Q is -CR 9 COR 6 .
  • R 6 is C C 6 alkyl optionally substituted with an aikoxy group.
  • R 6 is Cj-C* cycloalkyl.
  • R 9 is hydrogen.
  • R y is Cj-C (l alkyl. in another embodiment, R 9 is C(3 ⁇ 4R 81 . In another embodiment, R 9 is COR 81 .
  • Q is - €3 ⁇ 4- € ⁇ (0-( ) ⁇ ! R°.
  • R is C;.-Cg cycloalkyl
  • R is Ci-Cg alkyl optionally substituted with 1 -3 substiteunts selected from the group consisting of -CO2W or an ester thereof, ary!. or CYCg heterocyciyl. in a preferred embodiment within these embodiments, R 6 is C .-Q, alkyl.
  • Q is -O-CO-NHR ⁇ .
  • R lS is C t -Q alkyl, optionally substituted with -C(1 ⁇ 4H or an ester thereof, CyC% cycloalkyl, C . rC « heterocyciyl, C2-C10 aryl, or C Cie heteroaryJ.
  • R' 8 is Cj-Ci cycloalkyl, Cj-Cg heterocyciyl, CrCioaryl, or Cj-C 10 heteroaryl,
  • R.' is hydrogen.
  • R ' is Ci- ⁇ alkyl optionally substituted with a -CCVII or an ester thereof or a Ce-Cjo aryl optionally substituted with 1 -3 alkyl groups.
  • R" 1 is C 2 -Q, alkenyl.
  • R M is 2-C5 aikynyi
  • R' 4 is C3-Q cycloalkyl optionally substituted with 1 -3 alkyl groups.
  • R ! 4 is C 3 -Cg heterocyclvi optionally substituted with 1 -3 alkyl groups.
  • R 16 is hydrogen, in another embodiment, R 1 ' is CO2M or an ester thereof In another embodiment, R/ ? is C
  • R 10 and R 1 1 together with the intervening carbon atom and oxygen atoms form a heteroycle of formula
  • R 2lJ is C
  • q is 1. In another embodiment, q is 2. In another embodiment, p is 0. hi another embodiment, p is 1. In another embodiment, p is 2. In another embodiment, p is 3,
  • examples of compounds utilized by this in vention include certain compounds tabulated below.
  • the GGA derivative is of Formula (IV):
  • each R ! and 2 are independently Ci-Q alky], or R 1 and R 2 together with the carbon atom they are attached to form a Q5-C7 cyeloalkyl ring optionally substituted with 1 -3 Ci-Cfi aikyl groups: each of R ⁇ R 4 , and R 5 independently are hydrogen or Cj- aikyl, or R 3 and Q together with the intervening carbon atoms form a 6 membered aryi ring, or a 5-8 membered cycloalkenyl ring, or a 5- 14 membered heteroaryl or heterocycle, wherein each aryi, cycloalkenyl, heteroaryl. or heterocycle, ring is optionally substituted with 1 -2 substituenis selected from the group consisting of halo, hydroxy, oxo, ⁇ N(R lU ⁇ ? 5 and Ci-Ce aikyl group;
  • Q is a 6 -10 membered and or a 5- 14 membered heteroaryl or heterocycle containing up 10 6 ring hcteroaioms, wherein the heteroatom is selected from the group consisting of 0. N. S, and oxidized forms of N and S, and further wherein the aryi, heteroaryl, or heterocyeiyl ring is optionally substituted with 1 -2 substituenis selected from the group consisting of hydroxy, oxo, ⁇ N(R I9 )2, and C r C 6 aikyl group, wherein the aikyl group is optionally substituted with 3 -3 substituenis selected from hydroxy, N3 ⁇ 4, -CO 2 H or an ester or an amide thereof, a 5- 9 membered heteroaryl containing up to 3 ring heteroatoms, wherein the heteroaryl is optionally substituted with 1 -3 hydroxy, -N(R )2.
  • each R 10 independently is hydrogen or Ci-Cs aikyl
  • the compound f Formula (IV) includes tautomers and optical isomers such as enanliomers and diaslereomers.
  • an ester refers preferably to a phenyl or a Ci -Cj. aikyl ester, which phenyl or aikyl group is optionally substituted with a amino group.
  • an amide refers preferably to a moiety of formula -CQNfR ⁇ k wherein R !0 is defined as above.
  • Q is selected from a group consisting of oxazoie, oxadiazole, oxazoline, azalactone, imidazole, diazole, triazoie, and tlnazole. wherein each heteroaryl or heterocycle is optionally substituted as disclosed above,
  • the GGA derivative utilized is of formula:
  • the GGA derivative utilized is of formula: 2 R 4 R 5 wherein R ! , R", R " , R ⁇ and Q are defined as in any aspect and embodiment here.
  • Q is selected from the group consisting of;
  • R l ! is defined as above.
  • Q is phenyl, optionally substituted as described herein, in another embodiment, Q is benximidazole, benzindazole, and such other 5-6 fused 9-membered bicyclic heteroaryl or heterocyeie.
  • Q is quinoiine, isoqirinoline, and such other 6-6 fused 10 inembered heteroaryl or heterocyeie.
  • Q is benzodiazepine or a derivative thereof, such as, a benzodiazepinone.
  • benzodiazepine and derivatives thereof are well known to the skilled artisan,
  • rn is 0. In another embodiment, m is 1.
  • n is 0. In another embodiment, n is 1. In another embodiment, n is 2.
  • m+n is 1. In another embodiment, m-hi is 2. In another embodiment, m+n is 3.
  • R' and R 2 are independently Ci-Ce alky].
  • R 1 and R * independently are methyl, ethyl, or isopropyl.
  • K R " , and 3 are independently Ci-Q, alkyl.
  • one of R J , R 4 , and R 3 are alkyl and the rest are hydrogen.
  • two of R 3 , R 4 , and R 5 are alkyl, and the rest are hydrogen.
  • R " ⁇ II 4 , and R s are hydrogen.
  • R 3 , R 4 , and R 5 are methyl.
  • this invention provides a compound selected from the group consisting of:
  • n 0, 1, or 2;
  • each R and R * independently are Cj-Q alky!, or R * and R together with the carbon atom they are attached to form a Cj-C? cycloalkyl ring optionally substituted with 1 -3 CrQ alkyl groups;
  • each of R 3 , R 4 , and R 5 independently is hydrogen or Cj-Q all yl
  • Q is selected from the group consisting of:
  • X when X is bonded via a single bona, X is -0-, -NR -, or -CR " R and when X is bonded via a double bond, X is -CR 8 -;
  • Y l is hydrogen or -OR 1 ;
  • R 6 is Ct-Q alkyl, Ci-G$ alky! substituted with 1 -3 alkoxy or 1-5 halo groups, Cz-Ci alkenyl, C2-C alkynyl, C3- 0 cycioalkyi, C C s heterocyclyl C&-C10 aryl, CV Cio heteroaryi, or -MR 2l 'R 2! , wherein each cycioalkyi or heterocyclyl is optionally substituted with 1-3 Cj- C $ alkyl groups, and wherein each aryl or heteroaryi is optionally substituted independently with 1-3 nitro and Ci- Q alkyl groups;
  • R 7 is hydrogen or together with R 6 and the intervening atoms form a 5-7 membered ring optionally substituted with 3 -3 Cj-Gs alkyl groups;
  • each R s and R 9 independently are hydrogen, C r Cc alkyl, -COR 8 ' , -CO2R 41 , or -COhiHR 82 , or R 8 together with R° and the intervening atoms form a 5-7 membered cycioalkyi or heterocyclyl ring optionally substituted with 1 -3 C t -Gs alkyl groups;
  • R 10 is C]-C 6 alkyl
  • each R" and R 12 independently are Ci-Ce alkyl, Cs-Cio cycioalkyi, -CO2R"', or -CON(R i7 ) 2 ;
  • R i3 is;
  • R 14 is hydrogen or Ci-Gj alkyl;
  • R 1 '5 is CpQ aiky] or C3-C10 cycloalkyl optionally substitued with 1 -3 Cj-Q alkyl groups, or is:
  • R 16 is hydrogen, Cj-Q alkyl optionally substituted with a -CO ? !-] or an ester thereof or a Ce-Cio aiyl, C Ce alkenyl, Cj-Ce alkyn l, C Ci o cycloalkyl, or a CrCg hetcrocyclyl, wherein each cycloalkyl, heterocvclyl, or aryl, is optionally substituted with 1-3 alk l groups;
  • each R 17 independently are hydrogen, Cj-Cto cycloalkyl.
  • Cj- ⁇ 1 ⁇ 4 alkyl optionally substituted with 1 -3 substitetmts selected f om the group consisting of - €(1 ⁇ 2H or an ester thereof, aryi, C 3 -C ⁇ heieroeyciyl, or two R' 7 groups together with the nitrogen atom they are bonded to form a 5-7 raembered heterocycle;
  • R' 8 is hydrogen or Q-Cg alkyl
  • R i9 is hydrogen, Ci-C ⁇ alkyl substituted with 1 -3 hydroxy groups, -CHO, or is C(3 ⁇ 4H or an ester thereof;
  • R 21 ' and i! independently are hydrogen, C-. -C alkyl, optionally substituted with -C ⁇ 3 ⁇ 4H or an ester thereof, C 3 -CH, cycloalkyl, C 3 -Cs lieierocycivi, CV Cio aryl, or C2-C10 heteroaryl, or is C3-C10 cycloalkyl, Cs-Cg heterocvclyl, C&- Cioar i, or CVCio heteroaryl, wherein each cycloalkyl, heieroeyciyl, aryl, or heteroaryl is optionally substituted with 1-3 alkyl groups, or R *;J and gether with the nitrogen atom they are bonded to form a 5-7 raembered heterocycle, and if only one of R " ° and R"' are defined as above, then the other one is
  • R* 1 is C Q alkyl
  • R is: provided that, when X is bonded via a single bond, and R or R J is not
  • Y 2 is -0-CQ-NR !3 R 14 :
  • R* is not -CONHR 82 , Y 2 is -0-CO- R i3 R 14 ;
  • the GGA derivative utilized are of formula:
  • this invention is also directed to pharmaceutical compositions comprising at least one pharmaceutically acceptable excipieni and an effective amount of the trans-tsomer compound of GGA according to this invention.
  • Pharmaceutical compositions can be formulated for different routes of
  • compositions suitable for oral delivery will probably be used most frequently, other routes that raay be used include intravenous, intraarterial, pulmonary, rectal, nasal, vaginal, lingual, intramuscular, intraperitoneal, intracutaneous, transdermal, intracranial and subcutaneous routes.
  • Other dosage forms include tablets, capsules, pills, powders, aerosols, suppositories, parenterals, and oral liquids, including suspensions, solutions and emulsions.
  • Sustained release dosage forms raay also be used, for example, in a transdermal patch form, All dosage forms may be prepared using methods that are standard in the art (sec e.g., Remington's Pharmaceutical Sciences, 16 lh ed., A. Oslo editor, Easton Pa. 1980).
  • compositions are comprised of in general, GGA or a trans-isomer compound of GGA or a mixture thereof in combination with at least one pharmaceutically acceptable excipient.
  • Acceptable excipients are non-toxic, aid administration, and do not adversely affect the therapeutic benefit of the compound of this invention.
  • excipients may be any solid, liquid, semi-solid or, in the case of an aerosol composition, gaseous excipient that is generally available to one of skill in the art.
  • Pharmaceutical compositions in accordance with the invention are prepared by conventional means using methods known in the art.
  • compositions disclosed herein may be used in conjunction with any of the vehicles and excipients commonly employed in pharmaceutical preparations, e.g., talc, gum arabic, lactose, starch, magnesium stearate, cocoa butter, aqueous or non-aqueous solvents, oils, paraffin derivatives, glycols, etc. Coloring and flavoring agents may also be added to preparations, particularly to those for oral administration. Solutions can be prepared using water or physiologically compatible organic solvents such as ethanol, 1,2 -propylene glycol, poly glycols, dimethylsulfoxide, fatty alcohols, triglycerides, partial esters of glycerin and the like.
  • Solid pharmaceutical excipients include starch, cellulose, hydroxypropyl cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, dried skim milk and the like.
  • Liquid and semisolid excipients may be selected from glycerol, propylene glycol, water, ethanol and various oils, including those of petroleum, animal, vegetable or synthetic origin, e.g., peanut oil, soybean oil, mineral oil, sesame oil, etc.
  • the concentration of the excipient is one that can readily be determined to be effective by those skilled in the art, and can vary depending on the particular excipient used.
  • the total concentration of the excipients in the solution can be from about 0.001 % to about 90% or from about 0.001 % to about 10%.
  • a pharmaceutical composition comprising the compound of formula I and a-tocopheroi.
  • a related embodiment provides for a pharmaceutical composition comprising the compound of formula L a- tocopherol, and hydroxypropyi cellulose.
  • a pharmaceutical composition comprising the compound of formula I, a-tocopherol, and gum arabic.
  • a pharmaceutical composition comprising the compound of formula I, and gum arabic.
  • the compound of formula ⁇ , gum arabic and hydroxypropyi cellulose there is provided the compound of formula ⁇ , gum arabic and hydroxypropyi cellulose,
  • the concentration by weight can be from about 0.001 % to about i% or from about 0.001% to about 0.005%, or from about 0,005% to about 0.01%, or from about 0.01 % to about 0.015%. or from about 0,015% to about 0.03%, or from about 0,03% to about 0,05%, or from about 0.05% to about 0.07%, or from about 0.07% to about 0.1%, or from about 0.1% to about 0.15%, or from about 0.15% to about 0.3%, or from about 0.3% to about 0.5%, or from about 0.5% to about 1% by weight.
  • the concentration of -tocopherol is about 0.001% by weight, or alternatively about 0.005%, or about 0,008%, or about 0.01 %, or about 0.02%, or about 0.03%, or about 0.04%, or about 0.05% by weight.
  • the concentration by weight can be from about 0, 1 % to about 30% or from about 1% to about 20%, or from about 1 % to about 5%, or from about 1 % to about 10%, or from about 2% to about 4%, or from about 5% to about 10%, or from about 0% to about 15%, or from about 15% to about 20%, or from about 20% to about 25%, or from about 25% to about 30% by weight.
  • the concentration of hydroxypropyi cellulose is about 1% by weight, or alternatively about 2%, or about 3%, or about 4%. or about 5%. or about 6%, or about 7%. or about 8%, or about 30%, or about 15% by weight,
  • the concentration by weight can be from about 0.5% to about 50% or from about 1% to about 20%, or from about 1% to about 10%, or from about 3% to about 6%, or from about 5% to about 10%. or from about 4% to about 6% by weight, in some embodiments, the concentration of hydroxypropyl cellulose is about 1% by weight, or alternatively about 2%, or about 3%, or about 4%, or about 5%, or about 6%, or about 7%. or about 8%, or about 10%, or about 15% by weight.
  • the concentration of GGA, or the trans-geranylgeranyl acetone isomer can be from about 1 to about 99% by weight in the pharmaceutical compositions provided herein, In other embodiments, the concentration of the trans-geranylgeranyl acetone isomer can be from about i to about 75%, or alternatively, from about 1 to about 40%, or alternatively, from about 1 to about 30%, or alternatively, from about i to about 25%, or alternatively, from about 1 to about 2.0%, or alternatively, from about 2 to about 20%, or alternatively, from about 1 to about 10%, or alternatively, from about 10 to about 20%, or alternatively, from about 10 to about 15% by weight in the pharmaceutical composition, in certain
  • the concentration of geranylgeranyi acetone in the pharmaceutical composition is about 5% by weight, or alternatively, about 10%, or about 20%, or about 1%, or about 2%, or about 3%, or about 4%, or about 6%, or about 7%, or about 8%, or about 9%, or about 11%, or about 12%, or about 14%, or about 16%, or about 18%, or about 22%, or about 25%, or about 26%, or about 28%, or about 30%, or about 32%, or about 34%, or about 36%, or about 38%, or about 40%, or about 42%, or about 44%, or about. 46%, or about. 48%, or about 50%, or about 52%, or about 54%, or about 56%, or about 58%, or about 60%, or about 64%, or about 68%, or about 72%, or about 76%, or about 80% by weight,
  • this invention provides sustained release formulations such as drug depots or patches comprising an effective amount of GGA.
  • the patch further comprises gum Arabic or hydroxypropyl cellulose separately or in combination, in the presence of alpha-tocopherol.
  • the hydroxypropyl cellulose has an average MW of from 10,000 to 100,000.
  • the hydroxypropyl cellulose has an average MW of from 5,000 to 50,000.
  • the patch contains, in various embodiments, an amount of GGA. preferably the 5E, 9E, 13E isomer of it, which is sufficient to maintain a therapeutically effective amount GGA in the plasma for about 12 hours.
  • the GGA comprises at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the 5E, 9E, BE isomer of GGA.
  • co-administration When administered with another agent, the coadministration can be in any manner in which the pharmacological effects of both are manifest in the patient at the same time.
  • co-administration does not require that a single pharmaceutical composition, the same dosage form, or even the same route of administration be used for administration of both the compound of this invention and the other agent or that the two agents be administered at precisely the same time.
  • coadministration will be accomplished most conveniently by the same dosage form and the same route of administration, at substantially the same time. Obviously, such administration most advantageously proceeds by delivering both active ingredients simultaneously in a novel pharmaceutical composition in accordance with the present invention.
  • a compound of this invention can be used as an adjunct to conventional drug therapy.
  • This invention provides a synthetic method comprising one or more of the following steps;
  • R x and eac R 3 independently are alkyi or substituted or unsubstituted aryl under olef ation conditions to selectively provide a compound of formula VIII :
  • Vlil (v) reacting the compound of formula Vlil under reduction conditions to provide a compound of formuia iX
  • Compound ID is combined with at least an equimolar amount of a halogenating agent typically in an inert solvent
  • an "inert solvent” is a solvent that does not react under the reaction conditions in which it is employed as a solvent.
  • the reaction is typically ran at. a temperature of about 0°C to 20 °C for a period of time sufficient to effect substantial completion of the reaction.
  • Suitable solvents include, by way of example only, diethyl ether, aeeionitrile, and the like.
  • Suitable halogenating agents include PBri or PPhi/CBn.
  • Compound IV is combined with at least an equimolar amount of an alkyi acetoacetate, in the presence of a base and an inert solvent.
  • the reaction is typically run initially at (f'C, and then warmed up to room temperature for a period of time sufficient to effect substantial completion of the reaction.
  • Suitable solvents include, by way of example only, various alcohols, such as ethanol, dioxane, and mixtures thereof.
  • Suitable bases include, by way of example only, alkali metal alkoxides, such as sodium ethoxide.
  • Compound VI is combined with at least an equimolar amount, preferably, an excess of a compound of formula VII, and at least an equimolar amount, preferably, an excess of base, in an inert solvent.
  • the reaction is typically ran. initially at about -30°C tor about 1-2 hows, and at room temperature for a period of time sufficient to effect substantial completion of the reaction.
  • Suitable solvents include, by way of examples only tetrahydrofuran, dioxane, and the like.
  • Suitable bases include, by way of example only, alkali metal hydrides, such as sodium hydride, or potassium hexamethyldisilazide (KJIMDS), or potassium tertiary butoxide ( l BuOK).
  • Compound VIII is combined with a reducing agent in an inert solvent.
  • the reaction is typically ran at about 0°C for about 15 minutes, and at room temperature for a period of time sufficient to effect substantial completion of the reaction.
  • Suitable reducing agents include, without limitation, LiAlE$.
  • Suitable solvents include, by way of examples only diethyl ether, tetrahydrofuran, dioxane, and the like.
  • the method further comprises repeating steps (i). (ii), and (iii) sequentially with compound of formula VIII to provide the compound of formula L wherein m is 2.
  • the method or procedure further comprises repeating steps (i), (ii), (iii), (iv), and (v), sequentially, 1-3 times.
  • a method comprising one or more of the following steps:
  • R aikyl is substituted or unsubstituted alkyi
  • this invention provides a method comprising step (i) or step (ii) or steps (i) ⁇ * ⁇ (ii) :
  • Rl is as defined herein, and
  • this invention pro vides a method comprising reacting a ketai compound of formula XII:
  • each R 5 independently is Ci-C& alkyl, or two R 5 groups together with the oxygen atoms they are attached to form a 5 or 6 membered ring, which ring is optionally substituted with 1-3, preferably 1 -2.
  • Ci-Cg alkyl groups under hydrolysis conditions to provide a compound of formula IL
  • the ketai is combined with at least a catalytic amount, such as, 1 -20 mole% of an aqueous acid, preferably, an aqueous mineral acid in an inert solvent.
  • the reaction is typically run about 25°C to about 80°C, for a period of time sufficient to effect substantial completion of the reaction.
  • Suitable acids include, without limitation, liCL H2SO4, and tire like.
  • Suitable solvents include alcohols, such as methanol, ethanol, tetrahydrofuran. and the like.
  • this invention provides a method comprising reacting a compound of formula XI:
  • this invention provides a method comprising reacting a compound of formula X1C:
  • a compound of Formula (HI), where n is 2 is synthesized by repeating the reaction sequence of alkylation with a bela-keto ester, hydrolysis, decarboxylation, Wittig-Homer olefination, and LiAl3 ⁇ 4 reduction,
  • R ' is aikyl
  • Compound (ix.) with alcohol functionality is an intemieaiate useful for preparing the compounds utilized in this invention.
  • Compound (x), where L is an E ⁇ SO? ⁇ group is made by reacting compound (ix) with R'SOjCl in the presence of a base.
  • the transformation of compound (Hi) to compound (x) illustrates methods of adding isoprene derivatives to a compound, which methods are suitable to make compound (in) from compound (i).
  • m is 0 or 1 and R'-R 5 are as defined herein, and are preferably alkyl, or more preferably methyl
  • intermediate (ixa) prepared according to the scheme herein above, is converted to amino intermediate (ixb) via the corresponding bromide
  • intermediates (ixa) and (ixb) are converted to the compounds utilized in this invention by reacting with suitable isocyanaies or carbamoyl chlorides, which are prepared by art known methods.
  • GGA derivatives utilized herein are synthesized as schematically shown below. imines, ydazones, alkoxyirnines
  • Certain compounds utilized herein are obtained by reacting compound (x) with the anion Q(-), which can be generated by reacting the compound QH with a base.
  • bases include hydroxide, hydride, amides, alkoxid.es. and the like.
  • Various compounds utilized in this invention, wherein the carbonyl group is converted to an imine, a hydrazone, an aikoxyimine, an cnolcarbamate, a kctal, and the like, are prepared following well known methods.
  • the metal iation is performed, by reacting the ketone with a base such as dimsyl anion, a hindered amide base such as diisopropylamide, or hexamethyldisilazide, along with the corresponding metal cation, M.
  • a base such as dimsyl anion, a hindered amide base such as diisopropylamide, or hexamethyldisilazide
  • the amino carbonyl chloride or the isocyanate is prepared, for example, by reacting the amine (R' ⁇ NH with phosgene or an equivalent reagent well known to the skilled artisan.
  • the beta keto ester is hydro lysed while ensuring that the reaction conditions do not lead to decarboxylation.
  • the acid is activated with various acid activating agent well known to the skilled artisan such as carbonyi diirnodazo!e, or 0-Beii/.otria3 ⁇ 4ole-N,N,N', ! - teiTainethyl-uroniuni-hexailuoro-phospliate (HBTU) and reacted with the amine.
  • acid activating agent well known to the skilled artisan such as carbonyi diirnodazo!e, or 0-Beii/.otria3 ⁇ 4ole-N,N,N', ! - teiTainethyl-uroniuni-hexailuoro-phospliate (HBTU)
  • Compound (viii) is hydrolyzed to the carboxylic acid fx), which is then convened to the acid chloride (xi).
  • Compound (xi) is reacted with a suitable nucleophiie such as a hydrazide, a h droxylarainc, an amino aicohol, or an amino acid, and the intermediate dehydrated to provide a compound of Formula (I).
  • a suitable nucleophiie such as a hydrazide, a h droxylarainc, an amino aicohol, or an amino acid
  • the intermediate dehydrated to provide a compound of Formula (I).
  • the aliylic alcohol (ix) is oxidized to the aldehyde (xi), which is then reacted with a cyanohydrin or cyanotosyimethane to provide further compounds utilized in this invention.
  • GOA derivatives utilized in this invention can also be synthesized employing art known methods and those disclosed here b aJkene-aryl, alkene-heteroaryl, or alkene-akene couplings such as Heck, Stender, or Suzuki coupling. Such methods can use (vi) to prepare intermediate (xii) that can undergo Heck, Stender, or Suzuki coupling under conditions well known to the skilled artisan to provide compounds utilized in this invention. ilti olefinaion
  • L and Q are as defined herein, Ar is a preferably an aryl group such as phenyl the base employed is an alkoxide such as tertiarybutoxide, a hydride, or an alkyl lithium such as n-buty! lithium.
  • the metal lation is performed, by reacting the ketone with a base such as dimsyl anion, a hindered amide base such as diisopropylamide, or hexamethyidisiiazide, along i the corresponding metal cation, M.
  • a base such as dimsyl anion, a hindered amide base such as diisopropylamide, or hexamethyidisiiazide
  • the amino carbonyl chloride or the tsocyanate is prepared, ibr exampie, by reacting the amine R U R NH with phosgene or an equivalent reagent well known to the skilled artisan.
  • the beta keto ester is hydro!yzed while ensuring that the reaction conditions do not lead to decarboxylation.
  • the acid is activated with various acid activating agent well known to the skilled artisan such as carbonyl diimodazole, or tetramethyl-uronium-hexafluoro-phosphate (HBTU) and reacted with the amine.
  • acid activating agent well known to the skilled artisan such as carbonyl diimodazole, or tetramethyl-uronium-hexafluoro-phosphate (HBTU)
  • R is a memantine or a riluzole residue.
  • GGA is a known anti-ulcer drug used commercially and in clinical situations. GGA has also been sho wn to exert cytoprotective effects on a variety of organs, such as the eye. brain, and heart (See for example Ishii Y., et al dislike Invest Ophthalmol Vis So 2003; 44: 1982- 92; Tanito M, et al., J Neurosci 2005; 25:2396-404; Fujiki M, et al., J Neurotrauma 2006;
  • the isomeric mixture of GGA and/or compositions containing the 5-trans isomer of GGA can be used to inhibit neural death and increase neural activity in a mammal suffering from a neural disease, wherein the etiology of said neural disease comprises formation of protein aggregates which are pathogenic to neurons which method comprises administering lo said mammal an amount of GGA which will inhibit neural death and increase neural activity, or impede the progression of the neural disease.
  • this method is not intended to inhibit or reduce trie negative effect of a neural disease m which the pathogenic protein aggregates are intranuclear or diseases in which, the protein aggregation is related to SBMA.
  • Negative effects of neural diseases that are inhibited or reduced by GGA and the 5- trans isomer of GGA according to this invention include but are not limited to Alzheimer ' s disease, Parkinson's disease, multiple sclerosis, prion diseases such as uru, Creutzfeltdt- Jakob disease. Fatal familial insomnia, and Gerstmann-Straussler-Scheinker syndrome, amyotrophic lateral sclerosis, or damage to the spinal cord. GGA and the 5 -trans isomer of GGA are also contemplated to prevent neural death during epileptic seizure.
  • PBrj phosphorus tri bromide
  • Liquid chroinatography-iTiass spectrometry Rf retardation factor
  • Kp Ratio of AUC w u> AUC ⁇ fa,,,,,,,
  • Vcap 4000
  • the starting materials for the reactions described below are generally known compounds or can be prepared by known procedures or obvious modifications thereof.
  • many of the starting materials are available from commercial suppliers such as Aldrich Chemical Co. (Milwaukee, Wis., USA), Bachem (Torrance, Calif., USA), Emka- Chemce or Sigma (St. Louis, Mo.. US ).
  • Others may be prepared by procedures, or obvious modifications thereof, described in standard reference texts such as i ' ieser and Fieser's Reagents for Organic Synthesis, Volumes 1 15 (John Wiley and Sons, 3991). Rodd's Chemistry of Carbon Compounds.
  • the 2E,6E-farnesyl alcohol 3 (where the geometry at C2 and Co positions is already fixed as trans- or E) was designed and used as a commercially available starting material for the synthesis of 5E,9E,13E-geranylgeranyl acetone 1
  • the alcohol function of 2E, 6E- farnesyl alcohol 3 was converted to the corresponding bromide 4 by the treatment of phosphorus tribromide (PBr 3 ) in ethyl ether (EE) or with 13 ⁇ 4P and CBr 4 in acetonitrile (AC ) at 0°C.
  • keioester 6 The homologated keioester 6 after hydrolysis and decarboxylation using aqueous 5N KOH yielded the expected SE,9E-famesyl acetone 7, one of the key intermediate for the synthesis of 5E,9E, 13E-geranylgeranyi acetone 1 and 5Z,9E.13E-geranyigeranyl acetone 2.
  • the resulting 2Z-corijugated ester 12 was converted into the corresponding 2Z- alcohol 13 by means of a lithium aluminum hydride (LAH) treatment.
  • the 2Z-al.cohoi 13 was transformed into the corresponding 2Z,6E, 1 OE-geranylgeranyl bromide 14 by using
  • meftyiphosponrane bromide 1.7 under a basic conditions followed by treatment with formaldehyde (monomeric) can afford the 2Z.6E1 OE-geranylgeranyl alcohol 13 with cis (Z)- orientation at C2 (Ref.: Wiemer et an, Organic Letters, 2.005, 7(22), 4803-4806).
  • the conversion of bromide 14 to the ketoester 15 followed by hydrolysis and decarboxylation can afford she desired 5-cis (Z)-isomer; 5Z,9E, 13E-geranygerany! acetone (2).
  • the other synthon, namely the y!ide 21 can be synthesized from a commercially available starting material, ethyl levulinate 16, a sugar industry by-product.
  • the Retaliation of ethyl levuliaate 16 using conventional conditions ⁇ ethylene glycol, p-TsOH, azeotropic reflux) can yield the desired 2-oxo-ketal 17, which then can be reduced using LAH in THF at 0 °C to the corresponding alcohol 18. Furthermore, the alcohol IS then can be treated with PhjBr in diethyl ether at 0 °C to obtain the bromide 19, which then after treatment with PhiP can yield the phosphonium bromide salt 20.
  • the bromide salt 20 upon treatment with mild alkali (IN NaOH) can furnish the desired yiide 21, required to complete the synthesis of 5Z- GOA 2.
  • the 5E, 9E, BE-geranyl geranyl acetone (1) can be prepared by reacting 6E-10E- geranyl linalool (23) with diketene (24) catalyzed by DMAP in ethyl ether to give the ester 25.
  • the ester 25 in. the Carroll rearrangement using Al(OiPr)j at elevated temperature can afford the desired 5E, 9E, 13E-geranyl geranyl acetone (1).
  • the GGA ( ⁇ ) can be prepared by treating geranyi linalool (23) with the Meldrurn's acid 26 in the Carroll rearrangement using Al(QiPr)3 ⁇ 4 at 160 "C, Similar! ⁇ ', the use of ,'er.f-buiyl acetoaeetate (27) with geranyi linalool (23) in the Carroll rearrangement can also give the desired SE, 9E. 1 E-gerany! geranyi acetone (1 ).
  • compositions of 5E- and/or 5Z-GGA can be used in such preclinical research formulations.
  • Plasma concentrations and PK parameters were obtained from C S- 101 IV dosing and oral formulation PK studies.
  • PK PARAMETERS were calculated from noncompartmental analysis ( CA) model using WinNonlin software and the linear/log trapezoidal method.
  • Tmax (mm) Time to reach Cmax (directly taken from analytical data).
  • Example 7 GGA formuiatien using 5% Gum Arabic with 0,008% a-tocopheroi
  • HFC Hydroxypropyi Cellulose
  • Mrr 100,000; High Average Molecular Weight
  • 0.0OS% a-tocopherol
  • HPC Hydroxypropyi Cellulose
  • % ratios are based on volumes [0323] .4, Preparation of 5% Gum Arabic Solution: 1.25 g of Gum Arabic was suspended in DI water (23.75 mL; until the total volume was 25 mL) and agitated using agitator until all gum Arabic was miseible in DI water. To this solution was added a - tocopherol (2 ⁇ LL, 0.008%) and agitated for a minute to obtain 5% gum Arabic
  • Example 11 GGA formulation using 5% Gum Arabic + 3% Hydroxypropyl Cellulose (HPC; Av. Me- 10,262; Low Av. Mokcolar Weight) and with 0,008% a -tocopherol
  • Example 12 Culturing of primary motor neurons from rats.
  • Rat primary motor neurons were isolated from embryonic spinal cords in accordance with the method of Henderson et a!.; J Cohen and G P Wilkin (ed.), Neural Cell Culture, (1995) p69 «81 which is herein incorporated by reference in its entirety. Briefly, spinal cords were dissected from day 15 embryo (E15) and incubated in a trypsin solution, and followed by DNase treatment to release spinal cord cells from tissue fragments. The eel! suspension was centrifuged to remove tissue fragments. Then motor neurons were enriched by density gradient centrifugation.
  • Motor neurons were cultured in serum-free neurobasal medium containing insulin, forskolin, 3-isobutyl- 1 -meth Ixanthine, neurotrophic factors, Bovine serum, albumin, selenium, transferrin, putrescine, progesterone and B27 supplement in tissue culture plate coated with poly-omi thine and !aminin.
  • Rat eager motor neurons were prepared and cultured as described Example 1 1.
  • the ECJO is a measure of the effectiveness of a compound, and corresponds to the concentration at which the drug exhibits half its maximum effect.
  • Example 14 A large quantity of GGA isomer mixture (CNS-101) inhibited viability of neuroblastoma cells.
  • Example 15 A large quantity of GGA isomer mixture (CNS-101 ⁇ aad ds-isomer (CNS- 103) inhibited viability of seuro blastema cells.
  • Example 16 Effects of the GGA isomer mixture (CNS-101) on cells experiencing oxidative stress.
  • Neuro2A cells were cultured with CNS-101 , CNS- 102, or CNS-103 in the presence or absence of an inhibitor against a G-proiein (GGTI-298). After differentiation was induced, cells that extended neyrites were counted. These results are depicted in the table below;
  • Example 18 The GGA isomer mixture (CNS-101) activated neurite outgrowth of neuroblastoma cells.
  • Example 19 The GGA isomer mixture (CNS-101 ) and the trans-isomer (CNS-102) alleviated neurodegeneration induced by Kainic acid.
  • CNS- i 01 or CNS-102 were orally dosed to Sprague-Dawley rats, and Kainic acid was injected. Seizure behaviors were observed and scored (Ref. .J. Racine, Modification of seizure activity by electrical stimulation: II, Motor seizure, Eleeiroeneephaiogr. Clin.
  • Example 20 Comparison of the efficacy of O - 10 ⁇ and CNS-102 m alleviating neurodegeneration induced by Kainic acid.
  • CNS-101 , CNS- 102 or a vehicle only control were orally dosed to Sprague-Dawley rats, and Kainic ainic acid was injected. Seizure behaviors were observed and scored (Ref. R.J. Racine, Modification of seizure activity by electrical stimulation: II. Motor seizure, Eleeiroeneephaiogr. Clin. Neurophysiol. 32 (1972) 281 - 294. Modifications were made for the methods). Brain tissues of rats were sectioned on histology slides, and neurons in hippocampus tissues were stained by Nissl. Neurons damaged by ainic acid and behavior scores were quantified,
  • Ci S-103 25 mg/K rat
  • Example 21 GGA's effect on the activity of G proteins in a euroE, ⁇ 033 J Neuroblastoma cells can be obtained from the American Type Culture Collection (ATCC) and cultured according to the suggested culturing techniques of ATCC. The cultured cells will be contacted with an effective amount of GGA. The change in G protein activity will be monitored by a western blot of iysates obtained from subcellular fractionation of cells. Subcellular fractionation can be performed using commercially available kits (from Calbiochem for example) according to the manufacturer's protocol. The western analysis will be performed using subcellular fractions from the membrane and cytoplasmic compartments of cells.
  • the western blot will be performed according to standard molecular biology techniques using antibodies directed to the different G proteins: HOA, RAG , CDC42, RASD2. it is contemplated that reacting the neuroblastoma cells with an effective amount of GGA will modulate the active, membrane-bound portion of RHOA, ACI ,
  • TDP-43 depleted ceils mimic the effects of neurodegeneration related to ALS
  • TDP-43 depletion can be accomplished using the siRNA and/or shRNA technologies. It is contemplated that neurons which are susceptible to neurodegeneration by TDP-43 depletion will have a change in the G protein activity after said neurons are contacted with an effective amount of GGA. H is further contemplated that reacting said neurons with an effective amount of GGA will increase the active membrane- bound portion of the G proteins.
  • J0341 j The same assay will be performed using neuroblastoma cells or other neurons that are susceptible to neurodegeneration due to inhibition of geranylgeranylation of the G proteins.
  • GGTI-298 is a specific inhibitor of geranylgeranylation and increases neuronal ceil death through inhibiting the activation of G proteins by geranylgeranylation. Therefore, GGTI-298 and GGA will both be contacted with tissue cultures of neuroblastoma cells, it is contemplated that neurons which are susceptible to neurodegeneration by GGTI-298 will have a change in the G protein activity after said neurons are contacted with an effective amount of GGA. it is further contemplated that reacting said neurons with an effective amount of GGA will increase the active membrane- bound portion of the G proteins.
  • Example 22 GGA's effect on the pathogenicity of protein aggregates in neurons susceptible to neurodegeneration.
  • Cultured neuroblastoma cells can be made susceptible to neurodegeneration by mixing the cells with dopamine. The addition of dopamine to the cells will cause pathogenic protein aggregates in the cytoplasm.
  • an effective amount of dopamine will be first contacted with the neurons to induce pathogenic protein aggregate formation in the cells.
  • an effective amount of GGA will be contacted with said neurons. The change in the size and/or number of protein aggregates will then be measured using histological staining techniques and/or
  • contacting GGA with neurons susceptible to neurodegeneration due to dopamine-induced protein aggregation will sorubilize at least a portion of the protein aggregate, thus decrease the pathogenicity to the cell, it is further contemplated that contacting GGA with neurons susceptible to neurodegeneration due to dopamine-induced protein aggregation will alter the form of the pathogenic protein aggregate into a non-pathogenic form, thus decrease the pathogenicity to the ceil,
  • contacting GGA with neurons susceptible to neurodegeneratiori due to -amyloid peptide aggregation will solubilize at least a portion of the protein aggregate, thus decrease the pathogenicity to the cell. It is further contemplated that contacting GGA with neurons susceptible to neurodegeneration due to ⁇ - amyloid peptide aggregation will alter the form of the pathogenic protein aggregate into a non-pathogenic form, thus decrease the pathogenicity. The change in the size and/or number of protein aggregates will then be measured using histological staining techniques and or immunostaining techniques commonly known to one skilled in the art.
  • Example 23 GGA's effect ' in vivo in mammals susceptible to neurodegeneration.
  • Neurotoxins can be used to recapitulate the effect of AD in mice.
  • To test the effects of administering GGA to a mammal that is susceptible to AD neurotoxins will be administered systeraica!ly or by direct injection into the brain tissues of mice to induce the pathology associated with AD.
  • the neurotoxins will be administered either before, sim ultaneously, or after the administration of GGA.
  • the GGA may be administered to said mice mixed with a pharmaceutically acceptable e cipknt
  • These mice will then be monitored for survival rate, neuron density in brain tissues, arid learning, memory, and motor skills. The learning, memory, and motor skills are measured by techniques commonly known to one skilled in the art. It is contemplated that treating the animal with an effective amount of GGA wi ll attenuate some of the symptoms associated with the injection of the neuroto in.
  • mice There are a variety of mouse models available that are engineered to have the same pathology associated with different human diseases, One uch mouse model is a mouse that over-expresses the Amyloid beta Precursor Protein (APP). This mouse has a similar * pathology to that seen in human AD.
  • An effecti e amount of GGA will be administered to mice over-expressing APP.
  • the GGA may be administered to said mice mixed with a pharmaceutically acceptable excipient.
  • These mice will then be monitored for body weight, ⁇ -amyloid plaque formation, and learning, memory, and motor skills. Histology sections of these mice will also be analyzed by staining and immunohistochemical techniques to detect changes in the brain after GGA administration. It is contemplated thai treating the animal with an effective amount of GGA will attenuate some of the symptoms associated with AD.
  • mice expressing a Sodl mutant protein exhibit similar pathology to humans with ALS.
  • An effective amount of GGA will be administered to Sod l mutant mice.
  • the GGA may be administered to said mice mixed with a pharmaceutically acceptable excipient. These mice will then be monitored for survival rate, body weight, and motor skills. Histology sections of these mice will also be analyzed by histology staining and immunohistochemical techniques to detect changes in the brain, spinal cords, or muscles after GGA administration. It is contemplated that treating the Sodl mutant mice with an effective amount of GGA will increase the survival rate, body weights, and enhance the motor skills of these mice.
  • the percentage survival rate for each of the treated groups was calculated up to 150 days of age, and blood from animals in each group at PI 00 was analyzed for levels of alkaline phosphatase, alanine transaminase/seium glutamic pyruvic transaminase (ALT/SGPT);
  • albumin aspartate iransaminase/serum glutamic oxaloacetic transaminase (AST/SGOT).
  • albumin total protein, albumin, blood urea nitrate (BUN), creatinine cholesterol, and glucose, as well as the albumin/globulin ratio.
  • Example 25 Effects of 5-trans isomer of GGA's on s rvival, behavior, and pathology in S di mutant mice,
  • mice A total of 84 male mice (64 transgenic Sod 1 mutant mice; 20 wild type mice) ere included in the study. Dosing was administered daily beginning at postnatal day 38 (P3 ) via a single bolus oral gavage injection. Sod i mutant mice were randomized into experimental groups and administered vehicle, CNS-102 (i2mg/Kg), Riluzole (8mg Kg), . Wild type mice were randomized into two experimental groups and administered vehicle alone or CNS-102 (12mg/Kg).
  • the grip strength test assesses inotor function and control of the fore- and hincpaws. Mice were allowed to grab the bars on the Chatiilon DFIS-10 digital force gauge (Largo. FL), while being gently pulled parallel away from the bars by the tail. The maximum force measured prior to release of the subject's paw from tiie bar was recorded. [0356J Animals were scored clinical iy by allowing iliern to run in an exercise wheel and then scoring their gait using a scoring system adapted from Braestle et al, (Neurornoiecular Med, 2009 ; 1 1 (2); 58-62). Animals were scored using die scoring system tabulated below.
  • mice were scored neuro!ogica!ly using a scoring system adapted from Leitner et al. (Working with ALS mice; The Jackson Laboraiories/Prize4Life, Appendix B, 2009).
  • a score of 0 is given to mice that exhibit full extension of hind legs away from lateral midline when suspended by the tail
  • a score of 0.25 is given to mice that exhibit shaking or slight/partial collapse when suspended by the tail.
  • a score of 0.5 is given to mice that exhibit collapse or partial col lapse of leg extension towards the midline (weakness).
  • Example 26 Pharmacokinetics of 5-traos isomer of GGA's in rats.
  • Rats were administered a single dose of CNS-102 either intravenously (12mg/kg) or orally (24mg/kg), and blood samples were taken to measure the average plasma
  • the formulation for this study includes the following:
  • the ratio of total brain concentration to total plasma concentration (Kp) tor CNS- 102 was calculated. First, the total plasma concentration of CNS- 102 (in two different formulations) was calculated as described in Example 24. The total brain concentration of CMS- 102 in ng g for the same animals was also measured.
  • AUC(O-Iast) (min*ng ml ⁇ : area under the concentration-time curve from zero up to the last measurable time-point.
  • Terminal Half-fife (tl 12) - in ⁇ 2)/lz calculated using Lambda_z method to find best fit. If necessary, the concentration-time points were manually selected for use in the calculation, Bolded-italicized concentrations indicate points used for calculation.
  • AUCtot is the area under the concentration-time curve for total (bound and unbound) concentrations in brain or plasma.
  • Formulation 1 and Formulation 2 refer to the following: Formulation # 1 (Dose: 0.200 g/Kg)
  • Murine neuro2A neuroblastoma cells were treated with various concentrations of CNS-102 (all tram-GGAl CNS-101 (mixture of GGA isomers), and CNS-103 (ail cis-GGA ) for 48 hours. Differentiation was induced and cells incubated with geranyigeranyl transferase I inhibitor, GGTi-298. Cells were harvested and lysates were prepared and analyzed by western blot for HSP70 and 11SP90. Western signals in the absence of the compounds were normalized as 1.00. The results are tabulated below.
  • Murine neuro2A neuroblastoma cells were treated with various concentrations of CNS-101, CNS-102. or CNS-103 for 48 h. Differentiation was induced and cells incubated with geranyigeranyl transferase I inhibitor, GGT1-298. After 24 h digital images were taken and neurite outgrowth was quantified. The comparisons of the neuroprotective effect of these three compounds are shown below.
  • the median count is estimated to be 95.5 in the absence of inhibitor and 23.5 when ceils are treated with inhibitor and PBS. This inhibited outcome is 0,225 of the uninhibited count (Relative Performance), and represents 0% protection from the adverse effects of the inhibitor (% Protection).
  • the collected results are graphically plotted in FIG 8A. Increasing concentrations of CNS-102 (green) and CNS- 101 (red) results in increases in median counts with a maximum protection of -40% for CNS- 102 and 27% for CNS-101 -
  • the graph shows that both CNS-102 and the isomer mixture CNS-101 , give protection in a range from 10 nM to 10000 nM.
  • the CNS-102 treatment consistently pro vides more protection than does CNS- 101 , even at the highest dose, which has reduced counts over the maximum seen at 1000 nM,
  • the graph shows that both CNS-102 and the all-ay isomer, CNS-103, give protection in a ratige from 10 nM to 10000 nM, with the peak at about 100 tiM, and CNS-102 providing greater protection at the optimal, concentration of 100 nM

Abstract

This invention relates to the use of geranylgeranyl acetone (GGA), its isomers, and GGA derivatives in a method for for treating a disease in a subject mediated in part by miRNA-378 or miRNA-711 increased activity comprising administering to the subject a therapeutically effective amount of 5-trans-GGA or a derivative thereof.

Description

METHODS FOR TREATING NEURON DAMAGE
FIELD OF THE INVENTION
[0001] This invention relates generally to methods for treating a disease in a subject mediated in part by mieroRRA (raiRNA)-induced regulation of mRNA translation (e.g., miRNA-378 or miRNA-71 1 increased activity) comprising administering to the subject a therapeutically effective amount of 5-irans-gemaylgeraiiyl acetone (GGA) or a derivative thereof. This invention also relates to treating patient subgroups who are predisposed to showing improved therapy with GGA or a GGA derivative.
STATE OF THE ART
|0002| Geranylgeranyl acetone is an acyclic isoprenoid compound with a retinoid skeleton that has been shown to induce expression of heat shock proteins in various tissue types. GGA is a known anti-ulcer drug used commercially and in clinical situations.
[0003] GGA has also been shown to exert cytoprotective effects on a variety of organs, such as the eye, brain, and heart (See for example Ishii Y., et al., Invest Ophthalmol Vis Set 2003; 44: 1982-92; Tanito M, et al, J Neurosci 2005; 25:2396-404; Fujiki M, et al, J Neurotrauma 2006; 23 : 1 164-78; Yasuda H, et al., Brain Res 2005; 1032: 176-82; Ooie '!'. et al., Circulation 2001 ; 104: 1837-43; and Suzuki S. et al. Kidney Int 2005; 67:2220-20). The effects and cytoprotective benefits of GGA in these settings is less understood as is the relationship of isomers of GGA to these cytoprotective benefits. Of particular interest, is the effect of GGA on extranuclear neurodegeneration both on an intracellular or extracellular basis.
[0004] Neurodegeneration is often the result of increased age, sporadic mutations, environmental exposures, disease, and/or protein aggregation in CNScelis: pathogenesis may also be related to a combination of the aforementioned factors. Neurodegenerative diseases are often characterized by a progressive neurodegeneration of tissues of the nervous system and a change/loss of functionality of the neurons themselves as well as surrounding glial and immune cells. There is a need in the art for therapeutic compositions and methods that can treat these damaged cells without exerting toxic side effects.
[0005] GGA and derivatives thereof are described in US Patent Application No.
13/815,831 , US Patent Application Publication No. US 2006/0052347, US Patent Ho. 5,453,524, PCT Publication No. WO 2012/026813, PCT publication No. WO 2012/031028 and PCT application No, PCT/US2012/027147, each of which are incorporated herein by reference in its entirety.
[0006] Currently, the only approved therapy for amyotrophic lateral sclerosis (A1..S) is riluzoie. An American Academy of Neurology practice guideline publ ished in 2009 recommended riluzoie as being safe and effective for slowing disease progression of ALS, though only to a modest degree. In a Phase 3 trial involving 959 patients observed for an average of 18 months, the survival rate without tracheostomy was 57% of treated versus 50% of control patients. Due to the progressive nature of ALS, whether with or without riluzoie, symptomatic and palliative treatments contribute significantly to clinical management of patients with ALS.
[ 1007] Over the last few decades, a large number of experimental therapies have been tried and have failed in clinical trials. Recently, several agents have demonstrated evidence of benefits in Phase 2 trials, but none has successfully completed Phase 3. Dexpramipexoie, not long ago, failed a huge Phase 3 trial and only few new drugs remain in advanced clinical trial programs including CK-2017357, a fast skeletal muscle troponin activator, arimoclomol, another heat shock protein inducer, ozanezumab, an antibody against NOGO and anakinra, an anti-inflammatory lL-1 antagonist. Even if these agents are approved for treatment of ALS in the next few years, unless they halt progression of ALS, there will continue to be a significant unmet clinical need.
SUMMARY OF THE INVENTION
[0008] This invention relates to pharmaceutical methods and uses of geranylgeranyl acetone (GGA) and GGA derivatives. One aspect relates to a method for treating a disease in a subject mediated in part by regulating mR A translation via modulation of miRNA including but not limited to miRNA-378 or miRNA-711 increased activity comprising administering to the subject a therapeutically effective amount of 5-trans-GGA or a derivative thereof,
[0009] In another aspect, this invention provides a method for treating damaged cells involved in newodegeneration exhibiting altered miRNA (e.g., increased miRNA-378 and/or miRNA-71 1 activity), said method comprising administering an effective amount of 5-trans- GGA or a derivative thereof as defined herein so as to decrease regulatory miRNA activity (such as miRNA-378 or miRNA-71 1 ) specifically in damaged neurons and/or neuron support ce!is proximate to damaged neurons, if! a preferred embodiment, the decrease miRNA correlates to an increase in HSP 70 protein,
fOOlOj in another aspect, this invention provides a method for treating damaged ceils in a subject in need thereof, wherein the damage to the cells arises from repression of mRNA translation (including but not limited to increased miRNA-378 and/or miR A-11 activity, said method comprising decreasing mi. NA-378 or miRNA-71 1 activity) specifically in damaged neurons and/or neuron support cells proximate to damaged neurons said method comprising administering a therapeutically effective amount of 5-trans-GGA or a derivative thereof to the subject, in a related embodiment, the decrease miRNA correlates to an increase in HSP 70 protein.
[0O11J in other embodiments the effective amount of GGA is from about 1 mg/kg/day to about 12 mg/kg/day, or from about 1 mg/kg day to about 5 mg kg day, or from about 6 mg/kg/day to about 12 mg/kg/day, or preferably, about 3 mg/kg/day, about 6 mg kg day, or about 12 mg/kg/day.
(0012] In one aspect, provided herein is a method of treadng a neural disease comprising administering GGA or a derivative thereof in an amount of about 1 - about 2 mg/kg sublingual!}' to a patient, preferably a human patient, in need thereof. In one embodiment, the amount is administered as a daily amount, in another embodiment, the amount is administered once daily. In another embodiment, the neural disease is amyotrophic lateral sclerosis (ALS). In another embodiment, the patient has difficulty to swallow or is incapable of swallowing an oral dosage form, such as a solid oral dosage form. In another embodiment, the patient shows modulation of one or more of families of heat shock proteins such as heat shock protein 70 (HSP70), HSP1 10, HSP90, HSP60, HSP40 and HSP27. In another embodiment, GGA is administered in accordance with this invention. In another embodiment, the GGA is trans GGA, preferably trans GGA which is free of or substantially free of as GGA,
[0013] In one aspect, pro vided herein is a method of treating a damaged cell having altered miRNA such as increased miRNA-378 and/or miRNA-71 1 activity, the method comprising: contacting the damaged neuron with a therapeutically effective amount of 5-trans-GGA or a derivative thereof, and if miRNA-378 and/or miRNA-71 1 activity decreases in the contacted neuron, continuing to contact the damaged neurons with a therapeutically effective amount of 5-trans-GGA or a derivative thereof. In one embodiments, the damaged neuron is in a subject suffering from a neurodegenerative disease. In one embodiments, the neurodegenerative disease is ALS, Alzheimer's Disease (AD), or Parkinson's Disease (PD). In one embodiments, the 5-trans-GGA or a derivative thereof is administered sublingual!}' to the subject, in one embodiments, the damaged neuron has reduced HSP activity.
(001 j in one aspect, provided herein is a method of treating a damaged neuron having reduced HSP activity, the method comprising: contacting the damaged neuron with a therapeutically effective amount of 5-trans-GGA or a derivative thereof, and if HSP activity increases in the contacted neuron, continuing to contact the damaged neurons with a therapeutically effective amount of 5-trans-GGA or a derivative thereof, in one embodiment, the damaged neuron is in a subject suffering from a neurodegenerative disease. In one embodiment, the neurodegenerative disease is ALS, Alzheimer's Disease (AD), or Parkinson's Disease (PD). In one embodiment, the 5-trans-GGA or a derivative thereof is administered sublmgually. In one embodiment, the amount is administered as a daily amount. In one embodiment, the amount is administered once daily. It; one embodiment, the GGA or the derivative thereof is GGA, In one embodiment, the GGA is irons GGA free of or substantially free of cis GGA.
[0015] In one aspect, provided herein Is a method of modulating miRNA-378 or miRNA- 71 1 activity in a damaged neuron comprising contacting the damaged neuron with an effective amount of 5-trans-GGA or a derivative thereof. In one embodiment, the damaged neron is damaged at least in pari by ALS, Alzheimer's Disease (AD), or Parkinson's Disease (PD).
(0016] In one aspect, provided herein is a method of treating a damaged neuron comprising causing neuro-inflammation in the damaged neuron, wherein the neuroinflammation results at least in part from contacting the damaged cell with an effective amount of 5-trans-GGA or a derivative thereof.
[0017] In one aspect, provided herein is a method of treating a damaged neuron comprising increasing HSP acti ity in the damaged neuron, wherein the increasing HSP activity results at least in part from contacting the damaged cell with an effective amount of 5-trans-GGA or a derivative thereof.
[0018} In one aspect, provided herein is a a method of treating a damaged neuron having reduced GRP78 activity, the method comprising: contacting the damaged neuron with a therapeutically effective amount of 5-trans-GGA or a derivative thereof, and if GRP78 activity increases in the contacted neuron, continuing to contact the damaged neurons with a therapeutically effective amount of 5-trans-GGA or a derivative thereof.
[0019] In one aspect, provided herein is a method of treating a damaged neuron comprising increasing G .P78 activity in the damaged neuron, wherein the increasing GRP78activity results at least in part from contacting the damaged cell with an effective amount of 5-trans- GGA or a derivative thereof.
[0020] In another aspect, provided herein is a pharmaceutically acceptable composition, preferably in a unit dosage form, comprising about 50 - about 200 mg of GGA or a derivative thereof and at least one pharmaceutically acceptable excipieut. In one embodiment, the pharmaceutically acceptable composition is for sublingual administration. Pharmaceutically acceptable exciptents, including those suitable for sublingual delivery, are known to the skilled artisan for use in accordance with this invention. In another embodiment, the pharmaceutically acceptable composition comprises GGA. in another embodiment, the GGA is trans GGA, preferably trans GGA which is free of or substantially free of as GGA.
[0021] In one aspect, provided herein is a method of treating a neural disease comprising administering geranylgeranyl acetone (GGA) or a derivative thereof in an amount of about 1 - aboui 2 mg kg sublingual!}' to a human patient in need thereof. In one embodiment, the amount is administered as a daily amount. In one embodiment, the amount is administered once daily, in one embodiment, the neural disease is amyotrophic lateral sclerosis (ALS). In one embodiment, the GGA or the derivative thereof is GGA. in one embodiment, the GGA is tram GGA free of or substantially free oicis GGA. In one aspectt, provided herein is a pharmaceutically acceptable composition comprising about 50 - about 200 mg of GGA or a derivative thereof and at least one pharmaceutically acceptable excipient. In one
embodiment, the pharmaceutically acceptable composition is for sublingual administration. In one embodiment, the GGA or the derivative thereof is GGA. In one embodiment, the GGA is trans: GG A, free of or substantially free of cis GGA.
BRIEF DESCRIPTION OF THE DRAWINGS
[00221 FIG. 1 shows the time course of body weights of test animals.
[0023] FIG. 2 shows imputed clinical scores of in vivo tests,
[0024] FIG. 3 shows imputed neuroscores of in vivo tests. [0025] FIG. 4 shows a graph of phamiacokinetic data for CNS-102 administered to rats intravenously (see Example 26 for details).
[Θ026) FIG. 5 shows a graph of pharmacokinetic data for CNS-102 administered to rats orally (see Example 25 for details).
[00271 FIG. 6 shows a graph of total concentration of a first formulation of CNS-102 over time in brain and in plasma following administration to rats.
[00281 FIG. 7 shows a graph of total concentration of a second iorinuiation of CNS-102 over Lime in brain and in plasma following administration to rats (see Example 27 for details).
(0029} FIG. 8A illustrates the protection of cells by CNS-102 and CNS-101 in the presence ofGGTL
[0030] FIG. 8B illustrates the protection of cells by CNS- 102 and CNS- 103 in the presence of GGTL
[0031] FIG. 8C illustrates neurite outgrowth ratio for CMS- 102/CNS- 101 versus Log, 0 concentration.
[0032] FIG. 9 illustrates FISP expression in rat brain tissue after oral administration of CNS- 102.
[0033] FIG 10 illustrates concentration dependence of neuroprotection by CNS- 102.
[0034] PIG 11. illustrates survival plot, for drug treatment groups in SOD1 mice.
[0035] FIG. 12 illustrates comparative neurological motor function outcomes of SOD 1 mice.
[0036] FIG. 13 shows the HSP 70 expression (red) in CNSI 02/PBS Treated group with an objective lens of 4X.
[0037] FIG 14. A, Relative HSP70 mRNA expression levels in rat PBMCs Shrs post- dosing. Values are normalized to HSP70 mK A expression in PBMCs from vehicle treated rats. B. Dose-linearity plot for CNS-102 plasma exposure by AUG and Cn)8x in connection with data from A.
[0038] FIG. 15 Shows a schematic timeline of KA model study.
[0039] FIG 16. Correlation of mutant human SOD! copy numbers and survival 'in SOD1 mice. [0040] FIG 17 Shows Kaplan Meier survival curves. Analysis by PROC LIFETEST, SAS, gave 133.0 median, 126.7.5 mean, (N=16; CNS 102) vs. 125.0 median, 1 17.875 mean ( = 16; Vehicle), with a log-rank p = 0.015.
[0041) FIG 18 Shows daily average weight for treatment groups. [0042) FIG 19 Shows daily average body weighs from day 106 to end.
[0043] FIG 20 Shows the average neuroscore after treatment.
{0044] FIG 21 Shows average neuroscore vs. time.
[0045] FIG 22. Average 4-paw grip strength for surviving animals.
(0046] FIG 23. CMS 102 in the kainic acid rat experimental design. [0047] FIG 24, CNS102 enhances hippocampal HSP70 and GRP78.
(0048} FIG 25. CNS102 Dose-response of HSP related proteins in 24 h treated 2A cells by Western Blot,
[0049] FIG. 26 Shows dose-response in N2A Ceils Transfected with a 5'UTR. Luclferase Construct. {0050] FIG 27. CNS102 promotes prenylation and r.eurite outgrowth in the N2A model [005! ] FIG 28. Expression of microglia marker in rat hippocampus by western blot.
[0052 J FIG 29. Footprints and Quantitative Analysis of Stride Length from CatWalk Behavioral Test ai P127. FL: front left; FR: front right; HL: hind left; HR: hind right; AB: abdomen. [0053] FIG 30 graphically shows run time on catwalk. [0054] FIG 31 Shows mouse tail hang result.
[0055] FIG 32. Neuronal damage in the Hippocampus of Kainic Acid Treated Rats.
[0056] FIG 33. PK Parameters for C S 102 Administered IV and PO. [0057] FIG. 34 Compares P K f o r sublingual versus oral gavage. |0058] FIG. 35 Shows PK of CNS 102 repeat dose and brain penetration.
I [O0S9J FIG. 36 Shows a CNS102 dosing paradigm.
DETAILED DESCRIPTION
|OO60} it is to be understood that this invention is not limited to particular embodiments described. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.
|00f>ll it must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an excipient" includes a plurality of exeipients.
Definitions
[00621 Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein the foiiowing terms have the following meanings.
[0063] As used herein, the term "comprising" or "comprises" is intended to mean that the compositions and methods include the recited elements, but not excluding others.
"Consisting essentially of when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination for the stated purpose, Thus, a composition consisting essentially of the elements as defined herein would not exclude other materials or steps that do not materially affect the basic and novel characteristic(s) of the claimed invention. "Consisting of shall mean excluding more than trace elements of other ingredients and substantial method steps. Embodiments defined by each of these transition terms are within the scope of this invention.
[0064] The term "about" when used before a numerical designation, e.g., temperature, time, amount, and concentration, including range, indicates approximations which may vary by ( ) or ( - ) 10 %, 5 %, or 1 %.
[0065) The term "neuroprotective" refers to reduced toxicity of neurons as measured in vitro in assays where neurons susceptible to degradation are protected against degradation as compared to control. Neuroprotective effects may also be evaluated in vivo by counting neurons in histology sections.
[0066] The term "neuron'' or "neurons" refers to all electrically excitable cells that make up the central and peripheral nervous system. The neurons may be cells within the body of an animal or ceils cultured outside the body of an animal. The term '"neuron1" or "neurons" also refers to established or primary tissue culture cell lines that are derived from neural ceils from a mammal or tissue culture cell lines that are made to differentiate into neurons. "Neuron" or "neurons" also refers to any of the above types of cells that have also been 'modified to express a particular protein either extrachromosomally or intrachromosomally. "Neuron" or "'neurons'' also refers to transformed neurons such as neuroblastoma ceils. Examples of specific types of neurons include Basket ceils, Betz cells. Medium spiny neurons, Purkinje cells, Pyramidal cells, Renshaw cells, Granule cells, and Anterior horn cells.
(0067] The term "neuron support cells" refers to non-neuronal cells of the nervous system such as glial cells, astrocytes, and support cells within the brain such as glia. Other examples of glial cells include microglia, and microglia such as oligodendrocytes, ependymai cells, radial glia, Schwann cells, satellite cells, and enteric glial cells.
(0068] As used herein, the term '"ALS" refers to amyotrophic lateral sclerosis disease.
|0069j As used herein, the term "AD" refers to Alzheimer's disease.
(0070] As used herein, the term "treatment" or "treating" means any treatment of a disease or condition in a patient, including one or more of:
• preventing or protecting against the disease or condition, that is, causing the clinical symptoms not to develop, for example, in a subject at risk of suffering from such a disease or condition, thereby substantially averting onset of the disease or condition; * inhibiting the disease or condition, that is, arresting or suppressing the development of clinical symptoms; and/or
* relieving the disease or condition that is, causing the regression of clinical symptoms.
[0073 j The terms "neurodegenerative disease" or "neural disease" refer to diseases thai compromise the cell viability of neurons. Examples of neurodegenerative diseases include but are not limited to ALS, AD, Parkinson's Disease, multiple sclerosis, and prion diseases such as Kuru, Creutzfe tdt-Jakob disease, Fatal familial insomnia, and Gerstmann-Straussler- Scheinker syndrome. Neurodegenerative diseases can be recapitulated in vitro in tissue culture cells. For example, AD can be modeled in vitro by adding pre-aggregated p-amyloid peptide to the cells. ALS can be modeled by depleting an ALS disease-related protein, TDP- 43. Neurodegenerative disease can also be modeled in vitro by creating protein aggregates through providing toxic stress to the cell One way this can be achieved is by mixing dopamine with neurons such as neuroblastoma ceils. These Neurodegenerative diseases can also be recapitulated in vivo in mouse models. A transgenic mouse that expresses a mutant Sodl protein has similar pathology to humans with ALS. Similarly, a transgenic mouse that overexpresses APP has similar pathology to humans with AD.
|0 72] The term "miR A-378* refers to a miRNA with die following: an official symbol MIR378A (Gene ID 494327, GenBank Accession No: NC 000005.9) for human; and an official symbol Mir378 (Gene ID 723889, GenBank Accession No: NR .029879) for Mus musculus. Each of the sequences associated with the GeneBank and Gene ID Nos, are herein incorporated by reference in their entirety for all purposes.
[0073] The term "miRNA-71 H refers to a miRNA with the following: an official symbol IR7N (Gene ID 1003 13843, GenBank Accession No: NC_ 000003.1 1) for human; and an official symbol Mir71 1 (Gene ID 751536, GenBank Accession No: NC_000075.6) for Mus musculus. Each of the sequences associated with the GeneBank and Gene ID Nos. are herein incorporated by reference in their entirety for all purposes.
(00741 The term "HSP 70" refers to a family of ubiquitously expressed heat shock proteins. Proteins with simitar structure exist in virtually all living organisms. The Hsp70s are an important pan of the cell's machinery for protein folding, and help to protect ceils from stress. The protein and mRNA sequence for HSP 70 is known in the art, For example, GenBank Accession numbers CAA28382.1 , P08107.5, and NP_ 005337.2 represent the protein sequence for HSP 70 and GenBank Accession numbers NM_005346.4, N _005345.5, NM 006644.2 represent the nucleotide sequence of the protein. Also included within the definition of HSP 70 is HSP70.3, which has an official symble of Hspaia, Gene ID 1 3740, and GenBank Accession No.: NC_000083.6. Each of these GenBank sequences are herein incorporated by reference in their entirety, (00 5] The term "reporter" refers to a gene or label that is attached to a regulatory sequence of another gene of interest and can be used as an indication of whether a certain gene has been taken up by or expressed in the cell or organism population. Non-limiting examples of reports include green fluorescent protein and derivatives (i.e. yellow fluorescent protein and red fluorescent protein), luciferase, dsRed, GUS gene, and the like.
(0076] The term "alky!" refers to substituted or unsubstituted, straight chain or branched aikyl groups with Cj-C|2, |-Q, and preferably CpC? carbon atoms. (0077} The term "aryi" refers to a 6 to 10 membered, preferably 6 membered ary! group. An aryi group may be substituted with 1 -5, preferably 1 -3, halo, alkyl, and/or -O-alkyS groups.
[0078] An effective amount of GGA is the amount of GGA required to produce a protective effect in vitro or in vivo. In some embodiments the effective amount in vitro is about from 0, 1 nM to about i mlvl In some embodiments the effective amount in vitro is from about 0.1 nM to about 0.5 nM or from about 0.5 nM to about 1.0 nM or from about 1.0 nM to about 5.0 nM or from about 5.0 nM to about 10 nM or from about 10 nM to about 50 nM or from about 50 nM to about i 00 nM or from about ] 00 nM to about 500 nM or from about 500 nM to about 1 mM, In some embodiments, the effective amount for an effect in vivo is about 0.1 rng to about 100 mg, or preferably, from about 1 mg to about 50 mg, or more preferably, from about 1 mg to about 25 mg per kg/day. In some other embodiments, the effective amount in vivo is from about 10 rag/kg/day to about 300 mg/kg/day, about 20 mg/kg/day to about 00 mg/kg/day, about 30 mg/kg/day to about 80 mg/kg/day, about 40 mg/kg/day to about 70 mg/kg/day, or about 50 mg/kg/day to about 60 mg/kg/day. In still some other embodiments, the effective amount in vivo is from about 100 mg kg/day to about 1000 mg/kg/day.
[0079J In yet another embodiment, a patient suffering from acute CNS injuiry would be administered iv with a bolus concentration of 5-trans-GGA or its derivate wherein the bolus concentration provides for a Tmax of less than about 7 hours. In further embodiments, the bolus concentration provides for a Tmex of less than about 10 hours, less than about 9 hours, less than aboul 8 hours, less than about 7 hours, less than about 6 hours, less than about 5 hours, less than about 4 hours, less than about 3 hours, or less than about 2 hours.
{0080} Routes of administration refers to the method for administering GGA to a mammal. Administration can be achieved by a variety of methods. These include but are not limited to subcutaneous, intravenous, transdermal, sublingual, or intraperitoneal injection or oral administration,
{00811 The term "halogenating" is denned as converting a hydroxy group to a halo group, The term "halo" or "halo group" refers to fluoro, chloro, bromo and iodo. {0082 J The term "stereoselectively" is defined as providing over 90% of the E isomer for the newly formed double bond. (0083) "Geometrical isomer"" or "geometrical, isomers" refer to compounds that differ in the geometry of one or more oiefime centers. or "(E)" refers to the tram orientation and "Z" or "(Z)" refers to the cis orientation.
[0084] Geranylgeranyl acetone (GGA) refers to a compound or the formula:
Figure imgf000014_0001
wherein compositions comprising the compound are mixtures of geometrical isomers of the compound.
[0085] The 5-trans isomer of geranylgeranyl acetone refers to a compound of the formula I :
13E 9E 5E
O
I
wherein the number 5 carbon atom is in the 5-trans (5E) configuration.
(0086] The 5-cis isomer of geranylgeranyl acetone refers to a compound of the formula II:
Figure imgf000014_0002
Π
wherein the number 5 carbon atom is in the 5-cis (5Z) configuration.
(0087 j Unless otherwise indicated, all numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations. Each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
(0088 j As used herein, Cn C.„ such as Ci-Cio, CpC*, or Cj-Q when used before a group refers to that group containing m to n carbon atoms.
(0089] The term "about" when used before a numerical designation, e.g., temperature, time, amount, and concentration, including range, indicates approximations which may vary by ( + ) or ( - ) l 0 %, 5 % or l %. [0090] The term "alkoxy" refers to -O-alk l.
[0091] The term "alkyl" refers to monovalent saturated aliphatic hydrocarbyl groups having from 1 to 10 carbon atoms (i.e., CVC;o alky!) or I to 6 carbon atoms (i.e., Cj-Ce alkyl), or i to 4 carbon atoms. This term includes, by way of example, linear and branched hydrocarbyl groups such as methyl (CH3-), ethyl (CH3C¾-), «-propyl (CH3CH2C¾-), isopropyl ((CH^CH-), Λ-butyl (CH3CH2CH2CH2-), isobuty] ((CH3)2CHCH2-), m-butyl
((CH3XCH3C¾)CH-), /-butyl ((CH3)jC-), n-pentyl (CH3CH2CH2CH2CHr), and neopentyl i(C¾)3CC¾-).
[0092] The term "aryl" refers to a monovalent, aromatic mono- or bicyclic ring having 6-10 ring carbon atoms. Examples of aryl include phenyl and naphthyl. The condensed ring may or may not be aromatic provided that the point of attachment is at an aromatic carbon atom. For example, and without limitation, the following is an aryl group:
Figure imgf000015_0001
[00 3| The term "-CO2H ester" refers to an ester formed between the -~CCbH group and an alcohol, preferably an aliphatic alcohol. A preferred example included -CCbR1', wherein RL is alky] or aryl group optionally substituted with an amino group.
[Θ094] "Co-crystal," or as sometimes referred to herein "co-precipitate" refers to a solid, preferably a crystalline solid, comprising GGA or a GGA derivative, and urea or thiourea, more preferably, where, the GGA or the GGA derivative reside within the urea or thiourea lattice, such as in channels formed by urea or thiourea.
[0095] "Complexed" refers to GGA or a GGA derivative bound by certain quantifiable intermolecular forces, non-limiting examples of which include hydroge bonding and Van- Der Waais' interactions, and also by etitropic effects.
(§096] The term "chiral moiety" refers to a moiety that is chiral. Such a moiety can possess one or more asymmetric centers. Preferably, the chiral moiety is enantiomerically enriched, and more preferably a single enantiomer. Non limiting examples of chiral moieties include chiral carboxylic acids, chiral amines, chiral amino acids, such as the naturally occurring amino acids, chiral alcohols including chiral steroids, and the likes. |θ©97] The term "cycloalkyP refers to a monovalent, preferably saturated, hydrocarbyl mono-, bi-, or tricyclic ring having 3-12 ring carbon atoms. While cycioalkyl. refers preferably to saturated hydrocarbyl rings, as used herein, it also includes rings containing 1 -2 carbon-carbon double bonds. Nonlimiting examples of cycioalkyl include cyclopropyl. cyclobuiy h eyclopent l, cyciohexyl, cycloheptyl, adamentyl, and the like. The condensed rings may or may not be non-aromatic hydrocarbyl rings provided that the point of attachment is at a cycioalkyl carbon atom, For example, and without limitation, the following is a cycioalkyl group:
Figure imgf000016_0001
[0098] The term "halo" refers to F, CI, Br, and/or I,
[0099] The term "heteroaryl" refers to a monovalent, aromatic mono-, bi-, or tricyclic ring having 2-14 ring carbon atoms and 1-6 ring heteroatoms selected preferably from N, O, S, and P and oxidized forms of N, S, and P, provided that the ring contains at least 5 ring atoms. Nonlimiting examples of heteroaryl include furan, imidazole, oxadtazole, oxazole, pyridine, quinoline, and the like. The condensed rings may or may not he a heteroatom containing aromatic ring provided that the point of attachment is a heteroaryl atom. For example, and without limitation, the following is a heteroaryl group:
Figure imgf000016_0002
10100] The term "heterocyclyl" or heterocycle refers to a non-aromatic, mono-, bi-, or tricyclic ring containing 2-10 ring carbon atoms and 1 -6 ring heteroatoms selected preferably from N, O, S, and P and oxidized forms of N, S, and P, provided that the ring contains at least 3 ring atoms. While heterocyclyl preferably refers to saturated ring systems, it also includes ring systems containing 1 -3 double bonds, provided that they ring is non-aromatic.
Nonlimiting examples of heterocyclyl include, azalactones. oxazoline, piperidinyl, piperazinyl, pyrrolidinyl, tetrahydroruranyl, and tetrahydropyrany!. The condensed rings may or may not contain a non-aromatic heteroaiorn containing ring provided that the point of attachment, is a heterocyclyl group. For example, and without limitation, the following is a heterocyclyl group:
Figure imgf000017_0001
[01011 The term "hydrolyzing" refers to breaking an RH-0-CO, Rh-0-CS-. or an R -0
SO2- moiety to an R* -OH, preferably by adding water across the broken bond. A hydrolyzing is performed using various methods well known to the skilled artisan, non limiting examples of which include acidic and basic hydrolysis.
{0102] The term "oxo" refers to a OO group, and to a substitution of 2 gemiria! hydrogen atoms with a C-0 group,
[1)103 j The term "pharmaceutically acceptable" refers to safe and non-toxic for in vivo, preferably, human administration.
(0104] The term "pharmaceutically acceptable salt" refers to a salt that is pharmaceutically acceptable.
[0105] The terra "salt" refers to an ionic compound formed between an acid and a base. When the compound provided herein contains an acidic functionality, such salts include, without limitation, alkai metal, alkaline earth metal, and ammonium salts. As used herein, ammonium salts include, salts containing protonated nitrogen bases and alkylated nitrogen bases. Exemplary, and non-limiting cations useful in pharmaceutically acceptable salts include Na. , Rb, Cs, H4, Ca, Ba, imidazoiium, and ammonium cations based on naturally occurring amino acids. When the compounds utilized herein contain basic funetmaly, such sails include, without limitation, salts of organic acids, such as caroboxylic acids and sulfonic acids, and mineral acids, such as hydrogen halides, sulfuric acid, phosphoric acid, and the likes. Exemplary and non-limiting anions useful in pharmaceutically acceptable satis include oxalate, maleate, acetate, propionate, succinate, tartrate, chloride, sulfate, bisalfate, mono-, di-, and tribasic phosphate, mesylate, tosylate, and the likes.
The term "substantially pure warn isomer" refers to a trans isomer that is by molar amount 95%, preferably 96%, more preferably 99%, and still more preferably 99.5% or more a trans isomer with the rest being the corresponding cis isomer,
[0106] "TramT in the context of GOA and GGA derivatives refer to the GGA scaffold as illustrated beiow:
Figure imgf000018_0001
wherein R'-R5 is defined herein and q is 0-2. As shown, each double bond is in a trans or E configuration. In contrast, a cls form of GGA or a GGA derivative will contain one or more of these bonds in a cis or Z configuration,
Methods
jO 107] This invention describes methods and compositions for treating diseases mediated, at least in pari, by miRNA-378 and/or miRNA-71 1 increased activity. The increased activity may be due to an increase in the production of the raiRNA or an increase in the effectiveness of the iriiRNA. The increase in activity may be direct or indirect. Accordingly, one aspect of the invention relates to a method for treating a disease in a subject mediated in part by niiRNA-378 or miRNA-7i l increased activity comprising administering to the subject a therapeutically effective amount of 5 -trans -GGA or a derivative thereof.
[0108] In another aspect, this invention provides a method for treating damaged neurons exhibiting increased miRNA-378 and/or miRNA-71 1 activity, said method comprising administering 5-trans-GGA or a derivative thereof in an amount effective to decrease miRNA-378 or miRNA-71 1 activity specifically in damaged neurons and/or neuron support cells proximate to damaged neurons.
{0109] in another aspect, this invention provides a method for treating damaged neurons in a subject in need thereof, wherein the damage to the neurons is associated with increased miRNA-378 and/or raiR A- 11 activity, said method comprising decreasing miRNA-378 or miRNA-7 i i activity specifically in damaged neurons and/or neuron support cells proximate to damaged neurons said method comprising administering a therapeutically effective amount of 5-trans-GGA or a derivative thereof to the subject.
[0110] It was discovered that administration of 5- trans GGA or a derivati ve thereof may work by decreasing the activity and/or levels of miRNA-378 and miRNA-71 1 specifically in damaged regions of the nervous system. This specific decrease provides for a therapeutic with decreased off-target effects, since it's activities to the specified miRNAs are limited to diseased and/or damaged ceils, [01.11] In .some embodiments, the decrease in the mlRNA-378 and/or miRNA-71 1 activity correlates to an increase in HSP 70 expression. In certain embodiments of the above aspects, the subject has a disease mediated, at least in part, by miRNA-378 and/or miRNA-71 1. In another embodiment, the damaged neurons have increased miRNA-378 and/or miRNA-71 1 activity.
[0112] in further embodiments of the above method aspects, the method comprises decreasing miRNA-378 and/or miRNA-71 1 activity in neurons or neuron support ceils. In a specific embodiment, the miRNA-378 and/or miRNA-711 activity is decreased in neuron support cells, in a related embodiment, the neuron support cells are glial cells.
[0113] in certain embodiments, the subject being treated according to the methods described herein is a subject with a neurodegenerative disease, Non-limiting examples of neurodegenerative diseases but are not limited to Alzheimer's disease, Parkinson's disease, multiple sclerosis, prion diseases such as Ktiru, Creutzfeltdt-Jakob disease. Fatal familial insomnia, and Gerstmann-Straussier-Scheinker syndrome, amyotrophic lateral sclerosis, or damage to the spina! cord. GGA and the 5-trans isomer of GGA are also contemplated to prevent neural death during epileptic seizure. It is contemplated that either some or all of these neurodegenerative diseases may be mediated, at least in part, by increased miRNA-378 and/or miRNA-711 activity. In other embodiments, the subject being treated is one that has experienced acute CNS injuries, including but not limited to, CNS injury associated with the occurrence of. for example, stroke, aneurism, surgery, arteriovenus malformation (AVM), radiation, vascular dementia, epileptic seizures, cerebral vasospasm, acute or traumatic brain injury, and hypoxia of the brain as a result of, for example, cardiopulmonary arrest or near drowning,
[0114] Oral CNS- 102 or ail trans GGA ( 12 mg/kg/day) prolonged survival in the superoxide dis nutase 1 (SODl) mouse model of amyotrophic lateral sclerosis (ALS). Furthermore, CNS- 102 improved the clinical and neurological scores, reaching statistical superiority to scores in vehicle treated mice at certain time points. In addition, systemic CNS- 102 dosing has also provided neuroprotection in the hippocampus against locally injected kainic acid. The benefit is prolonged with peak protection at 72 hours following a single oral dose. In vitro, CNS- 102 protects neutite extension of the neuroblastoma cells (Neuro2A) from toxicity caused by an inhibitor of geranylgeranyl transferase.
[0115] Studies were conducted to lest that CNS- 102 acts through HSP induction. These studies have focused on HSP70 and have been conducted in the murine neuroblastoma cells and in the rat hippocampus and cerebral cortex..
[0116] Applicant has demonstrated thai CNS 102, the all-trans isomer, is substantially more potent than teprenone (by over an order of magnitude) as a neuroprotective agent. with efficacy noted using a daily oral dose of 12 mg/kg. Typical al!ometric scaling predicts a human equivalent dose of 1 -2 mg kg, making CNS102 a potential therapeutic,
[0117j Results provided herein provides evidence that that CNS 102 elicits
neuroprotective responses specifically via pathways involved in upregulation of heat shock proteins (HSPs), neurite outgrowth, prenylation of small G-proteins, and microglial activation.
[0118] Given that a complex etiology underlies ALS, an integrative treatment approach may be more effective. Compounds acting on a single mechanism of action may not consistently demonstrate efficacy across multiple model systems, including the SOD1 G93A familial transgenic mouse line (SODl ). in such a scenario, models do not always translate well to patients in the clinic, particularly in the case of ALS in which an
overwhelming majority of the cases are idiopathic. In contrast. CNS 102 likely targets multiple relevant pathways. It has the capacity to affect protein quality control and immune responses within the CNS, thereby slowing disease progression and possibly promoting neurite outgrowth and regeneration. [0119] it has been demonstrated that CNS-102 up-regulates expression of HSP70 in addition to other selected HSPs including HSP40, and HSP90. This has been demonstrated in vitro, in the murine neuroblastoma ceil neuroZA system, and in vivo in the rat cerebral cortex and hippocampus. The expression of HSP70 was detectable through 96 hours in the cortex and hippocampus of rats receiving a single dose of 12 mg/kg of CNS- 102. Comparative in- vitro studies demonstrate that CNS- 102 is a more potent inducer of HSPs than the cis-isomer CNS- 103.
{0120] It has been observed that CNS- 102 increases the survival in the SODl mouse model, which in part may be due to a molecular chaperone effect. HSPs are recognized to function as ATP-dependent chaperones, assisting in the folding of newly synthesized proteins and the sequestering of misfoided proteins. Protein misfolding leading to secondary cellular damages is postulated to be a significant component of the pathophysiology of ALS. Furthermore, a high threshold for induction of the stress response is thought to contribute to increased vulnerability to the toxicity of protein aggrega tes in motor neurons. HSPs gene induction may improve protein refolding in ALS. consistent with the reported protein refolding and survival benefits in a spinal and bulbar muscular atrophy (SMBA) mouse model dosed with teprenone.
[0121] The neuroprotective action for CNS-102 may be attributed in past to improved cytoprotection through an elevated heat shock response, HSP70 has been reported to play an important role in stabilizing axons and dendrites and supporting the growth cone. Therefore it may prevent the loss of motor neurons by its chaperonal activity on the cytoskeleton, supporting the development of a new therapeutic based on CNS-102 to treat neurodegenerative diseases.
Biomarker Development
10122 j From a clinical perspective, the peripheral, blood based biomarkers are preferable to matrices that are more difficult to obtain such as cerebrospinal fluid. The major source of HSPs in biood are peripheral blood monocytes (PBMCs), but HSPs can also be detected in serum. Initially PBMC derived HSPs were focused on, because the neuroprotective function is attributed to intracellular HSP70, while extracellular HSP70 is considered a proinflammatory mediator. In a first step the isolation procedure of PBMCs from blood and subsequent extraction of the total proteins was standardized. Next ELiSA kits for intracellular HSP70 and optimized assay conditions were identified. A yield of approximately 7 million PBMCs from a rat terminal bleed provides sufficient protein for an HSP70 ELISA assay. Spike and recovery experiments with PBMC lysates yielded 90-120% recovery of HSP70 indicating robust assay performance. An analysis of baseline HSP70 levels in PBMCs from human healthy volunteers showed HSP70 concentrations of l--3ng/mg total protein.
[0123| Since CNS-102 appears to regulate multiple HSPs qPC primers for the most important HSP family members for a broader characterization of the expression patterns of HSPs at the mRNA level were developed.
Figure imgf000022_0001
Primers for mouse, rat and human HSPs are tabuated. Left column shows new HSP terminology; right column shows old HS? terminology.
[0124] in a P /PD experiment over a wide dose-range it was demonstrated that a dose- dependent increase in H8P70 mRNA expression in rat PBMCs occurs 8hrs after a single dose of CNS-102 (Figure 14 A). This correlates with a dose dependent increase ofCNS-102 plasma exposure both by AUG and Cniax (Figure 14B), Relationships at substantially lower dose levels in sub-chronic repeat dose paradigms, consistent with the above presented efficacy data are contemplated.
[0125] The initial HSP biomarker development efforts demonstrate the basic feasibility of measuring HSP7Q and related HSPs in rat and human PBMCs.
Heat Shock Protein (HSP) biomarker development for preclinical am! clinical studies.
[0126] Since HSP70 acts in concert with a number of other HSP chaperones to mediate its beneficial effects on proteostasis, it is desirable to monitor the protein levels of multiple HSP family members. Due to the associated cost and the amount of protein required from a sample, it is challenging to accomplish this with multiple ELISA assays. However, proteomics platforms offer technology to multiplex the measurement of candidate biomarker proteins, even from multiple organisms, in one 'panel' by a method termed Multiple Reaction Monitoring or MRM. This obviates the need to develop expensive individual assays for the multiple proteins, as well as going from rats into human subjects. As MRM validation depends upon peptides isolated in the gas phase, a carefully selected set of tryptic peptides can be used as quantitative surrogates for a protein. Target biomarker proteins may be combined in a single assay without the need to design immune-affinity agents, or concerns about cross reactivity between reagents or interference from autoantibodies, This unbiased inclusion of markers based solely on their differentiating capacity, the wide dynamic range arid reproducibility and sensitivity are ail advantages in the MRM platform. Additional target biomarker proteins from existing biomarker panels can easily be included in the assay.
Development and basic qualification of a multiplexed I ISP MRM assay for rat and human PBMCs
[0127] The development of an MRM assay covering up to 8 heat shock proteins each (including HSP70, 1 10, 90, 60, 40 and 27) in human and rat PBMCs in a single panel is contemplated. Up to 20 other protein markers from existing panels may be added to the multiplex assay as potential mechanistic and disease biomarkers. qPCR primers developed for mRNA analysis are contemplated to be qualified in parallel.
[0128) Method; For the MRM assay, at least 5 peptides per protein marker unique to rat and human respectively, are chosen based on available databases of observed peptides. These are synthesized and their transitions characterized. A multiplexed assay with the ability to measure detectable markers are developed using these synthetic peptides and can be applied to both species/sample types. Assay development is followed by assay verification in up to 10 representative samples. The accuracy, precision and reproducibility of the MRM assays are determined from a pooled sample of rat PBMC lysates that are aiiquoted and spiked with I ISP standards. Three of these aliquots are analyzed in 3 separate runs. Accuracy is expressed as % deviation for the difference of the average HSP concentrations from the nominal HSP concentrations. Precision is expressed as %CV within an anaiylicai run and reproducibility is expressed as %CY between analytical runs,
|0129) To characterize stressors during sample handling, a number of experiments comparing HSP levels in the PBMC lysate from a pooled rat blood sample are contemplated by varying the following parameters: (i) time from sample collection to PBMC extraction, (ii) time from PBMC extraction to freezing of PBMC lysate, (iii) temperature change following sample collection until PBMC extraction, including heating to 42°C, keeping at 37°C, cooling to room temperature (20°C), and keeping on ice; (iv) mechanical stimulus following sample collection and prior to PBMC extraction including vortexing, vigorous manual shaking, carrying sample in a container by walking a defined distance and no movement. Any conditions causing more than a ± 15% change in HSP70 levels are defined as parameters that need to be maintained constant in the sample handling protocol. Sample handling experiments may be performed first by qPCR. before selecting the most critical sample handling conditions to be analyzed by MR.M. [0130] We also will determine the benchtop stability at room temperature (up to 8hrs) and the frozen stability (up to 2 months) of HSPs in spiked PBMC iysate samples. HSP stability is considered acceptable under conditions that lead to a loss of less than 15% of the nominal HSP concentration. The stability data are documented in the sample handling protocol,
C aracterization of biologkai variability of HSP7 in rat and human PBMCs
[0131 | It is contemplated to delect a 20% change of HSPs. as this is a typical change we observed with C S-102 treatment in in vitro and in vivo experiments. The intra- and inter- individual variability from these experiments will inform the detectable effect size, required sample size and/or expected power of future preclinical and clinical studies.
[0132] Method: The inter-individual variability is assessed for the MRM analysis of HSP proteins and the qPCR analysis of HSP famil .members in 3 male and 3 female rats as well as in human PBMC samples from 3 males and 3 females.
[0133] The infra-individual variability in humans is assessed from the same healthy volunteers, who provided samples for inter-individual assessment. Samples are collected two weeks after the first set of samples. This is the same duration as the planned Multiple Ascending Dose (MAD) Phase 1 study. The infra-individual variability assessment for HSP protein by MRM may not be feasible in rats, because a whole terminal blood sample may be required to obtain sufficient PBMC lysate for an MRM assay. The infra-individual variability in rats is determined at a minimum HSP rn NA with sample collection 1 week apart to reflect the duration of the kainic acid study described in Section 3.5.3. The intra- and inter- individual variability is expressed as %CV.
[0134] To compare the baseline HSP expression levels in PBMCs of healthy volunteers with ALS patients, we intend to obtain PBMC samples from approximately 3 ALS patients. Patient samples with medical history can be obtained from commercial tissue sources (e.g. Folio) or academic sample repositories such as the one maintained by the North East ALS (NEALS) Consortium.
Correlation of centra!, neuroprotective activity of CNS-102 with peripheral HSP biomarker response
[0135] As a surrogate animal model the use of the kainic acid (KA) model in rat is contemplated. Stereotactic injection of KA into the hippocampus leads to excitotoxic death of neurons, one of the mechanisms leading to motor neuron death in ALS, Furthermore, we demonstrated that CNS- 102 is efficacious in the KA model in the same dose range as in the SOD 1 model. A repeat dose paradigm will mimic chronic dose administration in the clinic and a 72 hour survival period following A injection encompasses the inflammatory response, including macrophage invasion into the CNS, which is also a component of ALS pathology,
[0136] Method: Rats are randomly assigned to treatment groups. Group 1 acts as a negative, sham control, Group 2 will show lesion size and biomarker response to KA insult without drug treatment, Group 3 will characterize the biomarker response elicited by the drug in combination with sham surgery and the remaining groups will characterize the dose response to CNS- 102. Dosing staff, the surgeon and the staff analyzing lesion areas are blinded to treatment assignments. Rats are dosed sublingually once daily for 6 days. Dose levels are bracketed around the plasma exposure corresponding to the 12mg kg oral dose, which was efficacious in the SOD1 and KA model, On day 0, following t e fourth dose, rats will undergo stereotactic surgery and receive bilateral injections of saline or KA according to treatment group assignments. 72 hours following the surgery, rats are exsanguinated and perfused with PBS. PBMCs are isolated from whole blood. Brains are collected and one hemisphere is immersion fixed in 4% paraformaldehyde and dehydrated in sucrose in preparation for cryo-sectionmg. From the second hemisphere the hippocampus is dissected and stored in RNA later for extraction of mRNA. Following the last dose, blood samples are collected from satellite PK animals for a 24 hour plasma concentration time profile. In addition terminal bleeds are collected for biomarker analysis from Group 7 animals on Day -3 to establish baseline HSP expression. A schematic timeline of the study is shown in Figure
15
Endpoirm
[0137! Frozen sections are stained with cresyl violet and the relative size of the lesion area in the CA3 region of the hippocampus is measured in a blinded fashion on digital microscope images in ImageJ according to an established protocol.
Experimental groups of KA model study
Croup # Rats" Drug treatment : Surgery am le collection
1 ' 9 M ; Vehicle : Saline Biomarker only
2 9 M Vehicle Kainic acid Brain & biomarker
3. 9+3* M ; CNS-lG2 high ; Saline Biomarker only
4 9+3* M 1 CNS- ! 02 low I Kainic acid Brain & biomarker 5 9+3* M CNS-102 mid Kainic acid ; Brain & biomarker
6 9+3* M CNS-102 high Kainic acid i Brain & biomarker
7 9 M None None Biomarfcers at baseline
"The group size may change depending on power calculations, * Satellite P animals; M: males
{01381 Terminal blood samples are analyzed for MRM target protein content in PB Cs and relative expression levels of HSP mRNA with established primers. mR A will also be extracted from the hippocampus of the second brain hemisphere and the relative expression pattern of HSPs analyzed with the same primer set. Plasma concentration of CNS-102 in the PK satellite animal samples are analyzed by Pharmaout laboratories. We plan to obtain the following key results from this study: (i) CNS- 102 plasma exposure at maximum and half maximum efficacy, i.e. neuroprotective effect of CNS- 1 02 in the CA3 region of the hippocampus, (ii) Fold-change of MRM-based HSP protein expression in PBMCs compared to baseline and compared to vehicle at maximum and half-maximum efficacy, (iii) Dose- dependent HSP mRNA expression profile differences between brain and PBMCs, and their correlation with drag exposure and efficacy. Overlap in HSP expression between CMS and PBMCs may be used to identify biomarker candidates for neuroprotective activity of CNS- 102, The non-HSP MRM target proteins are analyzed, in an exploratory manner for their potential as biomarkers for neuroprotection or disease pathology,
Screening Assays
[0139] Other aspects of the invention relate to assays and methods for identifying compounds that regulate HSP 70, said assay or methodcomprising: expressing, in a pool of cells, a nucleotide construct comprising the 3'UTR of HSP 70 and a reporter; adding a test compound to a first portion of the pool of cells; and determining the output level of the reporter; wherein an increase in the output level of the reporter in the first portion of cells indicates a compound that increases HSP 70 protein levels and a decrease in the output level of the reporter in the first portion of cells indicates a compound that decreases HSP 70 protein levels.
[0140] A further aspect provides for an assay or method for identifying compounds thai regulate HSP 70, aid assay or method comprising: expressing, in a pool of ceils, a nucleotide construct comprising the 3'UTR of HSP 70 ana a reporter; adding a test compound to a first portion of the pool of cells; adding a control to a second portion of the pool of ceils; and determining the output level of the reporter; wherein a higher output level of the reporter in the first portion of ceils compared to the second portion of ceils indicates a compound thai increases HSP 70 protein levels and a lower output level of the reporter in the first portion of cells compared to the second portion of cells indicates a compound that decreases HSP 70 protein levels.
[0141 j In certain embodiments, the nucleotide further comprises a promoter region for directing expression, of the 3'UTR. As used herein, the term "promoter" refers to a nucleic acid sequence sufficient to direct transcription of a gene, Also included in the invention are those promoter elements which are sufficient to render promoter dependent gene expression controllable for cell type specific, tissue specific or inducible by external signals or agents. Suitable promoters include, for example, cytomegalovirus, EFl-l ct, SV40, and PGK.
[0142] The 3'UTR of HSP 70 refers to the untranslated region on the 3 " end of the HSP 70 gene. In certain embodiments, the 3' UTR comprises about 2000, about 1800, about 1600, about 1400, about i 300, or about 1253 bp from the end of the protein coding sequence.
[0343] The term "control" can refer to a non-active compound, a mock solution (a solution with no compound), and the like,
[01441 The term "determining the output level" refers to quantitatively or qualitatively measuring the output of the reporter used in the assay. For example, one suitable reporter system is luciferase. Firefly luciferase catalyzes the oxidative carboxylation of luciferin, a reaction with the highest efficiency of any known bioluminescence reaction. At the optimal reaction pl l of 7.8. light emission peaks at 562 nm. This form of light emission yields a very sensitive non-radioactive assay. For measurement of expressed luciferase activity in vitro, luciferase can be extracted from transfected cells through eel! lysis. A typical firefly luciferase assay can then be carried out in an assay cuvette, ATP, Mg2+ and buffer are added to the iysate either separately or as a preform uiated solution. The luminescent reaction is then triggered by an injection of luciferin, and die emitted light is recorded.
[0145| Other suitable reporters are known in the art and include, for example, green fluorescent protein, red fluorescent protein, dsRed, yellow fluorescent protein, luciferin, arid aequorin. In a specific embodiment, the reporter is luciferase.
[0146] In other embodiments, of the assay aspects described herein, the cells are exposed to damage prior to addition of the test compound and/or control. Exposure to damage, especially neurons, may increase the sensitivity of the assay and/or allow for evaluation of the specificity of the assay.
[0147] Suitable vectors for expressing the 3'UT and reporter are well-known in the art, and include vectors capable of expressing a polynucleotide operative] linked to a regulatory element, such as a promoter region and/or an enhancer thai is capable of regulating expression of such DNA. Thus, an expression vector refers to a recombinant DNA or UNA construct, such as a p!asmtd, a phage, recombinant virus or other vector that, upon introduction into an appropriate host cell, results in expression of the inserted DNA, Appropriate expression vectors include those that repHcable in eukaryotic cells and/or prokaryotic cells and those that remain episomai or those which integrate into the host ce!l genome.
{0148) As used herein, the term "vector" refers to a non-chromosonia! nucleic acid comprising an intact rep] icon such that the vector may be replicated when placed within a cell, for example by a process of transformation. Vectors may be viral or non-viral. Viral vectors include retroviruses, adenoviruses, herpesvirus, baecuJoviruses. modified bacculoviruses, papovirus, or otherwise modified naturally occurring viruses. Exemplary non- iral vectors for delivering nucleic acid include naked DNA; DNA complexed with cationic lipids, alone or in combination with cationic polymers; anionic and cationic liposomes; DKA-protein complexes and particles comprising DNA condensed with cationic polymers such as heterogeneous polylysine, defmed-iength oligopeptides, and polyethylene imine, in some cases contained in liposomes: and the use of ternary complexes comprising a virus and polylysine-DNA.
[0149] Non-viral vectors may include a plasmid that comprises a heterologous polynucleotide capable of being delivered to a target ceil, either in vitro, in vivo or ex-vivo. The heterologous pol ynucleotide can comprise a sequence of interest and can be operably linked to one or more regulatory elements and may control the transcription of the nucleic acid sequence of interest, As used herein, a vector need not be capable of replication in the ultimate target cell or subject. The terra vector may include expression vector and cloning vector.
[0150] A "viral vector*" is defined as a recombinant ly produced virus or viral particle that comprises a polynucleotide to be delivered into a host cell, either in vivo, ex vivo or in vitro. Examples of viral vectors include retroviral vectors, adenovirus vectors, adeno-associated virus vectors, alphavirus vectors and the like. Alphavirus vectors, such as Semliki Forest virus-based vectors and Sindbis virus-based vectors, have also been developed for use in gene therapy and immunotherapy. In aspects where gene transfer is mediated by a retroviral vector, a vector construct refers to the polynucleotide comprising the retroviral genome or pari thereof, and a therapeutic gene. As used herein, "retroviral mediated gene transfer" or "retroviral transduction" carries the same meaning and refers to the process by which a gene or nucleic acid sequences arc stably transferred into the host ceil by virtue of the virus entering the ceil and integrating its genome into the host celt genome. The virus can enter the host cell via its normal mechanism of infection or be modi ied such thai it binds to a different host cell surface receptor or ligand to enter the cell. As used herein, retroviral vector refers to a viral particle capable of introducing exogenous nucleic acid into a cell through a viral or viral-like entry mechanism.
[01.51] A further aspect of the invention relates to a compound identified by an assay as described herein,
[0152] Also provided is a kit comprising a poo! of cells expressing a nucleotide construct comprising the 3 *UTR of HSP 70 and a reporter; a means for detecting the output of the reporter; and instructions for use. in one embodiment, the ceils are neurons. In a further embodiment, die reporter is selected from the group consisting of fluorescent protein, red fluorescent protein, dsRed, yellow fluorescent protein, luciferin, and aequorin and luciferase. Compo srids
Described in the following are compounds useful in the methods and assays of the disclosure.
GGA
(Ill 53] This invention relates to compounds and pharmaceutical compositions of isomers of geranylgeranyl acetone. In certain aspects, this invention relates to a synthetic 5-trans isomer compound of forrnula I:
Figure imgf000029_0001
wherein I is at least 80% in the 5E, 9E, 1 3E configuration. In some embodiments, the in vention provides for a compound of formula I wherein I is at least 85%, or at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or at least 99,5%, or at least 99.9% in the 5E. 9E» 13E configuration. In some embodiments the invention for the compound of formula 1 does not contain any of the cis-isomer of GGA.
[0154] Another aspect of this invention relates to a synthetic 5-cis isomer compound of formula SI:
Figure imgf000030_0001
II
wherein II is at least 75% in the 5Z, 9E, 1 E configuration, in certain embodiments, the invention provides for a compound of formula II wherein ΪΙ is at least 80% in the 5E, 9E, 13E configuration, or alternatively, at least 85%, or at least 90%, or at least 95%, or at least 96%r or at least 97%, or at least 98%, or at least 99%, or at least 99.5%, or at least 99.9% in the 5E, 9E, Ϊ3Ε configuration. In some embodiments of the invention, the compound of formula II does not contain any of the trans-isomer of GGA.
[0155] The configuration of compounds can be determined by methods known to those skilled in the art such as chiroptical spectroscopy and nuclear magnetic resonance spectroscopy.
{0156] The data contained in the examples herewith demonstrate at low concentrations the trans-isomer of GGA is pharmacologically active and shows a dose-dependent relationship. In corstrast, the cis-isomer of GGA does not demonstrate a dose dependent relationship and is deemed to be at best of minimal activity.
GGA derivatives
[0157] GGA derivatives useful in this invention include those described in PC f publication no. WO 2012/031028 and PCT application no. PCT/US2012/027147, each of which are incorporated herein by reference in its entirety. These arid other GGA derivatives utilized herein are structurally shown below.
{01581 In one aspect, the GGA derivative utilized herein is of Formula I:
Figure imgf000030_0002
(!) wherein
n is 1 or 2;
each R1 and R" are independently CVQ alkyi. or R' and together with the carbon atom they are attached to ibrrn a Cs-C6 cvcloalkvi ring optionally substituted with 1 -3 C i -Q, alkyi groups; each of R , R4, and R5 independently are hydrogen or Ci-C6 alkyi;
Figure imgf000031_0001
Q2 is hydrogen, R6, -0-Rc s -NR'R8. or is a chiral moiety;
R6 is:
Cj-Cf, alkyi, optionally substituted with -C0 H or an ester thereof, C |-Q, alkoxy, oxo, -CR=€R2, -OCR, CS-CB cycloalkyl. Cj-Cg heterocyclyl. Ce-Cio aryf, or Cj-Cio hctcroaryl wherein each R independently is hydrogen or C pCy, alkyi;
C3-C10 cycloalkyl;
Qj-Cs heterocyclyl;
Cfi-Cio aiyl; or
-Cj o heteroaryi;
wherein each cycloalkyl. heterocyclyl. aryl, or heteroaryl is optionally substituted with 1 -3 alkyi groups, optionally substituted with 1-3 halo, preferably, fluoro, groups, or R! g and R|9 together with the nitrogen atom they are attached to form a 5-7 merabered heterocycle;
each R .' and R8 are independently hydrogen or defined as R"; refers to a mixture of cis and trans isomers at the corresponding position wherein at least 80% and, preferably, no more than 95% of the compound of Formula (I) is present as a trans isomer;
or a salt or a tautomer thereof.
[0159 J In one embodiment, the GGA derivative utilized is of Formula (I-A):
Figure imgf000031_0002
(I-A)
as a substantially pure trans isomer around the 2,3 double bond wherein, n. R -R\ Q', and Q2 a defined as in Formula (!) above.
[0160] In another embodiment, a is 1. In another embodiment, n is 2.
{0161] In another embodiment, the GGA derivative utilized is of Formula (i-B):
2
,0·
CT
R2 F?3 R
ii-B)
as a substantially pure /raw? isomer around the 2.3 double bond wherein, R!-R*, Q , and Q* are defmed as in Formula (I) above,
{0162] In another embodiment, [he GGA derivative utilized is of Formula II:
Figure imgf000032_0001
(Π)
wherein Q1 and Q are defmed as in Formula (i) above.
(0163 j In another embodiment, the GG A derivative utilized is of Formula (ΙΙ-Λ), (II-B), or (11-Cj:
Figure imgf000032_0002
(Π-Α)
Figure imgf000032_0003
(II-B) ill-C) wherein K6-Rs are defined as in Formula (I) above.
{0164] In another embodiment, the GGA derivative utilized is of Formula i'il-D), fll-E), (II-F):
Figure imgf000033_0001
(ΐΙ-D)
Figure imgf000033_0002
(Π-Ε) (II-F) as a substantially pure tra isomer around the 2,3 double bond wherein R6-R*are defined as in Formula (!) above.
{0165] In a preferred embodiment, R6 is Q-Cioary), such as naphthyl. in another preferred embodiment, R" is a heieroaryl, such as quinolinyl.
[01661 In another aspect, the GGA derivative utilized in this invention is of Formula (II):
Figure imgf000033_0003
(II) or a pharmaceutically acceptable salt thereof, wherein
m is 0 or I :
n is 0, I , or 2;
each Rl and R2 are independently Ci-C6 aiky!, or R1 and R2 together with the carbon atom they are attached to form a C5-C7 eycioalkyl ring optionally substituted with 1-3 C|-C* aikyi groups;
each of RJ, R4, and 5 independently are hydrogen or Cj-Cg alkyi;
Q is -X-CO-NR ,eR,y, ~X-CS-NRI8R", or -X-S02-NR'¾iy:
X is -0-, -S-, -MR7-, or -CR8R9;
R7 is hydrogen or together with R!* or R^ and the intervening atoms form a 5-7 membered heterocyclic ring optionally substituted with i-3 C 1-C alkyl groups; each R* and R9 independently are hydrogen, CrC6 alkyl, -COR8i or -C02RSi . or R8 together with RsS or '" and the intervening atoms form a 5-7 membered heterocyciy! ring optionally substituted with 1 -3 -Cs alkyi groups; and
each. R18 and R19 independently is hydrogen: CJ-CA alkyl, optionally substituted with -CO2H or an ester thereof, Ci-Q alkoxy, oxo, -CR-CR2, -CCR, C3-C8 cycloalkyl, C C8 heteroeydyi, Ce-Cioaiyl, or Ca-Cio heteroaryl, wherein each R. independently is hydrogen or Ci- j alkyl; C3-CJ0 cycloalkyl; C3-Q heterocyclvl; C5-C10 aryl; or C2-C10 beteroaryl; wherein each cycloalkyl, heterocyclvl. aryl, or heteroaryl is optionally substituted with 1-3 alky! groups, optionally substituted with 1-3 halo, preferably, fluoro, groups, or R! 8 and 1 together with the nitrogen atom they arc attached to form a 5-7 membered heterocyele.
|0167] As used herein, the compound of Formula (II) includes optical isomers such as enantiomers and diastereomers. As also used herein, an ester refers preferably to a phenyl or a CpC;, alkyl ester, ich phenyl or alkyl group is optionally substituted with a amino group.
[0168] In one embodiment, the compound of Formula (II) is of formula:
Figure imgf000034_0001
wherein R\ , R-, R , R\ and Q are defined as in any aspect or embodiment herein.
[0169] In another embodiment, the GGA derivative utilized is of formula:
Figure imgf000034_0002
wherein R\ R2, R4, R5, and Q are defined as in any aspect and embodiment here.
[0170] In one embodiment, the compound of Formula (II) is of ibrmul
Figure imgf000034_0003
wherein R1, R" RJ, R4. R5, and Q ate defined as in any aspect or embodiment herein.
In another embodiment, the GGA derivative utilized is of formula:
Figure imgf000034_0004
wherein R', RA R", R\ ra, n, X, R' and R '" are defined as in any aspect and embodiment here.
|0172| in another embodiment, the GGA derivati ve uiilized is of formula :
Figure imgf000035_0001
wherein R1, R\ R\ R% m, n, and R's are defined as in any aspect and embodiment here.
[0173J In another embodiment, the GGA derivative utilized is of formula:
Figure imgf000035_0002
wherein R is defined as in any aspect and embodiment here.
[0174] In another embodiment, the GGA derivative utilized is of formula:
Figure imgf000035_0003
|()175j wherein Rl 8 is defined as in any aspect and embodiment here.
{0176] In another embodiment, the GGA derivative utilized is of formula:
Figure imgf000035_0004
wherein R! S is defined as in any aspect and embodiment here.
{0177J fn another embodiment, the GGA derivative utilized is of formula:
Figure imgf000035_0005
(0178] wherein R " is defined as in any aspect and embodiment here.
{01793 i another embodiment, the GGA derivative utilized is of formula:
Figure imgf000035_0006
wherein R18 and R19 are defined as in any aspect and embodiment here.
{0180] In another embodiment, ihe GGA derivative utilized is of formula:
Figure imgf000036_0001
wherein R is defined as in any aspect and embodiment here.
[0181] in another embodiment, the GGA derivative utilized is of formula:
Figure imgf000036_0002
wherein RI S and Rw are defined as in any aspect and embodiment here.
[0182] In one embodiment, m is Q. in another embodiment,, m is 1.
[0183] In another embodiment, n is 0. in another embodiment, n is 1. In another embodiment; n is 2.
[0184] In another embodiment, m+n is !. In another embodiment, m+rt is 2. In another embodiment, m+n is 3.
[0185] in another embodiment, R1 and R2 are independently \ -C(, aikyi. In another embodiment, R1 and R2 independently are methyl, ethyl, or isopropyl.
[0186] in another embodiment, R1 and together with the carbon atom the are attachec to form a C5-C7 cycioalkyl ring optionally substituted with 1 -3 CrC(, aikyi groups, in another embodiment, R! and R2 together with the carbon atom they are attached to form a ring that is:
Figure imgf000036_0003
[0187] In another embodiment, R\ R4 and R* are independently CrCi; aikyi. In another embodiment one of R1, R4, and R3 are aikyi, and the rest are hydrogen, in another embodiment, two of R3, R4, and R5 are aikyi, and the rest are hydrogen, in another embodiment, R-', R4, and * are hydrogen. In another embodiment, R3, R4, and R5 are fOJ88j In another embodiment. Q is -X-CO-NR R '. In another embodiment., Q is -X-CS-N '¾ 'g. In another embodiment, Q is -X-S02-NR%'9. in another embodiment, Q is -OCONHR18 ---OCONR18Ri9, -NHCONHR18, -NHCONR R!*, -OCSNHR,s -
OCSNR,8R19. -NHCS HR18, or -NHCSNR18R19.
[01.89] In another embodiment, X is -0-. In another embodiment, X is -NR.'-. In anoiher embodiment, X is or -CR'PJ',
|0190] in another embodiment, one of R"3 and ,? is hydrogen. In another embodiment, one or both of R1& and R19 are Ci-C6 aikyi. In another embodiment, one or both of R1* and R19 are C|-C¼ aikyi, optionally substituted with an R':0 group. In another embodiment, one or both of Rn and K'v are Cj-Qo cycioalkyl, In another embodiment, one or both ofR'8 and Ri9 are C3-C10 cycioalkyl substituted with 1-3 aikyi groups. In another embodiment, one or both of Ri8 and R! are Cj-Cg heterocyeiyl. In another embodiment, one or both of RlS and ''* are Cfi-Cto r l, In another embodiment, one or both of Ri S and R'9 are Ca-Cioheteroaryl. In another embodiment, R; and R1 together with the nitrogen atom they are attached to form a 5-7 membered heterocycle.
|0191 j In another embodiment, R 0 is -C02H or an ester thereof, In another embodiment, R20 is Cj-Cg. in another embodiment, Ri0 is cycioalkyl. In another embodiment, R20 is Cj-Cg heterocyeiyl. In another embodiment. R20 is Ce-Cio aryl. In another embodiment, R*° is or Cj-Cio heleroaiyl.
[0192] In another embodiment, examples of compounds utilized by this invention include certain com ounds tabulated below,
Figure imgf000037_0001
Figure imgf000038_0001
Figure imgf000039_0001
Figure imgf000040_0001
Figure imgf000041_0001
0
Figure imgf000041_0002
if "i
Figure imgf000041_0003
Figure imgf000041_0004
Figure imgf000041_0005
Figure imgf000042_0001
..0., it ~ N
Figure imgf000043_0001
Figure imgf000043_0002
Figure imgf000043_0003
H
Figure imgf000043_0004
Figure imgf000043_0005
Figure imgf000044_0001
> 18..
Π = 0, n = 1, R18
m = 1, n = 1, R'8
ETi = 1 , n = 2, cyc!ohexyi, and R19 = methyl m = 0. n = 1, R18 n-pentyl, and R19 = methyl m = 1 , n = 1, R18 n-pentyl, and R19 = methyl m = 1,n = 2,
in another aspect, the GGA derivative utilized is of formula
Figure imgf000045_0001
wherein m is 0 or 1 ;
n is 0, 1 , or 2;
each R' and R" are independently Ci-Q alkyi, or R1 and R2 together with the carbon atom they are attached to form a C5-C7 cycloalkyi ring optionally substituted with 1 -3 C1-C alkyi groups;
each of RJ, R\ and R:x independently are hydrogen or Ci-C-6 alkyi;
Q is -X-CO-NRl8R" or -X-S02-N Rl g 19;
X is -0-, -NR7-, or -CR8R9;
R' is hydrogen or together with R or R ' and the intervening atoms form a 5- 7 membered ring optionally substituted with 1 -3 C Cs alkyi groups:
each Rs and R9 independently are hydrogen, Q-Ce alkyi, -COR81 or -C0 R8', or R* together with R'8 or R19 and the intervening atoms form a 5-7 membered cycloalkyi or heterocyclyl ring optionally substituted with 1 -3 Ci-Q, alky! groups; each R18 and R'v independently is hydrogen, C C* alkyi. optionally substituted with -C02H or an ester thereof, Ci-Cg cycloalkyi, C3-C3 heterocyclyl, - Cie aryi, or is rCio heteroaryl or is C3-C10 cycloalkyi, Cj-Cg heterocyclyl, €(,-€■ aryl, or C2-Cio heteroaryl wherein each cycloalkyi, heterocyclyl, aryl, or heteroaryl is optionally substituted with 1-3 alkyi groups, or- R=» and R!9 together with the nitrogen atom they are attached to form a 5-7 membered heterocycle.
in another aspect, the GGA derivative utilized herein is of Formula H I:
Figure imgf000045_0002
nil)
wherein
m is 0 or 1 ; n is 0, 1 . or 2;
each R' and R2 are independently C)-C6 aikyl, or 1 and R2 together with the carbon atom tiiey are attached to form a C5-C7 cycloalkyl ring optionally substituted with i -3 Ci-Ci, a!kyl groups;
each of R3, R4, and R5 independently are hydrogen or Cj-Cs alkyi;
Q is selected from the group consisting of:
Figure imgf000046_0001
when X is bonded via a single bond, X is -0-, -NR7-, or -CR8R9-, and when X is bonded via a double bond, X is -CR8-;
Y1 is hydrogen or -O-R10, Y2 is -OR! ! or -NHR! \ or Y! and Y2 are joined to form an oxo group (=0), an inline group (=NRL'), a oxime group (:=::N-OR! " k or a substituted or unsubstitued vinylidene (=CR, eR17);
R" is C|-Q aikyl optionaliy substituted with 1 -3 alkoxy or 1 -5 halo group, C Cr, aikenyl, C Cs alkyny!, C3-C;o cycloalkyl, -CJO aryl, C3-CG heterocyclyl, or Cz-Cio heteroaryl, wherein each cycloalkyl or heterocyclyl is optionally substituted with i-3 Ci -Cs aikyl groups, or wherein each aryl or heteroaryl is independently substituted with 1 -3 CI-CG aikyl or nitro groups, or R6 is -NR R19;
R7 is hydrogen or together with R" and the intervening atoms form a 5-7 membered ring optionally substituted with i-3 C i -Ce aikyl groups; each R* and R9 independently are hydrogen, Cj-C6 alkyi, -COR8' or -C02R*\ or R together with R6 and the intervening atoms fonn a 5-7 membered cycloalkyl or heterocyclyl ring optionally substituted with i-3 Ci-Q aikyl groups;
R10 is d-C6 aikyl;
R" and R are independently CrC6 aikyl, C Cw cycloalkyl, -C02R!5 , or - CON( L S) , or RKl and R" together with the intervening carbon atom and oxygen atoms fonn a heterocycle optionally substituted with 1-3 Cj-C* alky! groups;
Ru is C| -C6 aikyl or C3-C10 cycloalkyl optionally substituted with 1 -3 C,-C6 aikyl groups; R'4 is hydrogen, Cj-Cs alkyl optionally substituted with a -CC½H or an ester thereof or a C6-Cio aryi, Cj- s alkenyl, Cr , alkynyl C3-C10 cycloaikyl or a C3-C8 heierocydyi, wherein each cycloaikyl, hetcrocyclyl, or aryi, is optionally substituted with 3 -3 aikyl groups; each R independently are hydrogen, GVC IQ cycloaikyl, LVCV. alkyl optionally substituted with 1-3 substituents selected frora the group consisting of -COiH or an ester thereof, aryi, or Cj-Ct heterocyclyl, or two groups together with the nitrogen atom they are bonded to form a 5-7 membered heterocycle:
R16 is hydrogen or CrC(, alkyl;
Rr is hydrogen, C CS alkyl substituted with 1-3 hydroxy groups, -CHO, or is CO2H or an ester thereof;
each R!8 and Rl 9 independently is hydrogen, Ci-C« aikyl apiiorially substituted with - CG2H or an ester thereof, CVCie cycloaikyl, C3-C:8 heterocyclyl, Cs-Cjo aryi, or C2~CVi heteroaryl, or is C3-C10 cycloaikyl, Cj-Cg heterocyclyl, C6-Cio aiyl, or C2-C10 heteroaryl wherein each cycloaikyl, heterocyclyl, aryi or heteroaryl is optionally substituted with 1-3 alkyl groups, or R! ¾ and Ri9 together with the nitrogen atom they are attached to form a 5-7 membered heterocycle; and
each R8 ! independently is (h-Q, alkyl,
[0195] In one embodiment, m is 0. In another embodiment, rn is 1. in another
embodiment, n is 0. in another embodiment, n is 1. In another embodiment, n is 2.
[0196] In one embodiment, the compound of Formula (01) is of formula:
Figure imgf000047_0001
wherein R1, R2, R3, R4, R\ R6. X, Υ', and Y" are defined as in any aspect or embodiment herein.
j J97| in one embodiment, the GGA derivative utilized is of formula:
Figure imgf000047_0002
wherein R1, R2, R'\ R", R5, R6, X, Y\ and Y" are defined as in any aspect and embodiment here.
[0198] In another embodiment, the GGA derivative utilized is of formula:
Figure imgf000048_0001
wherein R', R2, R',
Figure imgf000048_0002
Rs, R6, X, and Y2 are defined as in any aspect and embodiment here. {0199) In another embodiment, the GGA derivative utilized is of formula:
R2 R3 R'1 R5 R6 wherein R!, R2, R."\ R4, R3, R6 and X are defined as in any aspect and embodiment here. (0200] In another embodiment, the GGA derivative utilized is of formula:
Figure imgf000048_0003
wherein R\ R2, R4. R3, and Q are defined as in any aspect and embodiment here.
[0201] In another embodiment, the GGA derivative utilized is of formula:
Figure imgf000048_0004
wherein R\ R2, R4, R3, m, n, X, and R" are defined as in any aspect and embodiment her 10202] In another embodiment, the GGA derivative utilized Is of formula:
Figure imgf000048_0005
wherein R1.. R 3 R4, R5, m, n, and R!S are defined as in any aspect and embodiment here.
[0203] In another embodiment, the GGA derivati ve utilized is of formula:
Figure imgf000048_0006
wherein R ', R2, R4, R5, R*. m, n, and R" are denned as in any aspect and embodkneni here.
{0204] In another embodiment, each R! and W" are Cj- s a!kyi. In another embodiment, each R1 arid R~ are methyl, ethyl, or isopropyl. In another embodiment, R! and R2 together with the carbon atom they are attaclied to form a 5-6 membered ring optionally substituted with ! -3 Ci-Cs alkyl groups, in another embodiment, R1 and R2 together with the carbon atom they are attached to form a ring thai is:
Figure imgf000049_0001
[0205] In another embodiment, R'\ R4, and R5 are C|-C$ alkyi, in another embodiment, one of RJ, R !. and I are alkyl, and the rest are hydrogen, in another embodiment, two of R^, R4, and R5 are alkyl, and the rest are hydrogen. In another embodiment, R3, R4, and Ri are hydrogen. In another embodiment, R RA t and R5 are methyl.
[0206] in another embodiment, X is G. In another embodiment X is -NR' . in another embodiment, R' is hydrogen. In another embodiment, R' together with R6 and the intervening atoms form a 5-7 membered ring optionally substituted with 1-3 Cj-Ce alkyl groups. In another embodiment, X is -CR 'R -. In another embodiment, X is -CR -. In another embodiment, each R* and R9 independently are hydrogen, C|-C6 alkyl, -C()RS?. or - C02RSi. In another embodiment, R8 is hydrogen, and Ry is hydrogen, Ci-Ce alkyl, -CORg! , or -C02R81.
[0207] In another embodiment, Ry is hydrogen. In another embodiment, R9 C alkyl. In another embodiment, R9 is methyl. In another embodiment, R9 is -COaR*'. In another embodiment, R9 is -COR81.
{02Θ8] in another embodiment, R' together with R and the intervening atoms form a 5-7 membered ring. In another embodiment, the moiety: 6
Which is "Q," has the structure:
Figure imgf000049_0002
wherein is hydrogen, CrC6 alky], or -C02R8i and n is i , 2, or 3. Within these embodiments, in certain embodiments, R9 is hydrogen or C; -C6 alkyl. In one embodiment, R9 is hydrogen. in another embodiment, R'' is CrCe alkyl.
10209] In another embodiment, Rh is Ci-Cg alkyl. In another embodiment, R° is methyl, ethyl, butyl, isopropyi, or tertiary butyl In another embodiment. R6 is d-C6 alkyl substituted with 1-3 a!koxy or 1-5 halo group. In another embodiment, R6 is alkyl substituted with an aikoxy group, in another cmbodiraenr, R6 is alky! substituted with 1-5, preferably, 1 -3, halo, preferably fluoro, groups.
10210] In another embodiment, R* is WLV. In a preferred embodiment, R19 is H, In a preferred embodiment R'8 is Ci-Q alkyl, optionally substituted with a group selected from the group consisting of -CO„H or an ester thereof, C3-C10 cycloalkyl, C'3-C's heterocyclyl, i-V Cio aryl, or C Qo heteroaryl. In another preferred embodiment. R18 is C3-C10 cycloalkyl, C3- Cg heterocyclyl, C&-C )o aryi. or Cj-Cio heteroaryl. In a more preferred embodiment. RIS is C3-C10 cycloalkyl.
[0211] In another embodiment, R6 is C2-C6 alkenyl or C2- alkynyi. In another embodiment, R6 is C Cio cycloalkyl, in another embodiment, R° is C3-C10 cycloalkyl substituted with 1-3 Cj-Cg alkyl groups. In another embodiment, R6 is cyclopropyl, cyclobutyi, cycloperjlyl, cyclohexyl, or adamentyi. In another embodiment, R5 is aryl or C2-C jo heteroaryl. In another embodiment, R6 is a 5-7 membered heteroaryl containing at least 1 oxygen atom, in another embodiment, R6 is C(,-C:o ary!, Cj-Cg heterocyclyl. or C Cm heteroaryl, wherein each aryl. heterocyclyl, or heteroaryl is optionally substituted with 1 -3 C1-C& alky I groups,
[0212] In another embodiment, Y" is -O-R1 1. In another embodiment, Y1 and Y~ arc joined to form =NR13. In another embodiment, Y' and Y2 are joined to form =NOR'4. In another embodiment, Y1 and Y* are joined to form (=0). In another embodiment, Y1 and Y4 are joined to form =€RlbR'7.
[0213] In another embodiment, Q is -CR9COR6. In another embodiment, R6 is C C6 alkyl optionally substituted with an aikoxy group. In another embodiment, R6 is Cj-C* cycloalkyl. In another embodiment, R9 is hydrogen. In another embodiment, Ry is Cj-C(l alkyl. in another embodiment, R9 is C(¾R81. In another embodiment, R9 is COR81.
[0214] In another embodiment, Q is -€¾-€Η(0-( )ΝΗΙ ! R°. In another embodiment, R is C;.-Cg cycloalkyl In another embodiment, R is Ci-Cg alkyl optionally substituted with 1 -3 substiteunts selected from the group consisting of -CO2W or an ester thereof, ary!. or CYCg heterocyciyl. in a preferred embodiment within these embodiments, R6 is C .-Q, alkyl.
[0215] In another embodiment, Q is -O-CO-NHR^. ithing these embodiment, in another embodiment, RlS is Ct-Q alkyl, optionally substituted with -C(¼H or an ester thereof, CyC% cycloalkyl, C.rC« heterocyciyl, C2-C10 aryl, or C Cie heteroaryJ. in yet another embodiment, R'8 is Cj-Ci cycloalkyl, Cj-Cg heterocyciyl, CrCioaryl, or Cj-C 10 heteroaryl,
[0216] in another embodiment, R.' is hydrogen. In another embodiment, R ' is Ci- ^ alkyl optionally substituted with a -CCVII or an ester thereof or a Ce-Cjo aryl optionally substituted with 1 -3 alkyl groups. In another embodiment, R"1 is C2-Q, alkenyl. In another embodiment, RM is 2-C5 aikynyi In another embodiment, R'4 is C3-Q cycloalkyl optionally substituted with 1 -3 alkyl groups. In another embodiment, R! 4 is C3-Cg heterocyclvi optionally substituted with 1 -3 alkyl groups.
[0217] In another embodiment, preferably, R16 is hydrogen, in another embodiment, R1' is CO2M or an ester thereof In another embodiment, R/? is C|-Q alkyl substituted with 1 -3 hydroxy groups. In another embodiment, R1 ' is C|-(N alky! substituted with 1 hydroxy group. In another embodiment, R ' ' is -CHiOR
{0218] In another embodiment, R 10 and R1 1 together with the intervening carbon atom and oxygen atoms form a heteroycle of formula;
Figure imgf000051_0001
wherein q is 0 or 1 , p is 0, 1 , 2, or 3. and R2lJ is C |-C6 alkyl.
[0219] In another embodiment, q is 1. In another embodiment, q is 2. In another embodiment, p is 0. hi another embodiment, p is 1. In another embodiment, p is 2. In another embodiment, p is 3,
[0220] in another embodiment, examples of compounds utilized by this in vention include certain compounds tabulated below.
Figure imgf000052_0001
Figure imgf000053_0001
Chemical Structure
..0. o
0,
Figure imgf000054_0001
1°
Figure imgf000055_0001
Figure imgf000056_0001
Figure imgf000057_0001
SUBSTITUTE SHEET (RLILE 26)
Figure imgf000058_0001
Cliemicai Structure
0
Figure imgf000059_0001
Figure imgf000059_0002
Figure imgf000059_0003
,0
7
Figure imgf000060_0001
5S
SUBSTITUTE SHEET (RLTLE 26)
Figure imgf000061_0001
Figure imgf000062_0001
Chemical Structure
0
-0' ✓.0 0
Figure imgf000063_0001
COOEt
Figure imgf000063_0002
0
Figure imgf000064_0001
aspect, the GGA derivative is of Formula (IV):
Figure imgf000064_0002
(IV)
lereof, or a pharmaceutically acceptable salt of each thereof, wherein each R! and 2 are independently Ci-Q alky], or R 1 and R2 together with the carbon atom they are attached to form a Q5-C7 cyeloalkyl ring optionally substituted with 1 -3 Ci-Cfi aikyl groups: each of R\ R4, and R5 independently are hydrogen or Cj- aikyl, or R3 and Q together with the intervening carbon atoms form a 6 membered aryi ring, or a 5-8 membered cycloalkenyl ring, or a 5- 14 membered heteroaryl or heterocycle, wherein each aryi, cycloalkenyl, heteroaryl. or heterocycle, ring is optionally substituted with 1 -2 substituenis selected from the group consisting of halo, hydroxy, oxo, ~N(RlU}?5 and Ci-Ce aikyl group;
Q is a 6 -10 membered and or a 5- 14 membered heteroaryl or heterocycle containing up 10 6 ring hcteroaioms, wherein the heteroatom is selected from the group consisting of 0. N. S, and oxidized forms of N and S, and further wherein the aryi, heteroaryl, or heterocyeiyl ring is optionally substituted with 1 -2 substituenis selected from the group consisting of hydroxy, oxo, ~N(RI9)2, and CrC6 aikyl group, wherein the aikyl group is optionally substituted with 3 -3 substituenis selected from hydroxy, N¾, -CO2H or an ester or an amide thereof, a 5- 9 membered heteroaryl containing up to 3 ring heteroatoms, wherein the heteroaryl is optionally substituted with 1 -3 hydroxy, -N(R )2. and Ci -Ci, alky! group, and phenyl optionally substituted with 1 -3 substihients selected from the group consisting of C|-C<> aikyl, hydroxy, and halo groups; and wherein each R10 independently is hydrogen or Ci-Cs aikyl
[0222] As used herein, the compound f Formula (IV) includes tautomers and optical isomers such as enanliomers and diaslereomers. As also used herein, an ester refers preferably to a phenyl or a Ci -Cj. aikyl ester, which phenyl or aikyl group is optionally substituted with a amino group. As used herein, an amide refers preferably to a moiety of formula -CQNfR^k wherein R!0 is defined as above.
In some embodiment, Q is selected from a group consisting of oxazoie, oxadiazole, oxazoline, azalactone, imidazole, diazole, triazoie, and tlnazole. wherein each heteroaryl or heterocycle is optionally substituted as disclosed above,
[0223] In one embodiment, the GGA derivative utilized is of formula:
Figure imgf000065_0001
62241 In another embodiment, the GGA derivative utilized is of formula: 2 R4 R5 wherein R!, R", R", R\ and Q are defined as in any aspect and embodiment here.
[0225] In another embodiment, Q is selected from the group consisting of;
Figure imgf000066_0001
wherein Rl ! is defined as above. In another embodiment, Q is phenyl, optionally substituted as described herein, in another embodiment, Q is benximidazole, benzindazole, and such other 5-6 fused 9-membered bicyclic heteroaryl or heterocyeie. In another embodiment, Q is quinoiine, isoqirinoline, and such other 6-6 fused 10 inembered heteroaryl or heterocyeie. In another embodiment, Q is benzodiazepine or a derivative thereof, such as, a benzodiazepinone. Various benzodiazepine and derivatives thereof are well known to the skilled artisan,
|0226j In another embodiment, rn is 0. In another embodiment, m is 1.
PI227J In another embodiment n is 0. In another embodiment, n is 1. In another embodiment, n is 2.
[0228] in another embodiment, m+n is 1. In another embodiment, m-hi is 2. In another embodiment, m+n is 3.
[0229] In another embodiment, R' and R2 are independently Ci-Ce alky]. In another embodiment, R1 and R* independently are methyl, ethyl, or isopropyl.
[0230] In another embodiment, R1 and R~ together with the carbon atom they are attached to form a C5-C7 cycioalkyl ring optionally substituted with 1-3 Ci-C6 alk l groups. In another embodiment. R* and R together with the carbon atom they are attached to form a ring that is:
Figure imgf000066_0002
, 3 4
[0231] In another embodiment, K R" , and 3 are independently Ci-Q, alkyl. In another embodiment, one of RJ, R4, and R3 are alkyl and the rest are hydrogen. In another embodiment, two of R3, R4, and R5 are alkyl, and the rest are hydrogen. In another embodiment, R"\ II4, and Rs are hydrogen. In another embodiment, R3, R4, and R5 are methyl.
[02321 In another embodiment, this invention provides a compound selected from the group consisting of:
Figure imgf000067_0001
Figure imgf000067_0002
N^ / - ^ ^; •-•N---' -.^ wherein R" is defined as above.
In another aspect, GGA derivatives utilized herein asre of formula (V):
Figure imgf000067_0003
wherem
m is 0 or 1 ; n is 0, 1, or 2;
each R and R* independently are Cj-Q alky!, or R* and R together with the carbon atom they are attached to form a Cj-C? cycloalkyl ring optionally substituted with 1 -3 CrQ alkyl groups;
each of R3, R4, and R5 independently is hydrogen or Cj-Q all yl;
Q is selected from the group consisting of:
Figure imgf000068_0001
7 . 9
when X is bonded via a single bona, X is -0-, -NR -, or -CR "R and when X is bonded via a double bond, X is -CR8-;
Yl is hydrogen or -OR1 ;
Y2 is -OR11, -NHR12, or ~0-CO-NR!3R , or Y1 and Ψ arc joined to form an oxo group (=0), an inline group (=NR15), a oxime group (= -OR*°), or a substituted or unsubstituted vinylidene (=CR,8R19);
R6 is Ct-Q alkyl, Ci-G$ alky! substituted with 1 -3 alkoxy or 1-5 halo groups, Cz-Ci alkenyl, C2-C alkynyl, C3- 0 cycioalkyi, C Cs heterocyclyl C&-C10 aryl, CV Cio heteroaryi, or -MR2l'R2!, wherein each cycioalkyi or heterocyclyl is optionally substituted with 1-3 Cj- C$ alkyl groups, and wherein each aryl or heteroaryi is optionally substituted independently with 1-3 nitro and Ci- Q alkyl groups;
R7 is hydrogen or together with R6 and the intervening atoms form a 5-7 membered ring optionally substituted with 3 -3 Cj-Gs alkyl groups;
each Rs and R9 independently are hydrogen, CrCc alkyl, -COR8' , -CO2R41, or -COhiHR82, or R8 together with R° and the intervening atoms form a 5-7 membered cycioalkyi or heterocyclyl ring optionally substituted with 1 -3 Ct-Gs alkyl groups;
R10 is C]-C6 alkyl;
each R" and R12 independently are Ci-Ce alkyl, Cs-Cio cycioalkyi, -CO2R"', or -CON(Ri7)2;
Ri3 is;
Figure imgf000068_0002
R14 is hydrogen or Ci-Gj alkyl; R1 '5 is CpQ aiky] or C3-C10 cycloalkyl optionally substitued with 1 -3 Cj-Q alkyl groups, or is:
Figure imgf000069_0001
R16 is hydrogen, Cj-Q alkyl optionally substituted with a -CO?!-] or an ester thereof or a Ce-Cio aiyl, C Ce alkenyl, Cj-Ce alkyn l, C Ci o cycloalkyl, or a CrCg hetcrocyclyl, wherein each cycloalkyl, heterocvclyl, or aryl, is optionally substituted with 1-3 alk l groups;
each R17 independently are hydrogen, Cj-Cto cycloalkyl. Cj-<¼ alkyl optionally substituted with 1 -3 substitetmts selected f om the group consisting of -€(½H or an ester thereof, aryi, C3-C§ heieroeyciyl, or two R'7 groups together with the nitrogen atom they are bonded to form a 5-7 raembered heterocycle;
R'8 is hydrogen or Q-Cg alkyl;
Ri9 is hydrogen, Ci-C^ alkyl substituted with 1 -3 hydroxy groups, -CHO, or is C(¾H or an ester thereof;
one or both of R21' and i! independently are hydrogen, C-. -C alkyl, optionally substituted with -C<¾H or an ester thereof, C3-CH, cycloalkyl, C3-Cs lieierocycivi, CV Cio aryl, or C2-C10 heteroaryl, or is C3-C10 cycloalkyl, Cs-Cg heterocvclyl, C&- Cioar i, or CVCio heteroaryl, wherein each cycloalkyl, heieroeyciyl, aryl, or heteroaryl is optionally substituted with 1-3 alkyl groups, or R*;J and gether with the nitrogen atom they are bonded to form a 5-7 raembered heterocycle, and if only one of R"° and R"' are defined as above, then the other one is
Figure imgf000069_0002
R*1 is C Q alkyl; and
R is:
Figure imgf000070_0001
provided that, when X is bonded via a single bond, and R or RJ is not
CO I1R ", Y; and Y are joined to form an imine group (=NR'>), and IV ' is:
Figure imgf000070_0002
or Y2 is -0-CQ-NR!3R14:
or provided thai, when Q is;
Figure imgf000070_0003
and R* is not -CONHR82, Y2 is -0-CO- Ri3R14;
or provided that, when Q is -O-CO-NR20Ri! ( then at least one of R4U and R
Figure imgf000070_0004
] In one embodiment, the GGA derivative utilized are of formula:
Figure imgf000071_0001
Figure imgf000071_0002
Figure imgf000071_0003
Fh isrmaeeuf seal Compositions
[0234J in another aspect, this invention is also directed to pharmaceutical compositions comprising at least one pharmaceutically acceptable excipieni and an effective amount of the trans-tsomer compound of GGA according to this invention. [0235] Pharmaceutical compositions can be formulated for different routes of
administration. Although compositions suitable for oral delivery will probably be used most frequently, other routes that raay be used include intravenous, intraarterial, pulmonary, rectal, nasal, vaginal, lingual, intramuscular, intraperitoneal, intracutaneous, transdermal, intracranial and subcutaneous routes. Other dosage forms include tablets, capsules, pills, powders, aerosols, suppositories, parenterals, and oral liquids, including suspensions, solutions and emulsions. Sustained release dosage forms raay also be used, for example, in a transdermal patch form, All dosage forms may be prepared using methods that are standard in the art (sec e.g., Remington's Pharmaceutical Sciences, 16lh ed., A. Oslo editor, Easton Pa. 1980).
[0236] The compositions are comprised of in general, GGA or a trans-isomer compound of GGA or a mixture thereof in combination with at least one pharmaceutically acceptable excipient. Acceptable excipients are non-toxic, aid administration, and do not adversely affect the therapeutic benefit of the compound of this invention. Such excipients may be any solid, liquid, semi-solid or, in the case of an aerosol composition, gaseous excipient that is generally available to one of skill in the art. Pharmaceutical compositions in accordance with the invention are prepared by conventional means using methods known in the art.
[0237] The compositions disclosed herein may be used in conjunction with any of the vehicles and excipients commonly employed in pharmaceutical preparations, e.g., talc, gum arabic, lactose, starch, magnesium stearate, cocoa butter, aqueous or non-aqueous solvents, oils, paraffin derivatives, glycols, etc. Coloring and flavoring agents may also be added to preparations, particularly to those for oral administration. Solutions can be prepared using water or physiologically compatible organic solvents such as ethanol, 1,2 -propylene glycol, poly glycols, dimethylsulfoxide, fatty alcohols, triglycerides, partial esters of glycerin and the like.
[0238) Solid pharmaceutical excipients include starch, cellulose, hydroxypropyl cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, dried skim milk and the like.
Liquid and semisolid excipients may be selected from glycerol, propylene glycol, water, ethanol and various oils, including those of petroleum, animal, vegetable or synthetic origin, e.g., peanut oil, soybean oil, mineral oil, sesame oil, etc. [02391 The concentration of the excipient is one that can readily be determined to be effective by those skilled in the art, and can vary depending on the particular excipient used. The total concentration of the excipients in the solution can be from about 0.001 % to about 90% or from about 0.001 % to about 10%. [0240] In certain embodiments of this invention, there is provided a pharmaceutical composition comprising the compound of formula I and a-tocopheroi. A related embodiment provides for a pharmaceutical composition comprising the compound of formula L a- tocopherol, and hydroxypropyi cellulose. In another embodiment, there is provided a pharmaceutical composition comprising the compound of formula I, a-tocopherol, and gum arabic. In a further embodiment, there is a pharmaceutical composition comprising the compound of formula I, and gum arabic. in a related embodiment, there is provided the compound of formula Ϊ, gum arabic and hydroxypropyi cellulose,
J0241 j When a-tocopheroi is used alone or in combination with other excipients, the concentration by weight can be from about 0.001 % to about i% or from about 0.001% to about 0.005%, or from about 0,005% to about 0.01%, or from about 0.01 % to about 0.015%. or from about 0,015% to about 0.03%, or from about 0,03% to about 0,05%, or from about 0.05% to about 0.07%, or from about 0.07% to about 0.1%, or from about 0.1% to about 0.15%, or from about 0.15% to about 0.3%, or from about 0.3% to about 0.5%, or from about 0.5% to about 1% by weight. In some embodiments, the concentration of -tocopherol is about 0.001% by weight, or alternatively about 0.005%, or about 0,008%, or about 0.01 %, or about 0.02%, or about 0.03%, or about 0.04%, or about 0.05% by weight.
10242'| When hydroxypropyi cellulose is used alone or in combination with other excipients, the concentration by weight can be from about 0, 1 % to about 30% or from about 1% to about 20%, or from about 1 % to about 5%, or from about 1 % to about 10%, or from about 2% to about 4%, or from about 5% to about 10%, or from about 0% to about 15%, or from about 15% to about 20%, or from about 20% to about 25%, or from about 25% to about 30% by weight. In some embodiments, the concentration of hydroxypropyi cellulose is about 1% by weight, or alternatively about 2%, or about 3%, or about 4%. or about 5%. or about 6%, or about 7%. or about 8%, or about 30%, or about 15% by weight,
[0243] When gum arabic is used alone or in combination with other excipients, the concentration by weight can be from about 0.5% to about 50% or from about 1% to about 20%, or from about 1% to about 10%, or from about 3% to about 6%, or from about 5% to about 10%. or from about 4% to about 6% by weight, in some embodiments, the concentration of hydroxypropyl cellulose is about 1% by weight, or alternatively about 2%, or about 3%, or about 4%, or about 5%, or about 6%, or about 7%. or about 8%, or about 10%, or about 15% by weight.
[0244J The concentration of GGA, or the trans-geranylgeranyl acetone isomer can be from about 1 to about 99% by weight in the pharmaceutical compositions provided herein, In other embodiments, the concentration of the trans-geranylgeranyl acetone isomer can be from about i to about 75%, or alternatively, from about 1 to about 40%, or alternatively, from about 1 to about 30%, or alternatively, from about i to about 25%, or alternatively, from about 1 to about 2.0%, or alternatively, from about 2 to about 20%, or alternatively, from about 1 to about 10%, or alternatively, from about 10 to about 20%, or alternatively, from about 10 to about 15% by weight in the pharmaceutical composition, in certain
embodiments, the concentration of geranylgeranyi acetone in the pharmaceutical composition is about 5% by weight, or alternatively, about 10%, or about 20%, or about 1%, or about 2%, or about 3%, or about 4%, or about 6%, or about 7%, or about 8%, or about 9%, or about 11%, or about 12%, or about 14%, or about 16%, or about 18%, or about 22%, or about 25%, or about 26%, or about 28%, or about 30%, or about 32%, or about 34%, or about 36%, or about 38%, or about 40%, or about 42%, or about 44%, or about. 46%, or about. 48%, or about 50%, or about 52%, or about 54%, or about 56%, or about 58%, or about 60%, or about 64%, or about 68%, or about 72%, or about 76%, or about 80% by weight,
[0245] In one embodiment, this invention provides sustained release formulations such as drug depots or patches comprising an effective amount of GGA. In another embodiment, the patch further comprises gum Arabic or hydroxypropyl cellulose separately or in combination, in the presence of alpha-tocopherol. Preferably, the hydroxypropyl cellulose has an average MW of from 10,000 to 100,000. In a more preferred embodiment, the hydroxypropyl cellulose has an average MW of from 5,000 to 50,000. The patch contains, in various embodiments, an amount of GGA. preferably the 5E, 9E, 13E isomer of it, which is sufficient to maintain a therapeutically effective amount GGA in the plasma for about 12 hours. In one embodiment, the GGA comprises at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the 5E, 9E, BE isomer of GGA.
[0246] Compounds and pharmaceutical compositions of this invention maybe used alone or in combination with other compounds. When administered with another agent, the coadministration can be in any manner in which the pharmacological effects of both are manifest in the patient at the same time. Thus, co-administration does not require that a single pharmaceutical composition, the same dosage form, or even the same route of administration be used for administration of both the compound of this invention and the other agent or that the two agents be administered at precisely the same time. However, coadministration will be accomplished most conveniently by the same dosage form and the same route of administration, at substantially the same time. Obviously, such administration most advantageously proceeds by delivering both active ingredients simultaneously in a novel pharmaceutical composition in accordance with the present invention.
10247J In some embodiments, a compound of this invention can be used as an adjunct to conventional drug therapy.
Synthetic Methods
[0248] This invention provides a synthetic method comprising one or more of the following steps;
Figure imgf000075_0001
III
(i ) reacting a compound of formula III under haiogenation conditions to provide a compound of formula IV;
Figure imgf000075_0002
IV (ii) reacting the compound of formula IV with alky) acetoacetate under alkylation conditions to provide a compound of formula V, where the stereochemistry at sterogenic center can be a racemic, R or S configuration:
Figure imgf000075_0003
V
(iii) reacting the compound of formula V under hydrolysis and decarboxylation conditions to provide a compound of formula VI:
Figure imgf000076_0001
(iv) reacting the compound offormuia VI with a compound of formula VII:
Figure imgf000076_0002
M l wherein Rx and eac R3 independently are alkyi or substituted or unsubstituted aryl under olef ation conditions to selectively provide a compound of formula VIII :
Figure imgf000076_0003
Vlil (v) reacting the compound of formula Vlil under reduction conditions to provide a compound of formuia iX
Figure imgf000076_0004
!X
[0249] Compound ID is combined with at least an equimolar amount of a halogenating agent typically in an inert solvent As used in this application, an "inert solvent" is a solvent that does not react under the reaction conditions in which it is employed as a solvent. The reaction is typically ran at. a temperature of about 0°C to 20 °C for a period of time sufficient to effect substantial completion of the reaction. Suitable solvents include, by way of example only, diethyl ether, aeeionitrile, and the like. Suitable halogenating agents include PBri or PPhi/CBn. After reaction completion, the resulting product, compound IV, can be recovered under conventional conditions such as extraction, precipitation, filtration, chromatography, and the like or, alternatively, used in the next step of the reaction without purification and/or isolation.
[0250] Compound IV is combined with at least an equimolar amount of an alkyi acetoacetate, in the presence of a base and an inert solvent. The reaction is typically run initially at (f'C, and then warmed up to room temperature for a period of time sufficient to effect substantial completion of the reaction. Suitable solvents include, by way of example only, various alcohols, such as ethanol, dioxane, and mixtures thereof. Suitable bases include, by way of example only, alkali metal alkoxides, such as sodium ethoxide.
|02S11 Compound V is reacted with at least an equimolar amount, preferably, an excess of aqueous alkali. The reaction is typically run at about 40 to 80 °C and preferably about 80°C for a period of time sufficient, to effect substantial completion of the reaction. Suitable solvents include, by way of examples only, alcohols, such as methanol, ethanol, and the like,
[0252] Compound VI is combined with at least an equimolar amount, preferably, an excess of a compound of formula VII, and at least an equimolar amount, preferably, an excess of base, in an inert solvent. The reaction is typically ran. initially at about -30°C tor about 1-2 hows, and at room temperature for a period of time sufficient to effect substantial completion of the reaction. Suitable solvents include, by way of examples only tetrahydrofuran, dioxane, and the like. Suitable bases include, by way of example only, alkali metal hydrides, such as sodium hydride, or potassium hexamethyldisilazide (KJIMDS), or potassium tertiary butoxide (lBuOK).
[0253] Compound VIII is combined with a reducing agent in an inert solvent. The reaction is typically ran at about 0°C for about 15 minutes, and at room temperature for a period of time sufficient to effect substantial completion of the reaction. Suitable reducing agents include, without limitation, LiAlE$. Suitable solvents include, by way of examples only diethyl ether, tetrahydrofuran, dioxane, and the like.
[0254] AS will be apparent to the skilled artisan, after reaction completion, the resulting product, can be recovered under conventional conditions such as precipitation, filtration, chromatography, and the like or, alternatively, used in the next step of the reaction without purification and/or isolation,
[0255] In some embodiments, the method further comprises repeating steps (i). (ii), and (iii) sequentially with compound of formula VIII to provide the compound of formula L wherein m is 2.
Figure imgf000077_0001
in another embodiment, the method or procedure further comprises repeating steps (i), (ii), (iii), (iv), and (v), sequentially, 1-3 times. {0256J In another of its synthetic method aspects, there is provided a method comprising one or more of the following steps:
(i) reacting a compound of formula MB:
Figure imgf000078_0001
ilB
wherein m is 1-3, under haiogenation conditions to provide a compound of formula IVB:
« ^"- ^ ^r ^ I Br
i <' m
IVB
(it) reacting the compound of formula IVB with alky] acetoacetatcs, under alkylating conditions to provide a compound of formula VB. where the stereochemistry at sterogenic center can be a racemic, R or 8 configuration:
Figure imgf000078_0002
VB
herein R aikyl is substituted or unsubstituted alkyi
(iii) reacting a compound of formula VB under hydrolysis and decarboxylation conditions to provide a compound of formula VIB:
Figure imgf000078_0003
VIB
{0257] in another of its synthetic method aspects, this invention provides a method comprising step (i) or step (ii) or steps (i) ·*· (ii) :
(i) reacting a compound of formula XC:
Figure imgf000078_0004
XC with alkyl acetoacetate under alkylating conditions to provide a compound of formula XIC, , where the stereochemistry at sterogenic center can be a racemic, R or S configuration:
Figure imgf000079_0001
wherein Rl is as defined herein, and
(is) reacting the compound XIC obtained under hydrolysis and decarboxylation conditio! to provide a compound of formula II:
Figure imgf000079_0002
II As will be apparent to the skilled artisan, the various reaction steps leading to compound VIB or to the 5Z isomer are performed in the manner described hereinabove,
[0258] In another of its synthetic method aspects, this invention pro vides a method comprising reacting a ketai compound of formula XII:
Figure imgf000079_0003
wherein each R5 independently is Ci-C& alkyl, or two R5 groups together with the oxygen atoms they are attached to form a 5 or 6 membered ring, which ring is optionally substituted with 1-3, preferably 1 -2. Ci-Cg alkyl groups, under hydrolysis conditions to provide a compound of formula IL
10259] The ketai is combined with at least a catalytic amount, such as, 1 -20 mole% of an aqueous acid, preferably, an aqueous mineral acid in an inert solvent. The reaction is typically run about 25°C to about 80°C, for a period of time sufficient to effect substantial completion of the reaction. Suitable acids include, without limitation, liCL H2SO4, and tire like. Suitable solvents include alcohols, such as methanol, ethanol, tetrahydrofuran. and the like.
[0260} In another embodiment, this invention provides a method comprising reacting a compound of formula XI:
Figure imgf000080_0001
XI
under hydrolysis and subsequently decarboxylation conditions to form a compound of formula I:
Figure imgf000080_0002
I
Alternatively, reacting compound of formula Vtl with X fol lowed by in siiu hydrolysis and decarboxylation of compound with formula XI can afford the compound of formula I.
[0261] In another embodiment, this invention provides a method comprising reacting a compound of formula X1C:
Figure imgf000080_0003
XiC
under hydrolysis and subsequent decarboxylation conditions to form the compound of formula Π
Figure imgf000080_0004
II
Hydrolysis and decarboxylation conditions useful in these methods will be apparent to tfe skilled artisan upon reading this disclosure. It will also be apparent to the skilled artisan that the methods further employ routine steps of separation or purification to isolate the compounds, following methods such as
chromatography, distillation, or crystallization.
Synthesis of GGA derivatives
[0262] Methods for making GGA or certain GGA derivatives utilized herein are described in PCT publication no. WO 2012/031028 and PCT application no. PCT7US2Q 12/027147, each of which are incorporated herein by reference in its entirety. Other GG A derivatives can he prepared by appropriate substitution of reagents and starting materials, as will be well known to the skilled artisan upon reading this disclosure. 10263] The reactions are preferably carried out in a suitable inert solvent that will be apparent to the skilled artisan upon reading this disclosure, for a sufficient period oftime to ensure substantial completion of the reaction as observed by thin layer chromatography, Ή- NMR, etc. If needed to speed up die reaction, the reaction mixture can be heated, as is well known to the skilled artisan. The final and the intermediate compounds are purified, if necessary, by various art known methods such as crystallization, precipitation, column chromatography, and the likes, as will be apparent to the skilled artisan upon reading this disclosure.
[0264] The compounds utilized in this invention are synthesized, e.g., from a compound of formula (IH-Aj:
Figure imgf000081_0001
(ΠΙ-Α) wherein n, R'-R" and \ are defined as in Formula (I) above, following various well known methods upon substitution of reacianls and/or altering reaction conditions as will he apparent to the skilled artisan upon reading this disclosure. The compound of Formula (III-A) is itself prepared by methods well known to a skilled artisan, for example, and without limitation, those described in PCT Pat. App. Pub. No. WO 2012/031028 and PCT Pat. Λρρ. No. PCT/US20I2/027147 (each supra). An illustrative and non-limiting method for synthesizing a compound of Formula (Hi- A), where n is 1, is schematically shown beiow.
7Q
Figure imgf000082_0001
Figure imgf000082_0002
(v) PO;i(RE)., (vi)
' ",
Figure imgf000082_0003
(v i li) (ix)
[0265] Starting compound (iii), which is synthesized from compound (i) by adding isoprene derivatives as described here, is alkylated with a beta keto ester (iv), in the presence of a base such as an alkoxide, to provide the corresponding beta-ketoestcr (v). Compound (v) upon alkaline hydrolysis followed by decarboxylation provides ketone (vi). Keto compound (vi) is converted, following a Wittig Homer reaction with compound (vii), to the conjugated ester (viii). Compound (viii) is reduced, for example with L1AIH4. to provide alcohol (ix).
[0266] As will be apparent to the skilled artisan, a compound of Formula (HI), where n is 2, is synthesized by repeating the reaction sequence of alkylation with a bela-keto ester, hydrolysis, decarboxylation, Wittig-Homer olefination, and LiAl¾ reduction,
(0267] Certain illustrative and non-limiting synthesis of compounds utilized in this invention are schematically shown below. Compounds where Q1 is -{OS)- or -802- are synthesized by substituting the carbonyl group of the reactants employed, as wis! be apparent to the skilled artisan. Q2COCl Rz 3 R4 /' R5
Base ' m
Q2 is H or R6
Figure imgf000083_0001
ix) Formula i
As shown above, R ' is aikyl.
[0268] Compound (ix.) with alcohol functionality is an intemieaiate useful for preparing the compounds utilized in this invention. Compound (x), where L is an E\SO?~ group is made by reacting compound (ix) with R'SOjCl in the presence of a base. The transformation of compound (Hi) to compound (x) illustrates methods of adding isoprene derivatives to a compound, which methods are suitable to make compound (in) from compound (i).
intermediate (ix) containing various R'-ff substituents are prepared according So this scheme as exemplified herein below, The transformation of compound (iii) to compound (x) illustrates methods of adding isoprene derivatives to a compound, which methods are suitable to make compound (iii) from compound (i). [0269} .1 he intermediates prepared above are converted to the compounds utilized in this invention as schematically illustrated below:
R,8-NH-COC!
Figure imgf000084_0001
Figure imgf000084_0002
Secondaiy ureas: Cyclic or A!ic clic
As used herein, for example, and without limitation, m is 0 or 1 and R'-R5 are as defined herein, and are preferably alkyl, or more preferably methyl, intermediate (ixa), prepared according to the scheme herein above, is converted to amino intermediate (ixb) via the corresponding bromide, intermediates (ixa) and (ixb) are converted to the compounds utilized in this invention by reacting with suitable isocyanaies or carbamoyl chlorides, which are prepared by art known methods. The thioearbamates and thioureas of this invention are prepared according to the methods described above and replacing the isocyanates or the carbamoyl chlorides with isothiocyanates (R" -N=C=S> or thiocarbamoyl chlorides (R ' -NH-
C(=S)C1 oi R1 R 'N-C(=S)Ci). These and other compounds utilized in this invention are also prepared by art known methods, which may require optional modifications as will be apparent to the skilled artisan upon reading this disclosure. Intermediates for synthesizing compounds utilized in this invention containing various R'-R5 substituents axe illustrated in the examples section and/or are well known to the skilled artisan.
[0270] Certain GGA derivatives utilized herein are synthesized as schematically shown below. imines, ydazones, alkoxyirnines
enolcarbama tea
Figure imgf000085_0001
ketals
[0271] Certain compounds utilized herein are obtained by reacting compound (x) with the anion Q(-), which can be generated by reacting the compound QH with a base. Suitable nonlimiting examples of bases include hydroxide, hydride, amides, alkoxid.es. and the like. Various compounds utilized in this invention, wherein the carbonyl group is converted to an imine, a hydrazone, an aikoxyimine, an cnolcarbamate, a kctal, and the like, are prepared following well known methods.
[0272] Other methods for making the compounds utilized in this invention are schematically illustrated below:
Figure imgf000085_0002
The metal iation is performed, by reacting the ketone with a base such as dimsyl anion, a hindered amide base such as diisopropylamide, or hexamethyldisilazide, along with the corresponding metal cation, M. The amino carbonyl chloride or the isocyanate is prepared, for example, by reacting the amine (R'^NH with phosgene or an equivalent reagent well known to the skilled artisan.
Figure imgf000086_0001
activation of -COM group;
R82NH2
Figure imgf000086_0002
10273] The beta keto ester is hydro lysed while ensuring that the reaction conditions do not lead to decarboxylation. The acid is activated with various acid activating agent well known to the skilled artisan such as carbonyi diirnodazo!e, or 0-Beii/.otria¾ole-N,N,N', !- teiTainethyl-uroniuni-hexailuoro-phospliate (HBTU) and reacted with the amine.
Figure imgf000086_0003
{0274] Various other compounds utilized in this invention are prepared from the compounds made in the scheme above based on art known methods.
Figure imgf000086_0004
(viii) (X)
As shown above, " is aikyi.
[02751 The intermediates prepared above are converted to the compounds utilized in this invention as schematically illustrated below:
Figure imgf000087_0001
s
Figure imgf000087_0002
O azoie
Figure imgf000087_0003
Oxazofrtes
H
ocvdes
Figure imgf000087_0004
[0276] Compound (viii) is hydrolyzed to the carboxylic acid fx), which is then convened to the acid chloride (xi). Compound (xi) is reacted with a suitable nucleophiie such as a hydrazide, a h droxylarainc, an amino aicohol, or an amino acid, and the intermediate dehydrated to provide a compound of Formula (I). Alternatively, the aliylic alcohol (ix) is oxidized to the aldehyde (xi), which is then reacted with a cyanohydrin or cyanotosyimethane to provide further compounds utilized in this invention. [0277] GOA derivatives utilized in this invention can also be synthesized employing art known methods and those disclosed here b aJkene-aryl, alkene-heteroaryl, or alkene-akene couplings such as Heck, Stiile, or Suzuki coupling. Such methods can use (vi) to prepare intermediate (xii) that can undergo Heck, Stiile, or Suzuki coupling under conditions well known to the skilled artisan to provide compounds utilized in this invention. ilti olefinaion
Figure imgf000088_0001
(xii)
Heck StiNe Suzuki coupling
Figure imgf000088_0002
(xii)
[0278] Higher and lower isoprenyl homologs of intermediates (x), (xi), and (xii), which are prepared following the methods disclosed here, can be similarly utilized to prepare other compounds utilized in this invention.
[0279] Compounds utilized in this invention are also prepared as shown below
Q-CH,
Figure imgf000088_0003
Formation of Wittig reagent
Figure imgf000088_0004
Formula {
{0280] L and Q are as defined herein, Ar is a preferably an aryl group such as phenyl the base employed is an alkoxide such as tertiarybutoxide, a hydride, or an alkyl lithium such as n-buty! lithium. Methods of carrying out the steps shown above are well known to the skilled artisan, as are conditions, reagents, solvents, and/or additives useful for performing the reactions and obtaining the compound of Formula (I) in the desired stereochemistry.
[0281] Other methods for making the compounds utilized in this invention are
schematically illustrated below:
Figure imgf000089_0001
Figure imgf000089_0002
R !3R(4NC0C1 or R!3NCO
Figure imgf000089_0003
[0282] The metal lation is performed, by reacting the ketone with a base such as dimsyl anion, a hindered amide base such as diisopropylamide, or hexamethyidisiiazide, along i the corresponding metal cation, M. The amino carbonyl chloride or the tsocyanate is prepared, ibr exampie, by reacting the amine RUR NH with phosgene or an equivalent reagent well known to the skilled artisan.
Figure imgf000089_0004
activation of -CO?H group;
R82NH,
Figure imgf000089_0005
[0283] The beta keto ester is hydro!yzed while ensuring that the reaction conditions do not lead to decarboxylation. The acid is activated with various acid activating agent well known to the skilled artisan such as carbonyl diimodazole, or
Figure imgf000089_0006
tetramethyl-uronium-hexafluoro-phosphate (HBTU) and reacted with the amine. Certain other methods of preparing the conjugates are sho wn below,
Figure imgf000090_0001
R ' 5NH dehydrating agent
sieves
Figure imgf000090_0002
26, 6£, 10Ε· Geranyi geranyl aicoho! 9 Aikyl isocyanatss 23 2E, SE. lOE-Gsranylgeranyl aikyl carbamates
As shown above, R is a memantine or a riluzole residue.
Utility
[0284| GGA is a known anti-ulcer drug used commercially and in clinical situations. GGA has also been sho wn to exert cytoprotective effects on a variety of organs, such as the eye. brain, and heart (See for example Ishii Y., et al„ Invest Ophthalmol Vis So 2003; 44: 1982- 92; Tanito M, et al., J Neurosci 2005; 25:2396-404; Fujiki M, et al., J Neurotrauma 2006;
23 : 1 164-78; Yasuda II et al., Brain Res 2005; 1032: 176-82; Ooie T, et al, Circulation 2001 ; 104: 1837-43; and Suzuki 8, et al., Kidney Int 2005; 67:2210-20).
|0285| In certain situations, She concentration of GGA required to exert a cytoprotective effect is an excessive amount of more than 600 mg per kg per day (Katsuno et al., Proc. Natl. Acad, Sci. USA 2003, 100,2409-2414). The trans-isomer of GGA has been shown to be more efficacious at lower concentrations than a composition containing from 1 :2 to 1 :3 C!s:irans mixture of GGA, and a composition of the cis-isomer of GGA alone. Therefore, the trans-isomer of GGA is useful for exerting cytoprotective effects on cells at a lower concentration than the cis-isomer or the 1 :2 to 1 ;3 mixture of cis and trans isomers.
Surprisingly, increasing amounts of the cis-isomer was found to antagonize the activity of the trans-isomer, as exemplified below.
[02S6J it is contemplated that the isomeric mixture of GGA and/or compositions containing the 5-trans isomer of GGA. can be used to inhibit neural death and increase neural activity in a mammal suffering from a neural disease, wherein the etiology of said neural disease comprises formation of protein aggregates which are pathogenic to neurons which method comprises administering lo said mammal an amount of GGA which will inhibit neural death and increase neural activity, or impede the progression of the neural disease. As it relates to the isomeric mixture of GGA, this method is not intended to inhibit or reduce trie negative effect of a neural disease m which the pathogenic protein aggregates are intranuclear or diseases in which, the protein aggregation is related to SBMA.
[Θ287] Negative effects of neural diseases that are inhibited or reduced by GGA and the 5- trans isomer of GGA according to this invention include but are not limited to Alzheimer's disease, Parkinson's disease, multiple sclerosis, prion diseases such as uru, Creutzfeltdt- Jakob disease. Fatal familial insomnia, and Gerstmann-Straussler-Scheinker syndrome, amyotrophic lateral sclerosis, or damage to the spinal cord. GGA and the 5 -trans isomer of GGA are also contemplated to prevent neural death during epileptic seizure.
Examples
J0288] The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion. The present examples, along with the methods described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Changes therein and other uses which are encompassed within the spirit of the invention as defined by the scope of the claims will occur to those skilled in the art.
[0289] in the examples below as well as throughout the application, the following abbreviations have the following meanings. If not defined, the terms have their generally accepted meanings.
°c = = degrees Celsius
PBrj = phosphorus tri bromide
EE = ethyl ether
ΗΟΠ = Ethanol
½OEt = sodium ethoxide
Oet Ethoxide
N : :: Norma]
KOH -- potassium hydroxide aqueous
ho iiffs)
Room temperature
lithium aluminum hydride
Tetrahydrofuran
minute(s)
Ethyl
Methanol
sodium hydride
Overnight
Trans
Cis
thin layer chromatography
geranylgeranyi acetone .
Microliter
Milliliter
negative logarithm of the dissociation constant hydroxypropyi. cellulose
Deionized
number average molar niass
Average
p-toluenssulfoaic acid
Triphenylphosphine
bromide ion
Tetrabromomethane
Liquid chroinatography-iTiass spectrometry Rf = retardation factor
PEG-200 polyethyl ne glycol
HMDA - potassium hexametii lencdiamke
ACN = Acetomtrile
TBDMS - tert-butyldraielfayl si!yl
Kp = Ratio of AUC w u> AUC^fa,,,,,,
AUC - Area Under the curve
LC-MS Parameters for Analysis
System: Agilent 1100 LC-MSD
Parameters;
Sample Concentration: 7.2 Mg in 1.44 mL DMSO (5mg mL). Dilute lOuL to 0.5 mL acetonitrik (iOOug/niL)
EPIC Column: Xtena MS, CI 8, 50 x 2.1, 3.5 micron
Column Temperature: 40 oC
Mobile Phase A: 0.1% formic acid in water
Mobile Phase B: 0,1% formic acid in acetomtrile
Flow Rate: 0.3 mL/min
Injection Volume: 5 sL
Gradient LC-MS:
time (min) B (%)
0 5
15 100
25 100 25. J 5
30 5
MS Parameters;
Ion Source: Electrospray
Polarity: Positive
Mass Range: 100 - 1000 amu
Fragmentor: 80
Dry Gas: 10 3 min
Dry Gas temp: 350 oC
Vcap: 4000
Nebulizer Pressure: 35
Gain: 5
[Θ290] The starting materials for the reactions described below are generally known compounds or can be prepared by known procedures or obvious modifications thereof. For example, many of the starting materials are available from commercial suppliers such as Aldrich Chemical Co. (Milwaukee, Wis., USA), Bachem (Torrance, Calif., USA), Emka- Chemce or Sigma (St. Louis, Mo.. US ). Others may be prepared by procedures, or obvious modifications thereof, described in standard reference texts such as i'ieser and Fieser's Reagents for Organic Synthesis, Volumes 1 15 (John Wiley and Sons, 3991). Rodd's Chemistry of Carbon Compounds. Volumes 1 5 ana Supplemental (Elsevier Science Publishers, 1989), Organic Reactions, Volumes i 40 (John Wiley and Sons, 1991 ), March's Advanced Organic Chemistry, (John Wiley and Sons, 4.sup.th Edition), and Larock's Comprehensive Organic Transformations (VCH Publishers inc., 1989).
Example 1 : 5E,9E ,i 3E-Geranylgeranyl Acetone Synthesis
[0291] Synthesis of 5-fraRS-lsomer: 5E,9E, 1 3B-Geranylgeranyi acetone 1: The synthesis of 5-lrans isomer: 5E.9EJ 3E-geranylgeranyl acetone 1 can be achieved as per outlined in the scheme- 1. Scheme
,ύΗ
T
Figure imgf000095_0001
203E Farresyi Bromide (4)
Figure imgf000095_0002
2E. 6E .10E-Conjug£!»?d ir. ·:->¾? .··' et. l Oli-Geranylgfciaiiy!
Figure imgf000095_0003
HaOEt EEtiOH, Dioxane
2fc o Ut-Geranylg«rafiy: Bromide 0 °C .. 3D ST OM 5E . 8E, I · Gemnyweranyi Ketoester (1 1 j
5N KOH ««o.> 5E
MftOH, 30 ¾. 24ft
1
5E, 9£, 13E-G«rany!g»rany! acetone (1 )
|02921 The 2E,6E-farnesyl alcohol 3 (where the geometry at C2 and Co positions is already fixed as trans- or E) was designed and used as a commercially available starting material for the synthesis of 5E,9E,13E-geranylgeranyl acetone 1 The alcohol function of 2E, 6E- farnesyl alcohol 3 was converted to the corresponding bromide 4 by the treatment of phosphorus tribromide (PBr3) in ethyl ether (EE) or with 1¾P and CBr4 in acetonitrile (AC ) at 0°C. The resulting bromide was then reacted with carbanion (derived from the reaction of ethyl acetoacetate 5 and sodium ethoxide) to yield the desired 5Es9E-farnesyl ketoester 6, The homologated ketoester 6 after hydrolysis and decarboxylation using aqueous SN KOH yielded the expected 5E,9E-farnesyl acetone 7. A one pot conversion of bromide 4 to the corresponding farnesyl acetone 7 can be possible without isolating intermediate ketoester 6.
[0293] In order to generate the /raw-orientation of olefin at C2 of conjugated olefin 8 in a key step, the reaction of 5E.9ETamesyl acetone 7 with carbaoion [derived from the reaction of {EtO)2PO-CH COOEt and sodium hydride (NaH)J at -30°C was conducted to obtain the desired 2E.6E, i 0E- conjugated ester 8. The formation of the product 8 with the exclusive tram (E) geometry was observed when the reaction was conducted at -30°C or temperature below -30°C, where all the three olefins are set in a trans (E) orientation (Ref. : Kato ct al., J . Org. Chem. 1980, 45, 1 126-1130 and Wiemcr et al. Organic Letters, 2005, 7(22), 4803- 4S06). T!ie minor cis- (Z)-isomer was eliminated separated from the tram- (E)-isomer 8 by a careful silica gel column chromatographic purification. However, it was also noted that the formation the corresponding os-isomer (Z) was increased when the reaction was conducted at 0°C or at higher temperature. It was also noted that the mixture of cis (2Zj- and trans (2E)- isomer of 8 can be separated by a very careful column chromatographic separation,
[0294] The resulting 2E-conjugated ester 8 was reduced to the corresponding 2E-alcohol 9 by means of a lithium aluminum hydride (LAtI) treatment, which was then converted into the corresponding 2E,6ES 1 QE-geranylgeranyS bromide 10 by means of phosphorus tribromide (PBr3) treatment in ethyl ether (EE) or with Ph3P and CBr4 in acetonitriie (ACN) at 0°C. Furthermore, the interaction of carbanion (derived from ethyl acetoacetate 5 and sodium ethoxide) with the bromide 10 at 0°C afforded the desired 2E.6E, 10E-gerany{geranyl ketoester 11, a precursor needed for 5E.9E, 13E-geraByigeranyl acetone 1. The subsequent ester hydrolysis and decarboxylation of ketoester 11 using aq. 5N KOH at 80°C yielded the requisite 5E.9E, 13E-geranyJgeranyl acetone 1. TLC Rf: 0.28 (5% Ethyl Acetate in Hexanes); LC Retention time: 16.68 min; MS (m e): 313 [M - 18 + H]+, 331 (MH]+, 353 [M - Kj,
Example 2; 5-Z,9E,13E-GeraByIgeranyI Acetone Synthesis
Scheme 2
Figure imgf000096_0001
2E,et-Famesyi Alcohol (3j
Figure imgf000096_0002
Figure imgf000097_0001
NaOB. E;OH. Dexane
2Z, 6E, ΙΟΕ-Geranyigersnyi Alcohol (13) 2Z, 6E.1Qe-G«fartyigeiar:¥i Bfonii le (14) 3 eC . JO mm, RT ON
Figure imgf000097_0002
52, gp. ^f-Seran hjsrany! Ketoester {15! 5Z, 9E,13i-GaranyigMnyi AcsioneP)
[0295| The 2E,6E-famesyl alcohol 3 (where the geometry at C2 and C6 positions is already fixed as trans- or E) was used as a commercially available starting material for the synthesis of 5Z59E.13 E-geranyigeranyl acetone 2, The reaction of farnesyl alcohol 3 with phosphorus tribromide (PBr3) in ethyl ether (EE) or with Ph3P and CBr in acetonitrile (AC ) at 0°C afforded the requisite bromide 4 , which was then reacted with carbanion (derived from the reaction of ethyl acetoacetate 5 and sodium ethoxtde) to yield the desired 5E,9E-famesy! keioester 6. The homologated keioester 6 after hydrolysis and decarboxylation using aqueous 5N KOH yielded the expected SE,9E-famesyl acetone 7, one of the key intermediate for the synthesis of 5E,9E, 13E-geranylgeranyi acetone 1 and 5Z,9E.13E-geranyigeranyl acetone 2.
[0296] With a view to obtain product with ds-gcometry at C2 with the conjugated olefin 12, the reaction of 5E,9E- farnesyl acetone 7 with carbanion [derived from the reaction of (EtO)2PO-CH2-COOEt and sodium hydride (NaH)] at 0°C was conducted. This reaction afforded a mixture of 2E,6E,10E-conjugaled ester 8 and 2Z,6E,10E-conjugated ester 12, from which the C2-cis (Zj- isomer 12 was separated by a repeated and careful silica gel column chromatography (Ref. Kato et ah, J- Org. Chera., 1980, 5, 1 126- 1 130).
[0297] The resulting 2Z-corijugated ester 12 was converted into the corresponding 2Z- alcohol 13 by means of a lithium aluminum hydride (LAH) treatment. The 2Z-al.cohoi 13 was transformed into the corresponding 2Z,6E, 1 OE-geranylgeranyl bromide 14 by using
phosphorus tribromide (PBr3) treatment in ethyl ether (EE) or with PI13P and CBr4 acetonitrile (ACN) at 0°C, and then reacted with carbanion (derived from ethyl acetoacetate 5 and sodium ethoxide) at 0CC afforded the desired 2Z.6E.1 OE-geranylgeranyl ketoester IS, a precursor needed for 5Z,9E,13E-geranylgeranyl acetone 2. The subsequent ester hydrolysis and decarboxylation of ketoester 15 using aq. 5N KOH at 80°C yielded the requisite
5Z,9EJ 3E~gerany!geranyl acetone 2, Example 3: 57 ) E » f E-i; era ny Jgers > ί Acetone Synthesis
|029S] Alternative synthesis of 5-cis Isomer: 5Z.9E, 13£-Geranylgeranyl acetone 2 : The alternative synthesis of 52,9B,13E-geranylgeranyi acetone 2 can be achieved as shown in the scheme -3.
Si- h me 3
Figure imgf000098_0001
lOc-Conjugaisd £¾s¾r (12) 3<3¾in in*srnv3 ii-3te (18) i si fevers B» formaton of cfc o>eSn
LAH, THF« C ¾ .. 5 mh 1 -OH Sec-SuU.
T . J Formaldehyde {monomer}
Ν^ 2Ζ ·"
2Z, 6g, l0E-<¾ran ¾srar¾1 Aics oi (13)
Figure imgf000098_0002
22. 6E, 1CE-Gsranylgeranyl Bromide (14)
1. gthyl etstoacssafs (5) 2. SN OH <aq.)
NaOEt BOH, Dtexsns MsOH. SO ¾, 2-31-·
0 9C ... 30 niln, RT ..OH
52, 3E, 13£-Gsran¾4g«ranyi AcstoneiS)
[0299] The use of 5£,9£-famesyl acetone 7, as a key intermediate, ears be used to generate additional double bond with cis-(Z)-orieniation, to one approach, the reaction of 5E.9E- fantesyl acetone 7 with the witting reagent 16 can afford the conjugated ester 12 with eis-fZ)- geometry at C2 position. The subsequent reduction of ester 12 with lithium aluminum hydride (LAH) can generate the corresponding alcohol 13, which then can be converted into the corresponding bromide 14, The conversion of bromide 14 to the ketoester 15 followed by hydrolysis and decarboxylation can afford the desired 5-cis (Z) isomer: Z,9E,13.E- geranygeranyl acetone (2). In an alternative approach, the reaction of 5E,9E-faracsyl acetone ? with tripheny! meftyiphosponrane bromide 1.7 under a basic conditions followed by treatment with formaldehyde (monomeric) can afford the 2Z.6E1 OE-geranylgeranyl alcohol 13 with cis (Z)- orientation at C2 (Ref.: Wiemer et an, Organic Letters, 2.005, 7(22), 4803-4806). The conversion of bromide 14 to the ketoester 15 followed by hydrolysis and decarboxylation can afford she desired 5-cis (Z)-isomer; 5Z,9E, 13E-geranygerany! acetone (2). TLC Rf: 0,32 (5% Ethyl Acetate in Hexanes); LC: Retention time: 17.18 mm; MS (m c): 313 [M - I S + H]+, 331 (MH, very weak ionization]+, 339 [M - CH2 + Na], 353 [M + KJ.
[0300] All the intermediate products were purified by silica gel column chromatography and then used in the next step, except the bromides 4, 10 and 14. Due to the unstable nature of bromides 4, 16 and 14 towards silica gel column chromatography, these bromides were used in the next step without purification. Alternatively, all the intermediate products shown in the schemes 1, 2 and 3 are liquids and therefore can be separated and purified by a distillation process under appropriate levels of vacuum. Ail the intermediates and final products were characterized by LC-MS for mass along with the Thin Layer Chromatography (TLC) for Rf values.
Example 4: 5-Z,9E, 13E-Geran Igeranyl Acetone Synthesis
[0301] Alternative synthesis of 5-cis Isomer: 5Z,9E,13E-Geranylgeranyl acetone 2; The alternative synthesis of 5Z.9E, 13E-geranyigeranyl acetone 2 can be achieved as shown in the seheme-4
Scheme 4
( 17)
3}
Figure imgf000100_0001
YMs (21 )
SE.SE-f amesyl Acetone (7) Phosphcnium Salt (20)
XSi, RT
Figure imgf000100_0002
5Z.SE.13E-2-Oxo~ketaM3GA (22) 5Z,9E,13E-GGA (2)
[0302] The convergent synthesis of 5Z,9E, 13 E-GGA 2 has been shown in the above scheme and is outlined as follows.
[0303] The 2E,6E-farnesyl alcohol 3 (where the geometry at C2 and Co positions is already fi ed as trans- or E) was used as a commercially availabl stalling material for the synthesis of 5Z,9Es13E-geranylgeranyl acetone 2, The reaction of famesyl alcohol 3 with phosphorus tribromide (PBr3) in ethyl ether (EE) or with 1¾P and CBr4 in acetonitriie (ACN) at 0 °C afforded the requisite bromide 4 , which was then reacted with carbanion (derived from the reaction of ethyl acetoacetate 5 and sodium ethoxkle) to yield the desired 5E,9E-farrtesyi ketoester 6. The homologated ketoester 6 after hydrolysis and decarboxylation using aqueous 5N K.OH yielded the expected 5E,9E-famesyl acetone 7, one of the key intermediate for the synthesis of 5E,9E, 13 E-geranylgeranyl acetone 1 and 5Z.9E,13E-geranylgeranyl acetone 2. [0304) The other synthon, namely the y!ide 21 can be synthesized from a commercially available starting material, ethyl levulinate 16, a sugar industry by-product. The Retaliation of ethyl levuliaate 16 using conventional conditions {ethylene glycol, p-TsOH, azeotropic reflux) can yield the desired 2-oxo-ketal 17, which then can be reduced using LAH in THF at 0 °C to the corresponding alcohol 18. Furthermore, the alcohol IS then can be treated with PhjBr in diethyl ether at 0 °C to obtain the bromide 19, which then after treatment with PhiP can yield the phosphonium bromide salt 20. The bromide salt 20 upon treatment with mild alkali (IN NaOH) can furnish the desired yiide 21, required to complete the synthesis of 5Z- GOA 2.
[0305] With a view to obtain product with cis-geometry, the reaction of 5E,9E-farnesyl acetone 7 with the ylide 21 in DCM at T can afford the desired 5Z-oxoketai 22 (Ref.: Ernest et al. Tetrahedron Lett. 1 82. 23(2), 167-170). The protected oxo-function from 22 can be removed by means of a mild acid treatment to yield the expected 5Z,9E,i 3E-GGA 2.
Example 5: 5E,9E,13E«Geranylgeranyl Acetone Synthesis
[0306) Alternative synthesis of $-trans isomer: 5E,9E, 13E-Geranylgerany 1 acetone 1 : The alternative synthesis of 5E,9E, 13E-geranyigeranyl acetone 1 can be achieved as shown in the scheme -5.
Scheme 5;
Figure imgf000101_0001
6E, l0E.Gerany'liirialool {23) Dketsne(24)
AKOPrk 140-150 °C, 5-6h
Figure imgf000101_0002
Mefdrums Acid (28)
AI{OiPt¾, 160 °C. 6h
[Θ3Θ7] The 5E, 9E, BE-geranyl geranyl acetone (1) can be prepared by reacting 6E-10E- geranyl linalool (23) with diketene (24) catalyzed by DMAP in ethyl ether to give the ester 25. The ester 25 in. the Carroll rearrangement using Al(OiPr)j at elevated temperature can afford the desired 5E, 9E, 13E-geranyl geranyl acetone (1). In another approach, the GGA ( ί) can be prepared by treating geranyi linalool (23) with the Meldrurn's acid 26 in the Carroll rearrangement using Al(QiPr)¾ at 160 "C, Similar!}', the use of ,'er.f-buiyl acetoaeetate (27) with geranyi linalool (23) in the Carroll rearrangement can also give the desired SE, 9E. 1 E-gerany! geranyi acetone (1 ).
Example 6; S-Z,9E,13E-Geranylgeraayl Acetone Synthesis
[0308] The alternative synthesis of 5Z,9E,13E-geranylgeranyl acetone 2 can be achieved as shown in the scheme-6,
Sc ense 6:
Ph3P, CBr,
THF, 0 °C
.^" -OTBDMS /•OTBDMS
HO'
28 Bro
Figure imgf000102_0001
3r
HfviOS, THF, RT, 1h
ph3P
Phos onium sait 30
5E, 9E-Farnesy Ύi acetone (?) Yiide 31
Figure imgf000102_0002
27,- ', BDMS Bher 32 22-Alcohol 1i
Figure imgf000102_0003
2Z-Bfcrnidel U 5Z- stoester 15
Figure imgf000102_0004
5Z-Gerany¾eranyl acetone 2
(0309] Alternative synthesis of 5-cis Isomer: 5Z,9E,13E-Geranylgeranyl acetone 2: The 2E,6£-farnesyl alcohol 3 (where the geometry at C2 and€6 positions is already fixed as tram- or E) was used as a commercially available starting materia] for the synthesis of 5Z.9E, 13E-geranylgeranyI acetone 2. The reaction of famesyl alcohol 3 with phosphorus tribromide (PBr in ethyl ether (EE) or with PlijP and€Β¾ in aeetonitrile (ACM) at 0 °C afforded the requisite bromide 4 , which was then reacted with carbanion (derived from the reaction of ethyl acetoaeetate 5 and sodium ethoxide) to yield the desired 5E,9E-farnesyl ketoester 6. The homologated ketoester 6 after hydrolysis and decarboxylation using aqueous 5N KOH yielded the expected 5E,9E-farnesyI acetone 7, one of the key intermediate for the synthesis of 5E,9E,!3E-geranylgeranyl acetone 1 and 5Z, E, 13E-geranylgerarsyl acetone 2.
[031.01 The yllde 31 synthesized from a commercially available mono-TBDMS protected ethylene glycol. 28, The conversion of alcohol function of 28 by using P1¾P and CBr,( in acetonitrile can afford the corresponding bromide 29, which, then can be used to make a phosphonium bromide salt 30 by treatment with P1¾P at elevated temperature. The bromide salt 30 upon treatment with HMDS in THF can afford the ylide 31, which then can be reacted in-situ with ketone 7 in a key step to establish cis geometry with the newly created double bond at C2 position and obtain the 2Z-TBDMS ether 32 (ref: Still et al. J. Org. Chem., 1980, 45, 4260-4262 and Donetti et al. Tetrahedron Lett. 1982, 23(21), 2219-2222). The deprotection of TBDMS with aqueous HCl to afford the corresponding alcohol 13 followed by conversion of alcohol to bromide using Ph-jP and CBr.; can afford the desired bromide 1.4. The bromide 14 upon reaction with ethyl acetoacetate can give ketoester 15, which then upon hydrolysis followed by decarboxylation can yield the desired 5-Z-GGA (5-cis) 2.
Formulations and Pharmacokinetics (PK) Studies
[0311 ] With a view to administer the geranylgeranyl acetone (GGA) effectively to determine its PK and efficacy, preclinical research formulations, exemplified in Example 6- 20, have been developed. The use of isomeric mixture of 5B-and 5Z- geranylgeranyl acetone (referred as GGA) was employed during the development of preclinical research
formulations, it is contemplated that synthesized compositions of 5E- and/or 5Z-GGA can be used in such preclinical research formulations.
[0312] In the following examples, Plasma concentrations and PK parameters were obtained from C S- 101 IV dosing and oral formulation PK studies. PK PARAMETERS were calculated from noncompartmental analysis ( CA) model using WinNonlin software and the linear/log trapezoidal method.
f 03131 Definitions for PK parameters :
Parameters that do not require lz: Tmax (mm): Time to reach Cmax (directly taken from analytical data).
Parameters that requires l z: Terminal Half-Life (t z) ~- in(2)/lz. Calculated using Lambda_z method to find best fit. If necessary, the concentration-time points were manually selected for use in the calculation. Bolded-italicized concenirations indicate points used for calculation .
301 Bioavailability
AUC (PO) /Dose (PO)
F (%) = Bioavailability X 100
AUC (PO) /Dose (PO)
Example 7: GGA formuiatien using 5% Gum Arabic with 0,008% a-tocopheroi
Using 5% Gum Arabic with 0.008% a-tocopheroi
Entry Gum Arabic (μί..) GGA (μΐ) GGA (%)*
190 uL 10 uL 5%
20 uL 10%
160 uL 40 uL 20%
140 μΐ 60 μΐ,
The % ratios are based on volumes
{0314J Preparation of 5% G am Arabic Solution: 1.25 g of Gum Arabic was suspended in DI water (23.75 mL; until the total volume was 25 mL) and agitated using agitator until all Gum Arabic was miscible in Dl water. To this solution was added a -tocopherol (2 μΐ, final concentration = 0.008) and agitated to obtain a solution of 5% gum Arabic, which was then used as a stock solution to formulate GG A.
{03.15] Preparation of GGA sospensioe in 5% Gum Arabic Aqueous Solution: To a respective amount of 5% of Gum Arabic solution from the stock, the corresponding amount of GGA was added and the resulting mixture was agitated to obtain an aqueous suspension formulation.
[0316] The in-vivo P studies by using rat species with GGA in 5% Gam Arabic as an aqueous suspension formulation resulted in 37.3% oral bioavailability (%F) with tl/2== 3.43h and Tmax- 7.33h. The in-vivo studies to obtain Kp, which is a ratio of AUCiTOOT to AUCptowo* was done in rat species at 6h and Sh time points and found that the GG A has Kp from 0.08 to 0.1 1. Example 8: GGA formulation using Hydroxypropyi Cellulose (HFC; Av. Mn=
100,000; High Average Molecular Weight) with 0.008% a-tocopherol
Hydroxypropyi Cellulose (HFC; Av, Mrr= 100,000; High Average Molecular Weight) with 0.0OS% a-tocopherol
Entry 3% I-IPC (μΐ) GGA (fiL) GGA (%)*
1 , 475 ,uL 25 uL 5%
·? 450 pL 50 pL 10%
3. 425 pL 75 pL 15%
4. 400 pL 100 pL 20%
The % ratios are based on volumes
|0317] Preparation of 3% Hydroxypropyi Cellulose Solution: To a mixture of 3g of hydroxypropyi cellulose (Av. Mn= 100,000) and a -tocopherol (8 pL, final concentration = 0.008%) was added DI water (-97 mL) until the total volume reached 100 mL. The resulting mixture was agitated to obtain a stock solution to formulate the GGA.
|03 8] Preparations of GGA suspeesioe in 3% Hydroxypropyi Cellulose Aqueous Solution: To a respective amount of 3% of hydroxypropyi cellulose solution from the stock, the corresponding amount of GGA was added and the resulting mixture was agitated to obtain an aqueous suspension formulation.
[0319] The iri-vivo PK studies by using rat species with GGA in 3% Hydroxypropyi
Cellulose (HPC, Av, Mn = 100,000) as an aqueous suspension formulation resulted in 41.8% oral bioavailability (%F) with tl/2= 3.13 h and Traax= 8.66h.
Example 9: GGA farrau tion using Hydroxypropyi Ce!laiose (HPC; Av. Mn= 10,262; Low Average Molecular Weight) with 0 J08% ra-tecopherol
Hydroxypropyi Cellulose (HPC; Av. n= J 0,262; Low Average Molecular Weight) with 0.008% a-tocop erol
Entry 3% HPC (pL) GGA (pL) GGA (%)·
1. 475 pX 25 μΐ, .5% . - 450 pL 50 pL 10% 425 pL 75 μ ΐ, 15%
4. 400 pL 100 μΐ 20%
* The % ratios are based on volumes
J0320] Prepar&tioa of 3% Hydroxypropyl Cellulose Setatios: To a mixture of 3g of hydroxypropyl cellulose (A v. Mix* 10,262} and a -tocopherol (8 pL, final concentration = 0.008%) was added Di water (-97 mL) until the total volume reached 100 mL. The resulting mixture was ajptated to obtain a stock solution to formulate the GGA.
0321j Preparation of GGA suspension/solution in 3% Hydroxypropyl Cellulose Solution: To a respective amount of 3% of hydroxypropyl cellulose solution from the stock, the corresponding amount of GG A was added and the resulting mixture was agitated to obtain an aqueous suspensi on formulation..
[0322] The in-vivo PK studies by using rat species with GGA in 3% Hydroxypropyl Cellulose (HPC, Av, Mn = 10,262) as an aqueous suspension formulation resulted in 35% oral bioavailability (%F) with tI/2- 18,73h and Tmax= 9,33h.
Example 10: GGA formulation using 5% Gam Arabic + 3% Hydroxypropyl Cellulose (HP C; Av, MB= 100,000; High Average Molecular Weight) and with 0.008% a - tocopherol
5% Gum Arabic + 3% Hydroxypropyl Cellulose (HPC; Av. n» 100,000; High Average Molecular Weight) and with 0.008% a -tocopherol
5% Gum
Arabic (0.008% GGA
Entry 3% HPC GGA ( i)
a -tocopherol) (%)*
(pL)
I. 460 pL 15 mg 25 pL 5%
2. 450≠ 15 me 50 μΐ 30%
3. 410 pL 15 mg 75 pL 15%
4. 385 pi. 15 mg 100 pL 20%
The % ratios are based on volumes [0323] .4, Preparation of 5% Gum Arabic Solution: 1.25 g of Gum Arabic was suspended in DI water (23.75 mL; until the total volume was 25 mL) and agitated using agitator until all gum Arabic was miseible in DI water. To this solution was added a - tocopherol (2 \LL, 0.008%) and agitated for a minute to obtain 5% gum Arabic
[0324 j Preparation of GGA suspension/solution in 5% Gum Arabic + 3%
Hydroxypropyl Cellulose (Av. Mn= 100,000): To a respective amount of 5% of Gum Arabic solution from the stock, the corresponding amount of GGA and hydroxyprop l cellulose (Av, Mn= 100,000] were added and the resulting mixture was agitated to obtain an aqueous suspension formulation.,
[0325] The irt-vivo PK studies by using rat species with GGA in 5% Gum Arabic + 3% Hydroxypropyl Cellulose (Av. Mn= 100.000) as an aqueous suspension formulation resulted in 58% oral bioavailability (%F) with 11/2= 10.2h and Tmax- 5.33k
Example 11: GGA formulation using 5% Gum Arabic + 3% Hydroxypropyl Cellulose (HPC; Av. Me- 10,262; Low Av. Mokcolar Weight) and with 0,008% a -tocopherol
Table 5: 5% Gum Arabic + 3% Hydroxypropyl Cellulose (HPC; Av, n= 10,262; Low Av. Molecular Weight) and with 0.008% a -tocopherol
5% Gum
Arabic (0.008% GGA
Entry 3% UPC GGA (uL)
a-tocopherol) (%)*
(pL)
1. -60 ui . 15 mg 25 μΐ, 5%
2. 450 μΤ 15 mg 50 ί . 10%
3. 4 ί ϋ μΙ, 1 5 rng 75 μΐ, 15%
4. 385 μΐ. 35 mg Ι ΟΟ μΙ. 20%
* The % ratios are based on volumes
|03261 A. Preparation of 5% Gum Arabic Solution: 1.25 g of Gum Arabic was suspended in DI water (23.75 mL; until the total volume was 25 mL) and agitated until all gum Arabic was miseible in DI water. To this solution was added a -tocopherol (2 μΧ. final concentration = 0.008%) and agitated for a minute to obtain a solution of 5% gum Arabic, which was then used as a stock solution to formulate GGA.
[0327] Preparation of GGA suspension/solution in 5% Gum Arabic + 3%
Hydroxypropyl Cellulose (Av, Mn= 100,000); To a respective amount, of 5% of Gum Arabic solution from the stock, the corresponding amount of GGA and hydroxypropyl cellulose (Av. Mn= 10,262) were added and the resulting mixture was agitated to afford an aqueous suspension.
{0328J The -vivo P studies by using rat species with GGA in 5% Gum Arabic + 3% Hydroxypropyl Cellulose (Av. Mn~ J O. 262) as an aqueous suspension formulation resulted in 36.5% oral bioavailability (%F) with 11/2= 6.73h and Tmax= i 3.3h.
Example 12: Culturing of primary motor neurons from rats.
[0329] Rat primary motor neurons were isolated from embryonic spinal cords in accordance with the method of Henderson et a!.; J Cohen and G P Wilkin (ed.), Neural Cell Culture, (1995) p69«81 which is herein incorporated by reference in its entirety. Briefly, spinal cords were dissected from day 15 embryo (E15) and incubated in a trypsin solution, and followed by DNase treatment to release spinal cord cells from tissue fragments. The eel! suspension was centrifuged to remove tissue fragments. Then motor neurons were enriched by density gradient centrifugation.
[0330] Motor neurons were cultured in serum-free neurobasal medium containing insulin, forskolin, 3-isobutyl- 1 -meth Ixanthine, neurotrophic factors, Bovine serum, albumin, selenium, transferrin, putrescine, progesterone and B27 supplement in tissue culture plate coated with poly-omi thine and !aminin.
Example 13; 5-Trans isomer of GGA (CNS-102) is more efficacious in vitro than the isomer mixture of GGA (C S 01),
Rat primar motor neurons were prepared and cultured as described Example 1 1. Various concentration of CNS- 101, which is a mixture of 5-trans and 5-cis isomer (cisrtrans ratio = 1 :2- 1 :3). CNS- 102 (herein also referred to as 5-trans isomer of GGA), and CNS-103 (herein also referred to as 5-cis isomer of GGA) were added to the culture at the time of plating the cells. The cells extending axons were counted in five different fields for each treatment after 72 hrs. Percentage of positive cells relative to total cells in the same magnification field was calculated and the results were expressed as means +/- standard deviations, rr-=5. The ECJO is a measure of the effectiveness of a compound, and corresponds to the concentration at which the drug exhibits half its maximum effect. These results are depicted in the table below:
GGA ECso
CNS- 101 6.1 nM (4-7 nM)*
CNS-102 0.92 nM (0.5-2.0 nM)*
CNS- 103 9.49 nM (8-12 n )*
* values in parenthesis indicate a reasonable range expected for the FC50
Example 14: A large quantity of GGA isomer mixture (CNS-101) inhibited viability of neuroblastoma cells.
[0331] Human SH-SY5Y neuroblastoma cells were culture in DMEM/HAM F12 supplemented with 10% fetal bovine serum (FBS) for 24 hrs. The ceils were treated with rctinoic acid in DMEM/HAM Fl 2 medium supplemented with 5% FBS for 48 hrs. Then the cells were treated with CNS-101 ( 100 micro molar (μΜ)) or vehicle, dimethyl sulfoxide for 48 hrs. Ceil viability was determined using ATP detection assay (Promega). These results are depicted in the table below:
GGA Mean of cell viability SE
(arbitrary units)
100 μ CNS- 101 1 181020 2581 5
Vehicle 1340600 23409
PO.001
Example 15: A large quantity of GGA isomer mixture (CNS-101 } aad ds-isomer (CNS- 103) inhibited viability of seuro blastema cells.
[0332 j Mouse Neuro2A neuroblastoma ceils were cultured in DMEM supplemented with 10% FBS for 24 hrs. The cells were treated with various concentrations of CNS- 101 , CNS- 102, and CNS- 103 as indicated for 48 hrs. Then differentiation was induced by relinoic acid in DM EM supplemented with 2% FBS. An inhibitor against a G-protein, GGTI-298, was incubated. After 24 hrs incubation, cells with neurites were counted. A large quantity of GGA isomer mixture (CNS-101) and the cis-isorner (C S- 103) inhibited viabiiiiy of neuroblastoma cells. These results are depicted in the table below:
GGA Mean of cell numbers SE
(Arbitrary units)
CNS-101 (10 μΜ) 0.551 0.1333
CNS-102 (10 μΜ) 0,738 0,0018
CNS- 103 (10 μΜ) 0.195 0.0933
P<0.03
The data in examples 1 3 and 14 support the conclusion that the cis isomer, CNS-103, has a deleterious effect on cell viability and that the trans isomer has a positive effect. The two examples taken together suggest that at higher concentrations, the cis isomer has an inhibitor) effect, on the trans isomer. Example 16 further elaborates upon these findings.
Example 16: Effects of the GGA isomer mixture (CNS-101) on cells experiencing oxidative stress.
(0333 J Human SH-SY5Y neuroblastoma cell s were culture in DMEM/HAM F 12 supplemented with 10% fetal bovine serum (FRS) for 2 days. The cells were treated with retinoie acid in DMEM/HAM F12 medium supplemented with 5% F BS for 48 hrs. Then the cells were treated with various conceniraiions of CNS101 for 48 hrs. Cells were exposed to hydrogen peroxide (75 micro M) or DMEM/HAM F12(eontrol) for 2 hrs, then cell viability was determined using ATP assay (Proinega). These results are depicted in the table below:
GGA Mean of cell viability
CNS-101 (10 μΜ) 640%
Vehicle 3-5%
100% of cell viability was evaluated in the absence of hydrogen peroxide and CNS-101. Eissaple 17: Effects of the GGA isomer mixture (CNS-101), the trans-isoraer (CNS- 102) and the eis-lsomer (CNS-103), and an inhibitor of a G-profein (GGTI-298), en the viability of cells.
[0334] Neuro2A cells were cultured with CNS-101 , CNS- 102, or CNS-103 in the presence or absence of an inhibitor against a G-proiein (GGTI-298). After differentiation was induced, cells that extended neyrites were counted. These results are depicted in the table below;
GGA Mean of cell numbers
(Arbitrary units)
CNS-101 (0.1-ΙμΜ) 0,45-0,65
CNS-102 (0.1-1 μΜ) 0,45-0.65
CNS-103 (0.1-1 μΜ) 0,20-0.45
Vehicle 0.0-0.20
Example 18: The GGA isomer mixture (CNS-101) activated neurite outgrowth of neuroblastoma cells.
[03351 Human SH-SY5Y neuroblastoma ceils were cultured in DMEM/HAM F 12 supplemented with 10% fetal bovine serum (FBS) for 24 hrs. The cells were treated with retinoic acid in DMEM HAM F12 medium supplemented with 5% FBS. Then the ceils were treated with various concentrations of CNS-iOl, Total length of neurites for each treatment was measured. These results are depicted in the table below:
GGA Mean. of neurite
outgrowth
CNS-101 (0.1 uM) 125%
CNS-101 (ΙμΜ) 166%
CNS-101 (Ί ΟμΜ) 194%
Vehicle 100% Data were spread in a range of +/- 10% from each mean. Example 19: The GGA isomer mixture (CNS-101 ) and the trans-isomer (CNS-102) alleviated neurodegeneration induced by Kainic acid.
[0336] CNS- i 01 or CNS-102 were orally dosed to Sprague-Dawley rats, and Kainic acid was injected. Seizure behaviors were observed and scored (Ref. .J. Racine, Modification of seizure activity by electrical stimulation: II, Motor seizure, Eleeiroeneephaiogr. Clin.
Neurophysi l 32 (1972) 281- 294. Modifications were made for the methods), Brain tissues of rats were sectioned on histology slides, and neurons in hippocampus tissues were stained by Nissl. Neurons in dentate gyrus tissues damaged by ainic acid were quantified. The Memantine composition used in comparison refers to a commercially available NMDA receptor agonist These results are depicted in the tables below:
GGA Hippocampus dentate
gyrus neurons damaged
(Arbitrary units)
CNS- 101 0.725
Vehicle 20.9
Memantine 3 ,53
P-valuc to vehicle data P<0.05
GGA Seizure behaviors scores
CKS- 102 18.8
Vehicle 34
Memantine 36,2
P-value to vehicle data P<0.1 1
Example 20: Comparison of the efficacy of O - 10} and CNS-102 m alleviating neurodegeneration induced by Kainic acid.
[0337] CNS-101 , CNS- 102 or a vehicle only control were orally dosed to Sprague-Dawley rats, and Kainic ainic acid was injected. Seizure behaviors were observed and scored (Ref. R.J. Racine, Modification of seizure activity by electrical stimulation: II. Motor seizure, Eleeiroeneephaiogr. Clin. Neurophysiol. 32 (1972) 281 - 294. Modifications were made for the methods). Brain tissues of rats were sectioned on histology slides, and neurons in hippocampus tissues were stained by Nissl. Neurons damaged by ainic acid and behavior scores were quantified,
[0338] These results indicate thai a lower concentration of the trans-isomer of GO A is more efficacious at protecting neurons from neuronal damage than a higher concentration of either the isomer mixture of the cis-isomer of GGA. Furthermore, it is contemplated that such effects of trans- GGA also renders it useful for protecting tissue damage during seizures, ischemic attacks, and neural inipairraeni such as in glaucoma.
Mean of Hippocampus Mean of
CA3 neurons damaged behaviors scores
(Arbitrary units)
CNS-102 (3 mg/Kg rat) 10.25 27,5
CNS-102 (12 mg/Kg rat) 10.16 22,5
Vehicle 37.67 50.5
P value to vehicle data PO.085 P<0.165
GGA Mean of Hippocampus Mean oi Seizure
CA3 neurons damaged behaviors scores
(Arbitrary units)
CNS- 102 (25 ffig/Kg rai) 1.97 25.75
Vehicle 9.43 38
P value to vehicle date P<0. !42 P<0.025 GGA Mean of Hippocampus Mean. of Seizure
CAS neurons damaged behaviors scores
(Arbitrary units)
CNS-101 (25 mg i vg rat) 37.38 33.83
Vehicle 38.77
GGA Mean of Hippocampus
CA3 neurons damaged
(Arbitrary units)
CNS-103 (12 rag/Kg rat) 8.16
Ci S-103 (25 mg/K rat) 10.64
Vehicle 10.71
Example 21: GGA's effect on the activity of G proteins in a euroE, {033 J Neuroblastoma cells can be obtained from the American Type Culture Collection (ATCC) and cultured according to the suggested culturing techniques of ATCC. The cultured cells will be contacted with an effective amount of GGA. The change in G protein activity will be monitored by a western blot of iysates obtained from subcellular fractionation of cells. Subcellular fractionation can be performed using commercially available kits (from Calbiochem for example) according to the manufacturer's protocol. The western analysis will be performed using subcellular fractions from the membrane and cytoplasmic compartments of cells. The western blot will be performed according to standard molecular biology techniques using antibodies directed to the different G proteins: HOA, RAG , CDC42, RASD2. it is contemplated that reacting the neuroblastoma cells with an effective amount of GGA will modulate the active, membrane-bound portion of RHOA, ACI ,
CDG 2, and/or RASD2. interaction of those small G-proteins with gene products involved in protein aggregations will also be tested. Those gene products include Huntington gene product (Hit), sumoylation machinery, etc. jO340] The same assay will be performed using neuroblastoma cells or other neurons that are depleted for the TDP-43 protein. TDP-43 depleted ceils mimic the effects of neurodegeneration related to ALS, TDP-43 depletion can be accomplished using the siRNA and/or shRNA technologies. It is contemplated that neurons which are susceptible to neurodegeneration by TDP-43 depletion will have a change in the G protein activity after said neurons are contacted with an effective amount of GGA. H is further contemplated that reacting said neurons with an effective amount of GGA will increase the active membrane- bound portion of the G proteins.
J0341 j The same assay will be performed using neuroblastoma cells or other neurons that are susceptible to neurodegeneration due to inhibition of geranylgeranylation of the G proteins. GGTI-298 is a specific inhibitor of geranylgeranylation and increases neuronal ceil death through inhibiting the activation of G proteins by geranylgeranylation. Therefore, GGTI-298 and GGA will both be contacted with tissue cultures of neuroblastoma cells, it is contemplated that neurons which are susceptible to neurodegeneration by GGTI-298 will have a change in the G protein activity after said neurons are contacted with an effective amount of GGA. it is further contemplated that reacting said neurons with an effective amount of GGA will increase the active membrane- bound portion of the G proteins.
Example 22: GGA's effect on the pathogenicity of protein aggregates in neurons susceptible to neurodegeneration, [0342] Cultured neuroblastoma cells can be made susceptible to neurodegeneration by mixing the cells with dopamine. The addition of dopamine to the cells will cause pathogenic protein aggregates in the cytoplasm. To test the effect of GGA on neurons susceptible to neurodegeneration, an effective amount of dopamine will be first contacted with the neurons to induce pathogenic protein aggregate formation in the cells. Next, an effective amount of GGA will be contacted with said neurons. The change in the size and/or number of protein aggregates will then be measured using histological staining techniques and/or
immunostatning techniques commonly known to one skilled in the art. It is contemplated that contacting GGA with neurons susceptible to neurodegeneration due to dopamine-induced protein aggregation will sorubilize at least a portion of the protein aggregate, thus decrease the pathogenicity to the cell, it is further contemplated that contacting GGA with neurons susceptible to neurodegeneration due to dopamine-induced protein aggregation will alter the form of the pathogenic protein aggregate into a non-pathogenic form, thus decrease the pathogenicity to the ceil,
[0343] Contacting neurons in vitro with β-arayioid peptide aggregates will recapitulate the toxic effects of AD due to β-amyioid peptide aggregates in vivo, To test if GGA reduces the pathogenicity of jj-amyloid peptide aggregates in cultured neuroblastoma cells, the p- amyloid peptide aggregates will be added directly to the cell culture medium of the cultured cells. The β-amyloid peptide can be purchased commercially and aggregated in vitro. An effective amount of GGA will then be added to the ceil culture to test for a modulation of the pathogenicity to the cells. It is contemplated that contacting GGA with neurons susceptible to neurodegeneratiori due to -amyloid peptide aggregation will solubilize at least a portion of the protein aggregate, thus decrease the pathogenicity to the cell. It is further contemplated that contacting GGA with neurons susceptible to neurodegeneration due to β- amyloid peptide aggregation will alter the form of the pathogenic protein aggregate into a non-pathogenic form, thus decrease the pathogenicity. The change in the size and/or number of protein aggregates will then be measured using histological staining techniques and or immunostaining techniques commonly known to one skilled in the art.
Example 23: GGA's effect' in vivo in mammals susceptible to neurodegeneration.
[0344] Neurotoxins can be used to recapitulate the effect of AD in mice. To test the effects of administering GGA to a mammal that is susceptible to AD, neurotoxins will be administered systeraica!ly or by direct injection into the brain tissues of mice to induce the pathology associated with AD. The neurotoxins will be administered either before, sim ultaneously, or after the administration of GGA. The GGA may be administered to said mice mixed with a pharmaceutically acceptable e cipknt These mice will then be monitored for survival rate, neuron density in brain tissues, arid learning, memory, and motor skills. The learning, memory, and motor skills are measured by techniques commonly known to one skilled in the art. It is contemplated that treating the animal with an effective amount of GGA wi ll attenuate some of the symptoms associated with the injection of the neuroto in.
[0345] There are a variety of mouse models available that are engineered to have the same pathology associated with different human diseases, One uch mouse model is a mouse that over-expresses the Amyloid beta Precursor Protein (APP). This mouse has a similar* pathology to that seen in human AD. An effecti e amount of GGA will be administered to mice over-expressing APP. The GGA may be administered to said mice mixed with a pharmaceutically acceptable excipient. These mice will then be monitored for body weight, β-amyloid plaque formation, and learning, memory, and motor skills. Histology sections of these mice will also be analyzed by staining and immunohistochemical techniques to detect changes in the brain after GGA administration. It is contemplated thai treating the animal with an effective amount of GGA will attenuate some of the symptoms associated with AD.
(0346) Mice expressing a Sodl mutant protein exhibit similar pathology to humans with ALS. An effective amount of GGA will be administered to Sod l mutant mice. The GGA may be administered to said mice mixed with a pharmaceutically acceptable excipient. These mice will then be monitored for survival rate, body weight, and motor skills. Histology sections of these mice will also be analyzed by histology staining and immunohistochemical techniques to detect changes in the brain, spinal cords, or muscles after GGA administration. It is contemplated that treating the Sodl mutant mice with an effective amount of GGA will increase the survival rate, body weights, and enhance the motor skills of these mice.
Example 24s Effects of 5-trans isomer of GGA on neurological function arid clinical score in Sodl mutant mice,
[0347] Wild type and Sodl mutant mice (mice carrying multiple copies of the SODI mutant gene) were treated with 12 mg/kg of CNS-102 (5-trans GGA; N=T6 Sodl mutant mice and N=10 WT mice), 8 mg/kg of Riluzole ( = 16 Sodl mutant mice), or with vehicle alone (N=T6 Sodl mutant mice and N=l 0 WT mice). Drug or vehicle alone was administered once a day continuously for 62 days, from age 38 days to age 150 days. The percentage survival rate for each of the treated groups was calculated up to 150 days of age, and blood from animals in each group at PI 00 was analyzed for levels of alkaline phosphatase, alanine transaminase/seium glutamic pyruvic transaminase (ALT/SGPT);
aspartate iransaminase/serum glutamic oxaloacetic transaminase (AST/SGOT). albumin, total protein, albumin, blood urea nitrate (BUN), creatinine cholesterol, and glucose, as well as the albumin/globulin ratio.
[0348] Effect of CNS-102 on Neurological Function and Clinical Score are described below. In clinical and neuroscore testing, shown in FTGs. 2 and 3, group A (CNS-102) and B (riluzole) tended to outperform group D (vehicle). At several timepoints around and after PI 00, group A significantly outperforms group D in neuroscore testing, suggesting treatment with C S-102 might be more effective in delaying onset or progression of symptoms. A similar trend was seen in clinical score testing.
(034 1 Animals that did not survive until the terminal date (Pi 50) were assigned their last score before death on subsequent testing days. No significant interaction (p=0.9987) in two- way repeated measure ANOVA analysis is found in the clinical scores test. Interaction between time and ireaiment is significant (p-0.0012) in neurological score performance. Group A and D differ significantly at timepoint PI i 1 (p<0.05). Values represent means ± SEMs.
|0350j Blood tests were performed on blood samples from animals in each group at PI 00 to evaluate the safety and efficacy of CNS-102. These results are tabulated below:
Figure imgf000118_0001
Figure imgf000118_0002
[0351] There were no abnormalities in blood and neurology examinations after more than two months continuous once daily oral dosing of 12mg/ g CNS-102. Surprisingly, cholesterol levels were reduced after administration of CNS-102, suggesting that 5-trans GGA may provide benefits other than neuroprotection, such as the reduction of cholesterol levels.
|0352] Thus, the 5-trans-isomer of GGA administered to Sodl mice increases their survival over mice treated with Riluzoie and with vehicle alone and has been shown to be safe for continuous daily administration, in addition, these results suggest that daily administration of CNS-102 ma reduce cholesterol levels in mice,
Example 25: Effects of 5-trans isomer of GGA's on s rvival, behavior, and pathology in S di mutant mice,
(0353] A study was undertaken to examine the effect of treatment with CNS- 102 (a 5 rans isomer of GGA) and/or Ri!uzo!e on survival, behavior, and pathology of Sodl transgenic mice.
Materials and Methods
[0354] A total of 84 male mice (64 transgenic Sod 1 mutant mice; 20 wild type mice) ere included in the study. Dosing was administered daily beginning at postnatal day 38 (P3 ) via a single bolus oral gavage injection. Sod i mutant mice were randomized into experimental groups and administered vehicle, CNS-102 (i2mg/Kg), Riluzole (8mg Kg), . Wild type mice were randomized into two experimental groups and administered vehicle alone or CNS-102 (12mg/Kg).
[0355] Subjects were weighed and tested for Grip Strength, and assigned Clinical and Neurological scores three times a week beginning at P90 and five times a week beginning al PI 00. The table below summarizes the experimental design of the study.
Figure imgf000119_0001
The grip strength test assesses inotor function and control of the fore- and hincpaws. Mice were allowed to grab the bars on the Chatiilon DFIS-10 digital force gauge (Largo. FL), while being gently pulled parallel away from the bars by the tail. The maximum force measured prior to release of the subject's paw from tiie bar was recorded. [0356J Animals were scored clinical iy by allowing iliern to run in an exercise wheel and then scoring their gait using a scoring system adapted from Braestle et al, (Neurornoiecular Med, 2009 ; 1 1 (2); 58-62). Animals were scored using die scoring system tabulated below.
Figure imgf000120_0001
j0357j Animals were scored neuro!ogica!ly using a scoring system adapted from Leitner et al. (Working with ALS mice; The Jackson Laboraiories/Prize4Life, Appendix B, 2009). A score of 0 is given to mice that exhibit full extension of hind legs away from lateral midline when suspended by the tail A score of 0.25 is given to mice that exhibit shaking or slight/partial collapse when suspended by the tail. A score of 0.5 is given to mice that exhibit collapse or partial col lapse of leg extension towards the midline (weakness).
Results
[0358J Wildtype groups displayed a steady increase in weight throughout the study, with no noticeable differences between treatment groups E (vehicle) and F (CNS-102). See, FIG. 1 . Body weight data for all transgenic groups showed a similar steady growth until P 85. After a plateau, body weights begin dropping rapidly around P101. This rapid decline may used as a marker for the onset of disease.
[0359] There was little to no difference observed between the transgenic groups, A similar pattern was seen in body condition scores. in grip strength testing, there was a rapid decline in strength in all of the transgenic groups between P93 and PI 07, also corresponding to the onset of disease.
[0360] Blood tests were performed on blood samples from animals in each group to e valuate the safety and efficacy of CNS-102. These results are tabulated below (X indicates incomplete test for analyte):
18
Figure imgf000121_0001
Figure imgf000121_0002
Figure imgf000121_0003
Figure imgf000121_0004
Figure imgf000121_0005
' "GROU P " CALCIUM PHOSPHORUS j BICARBONATE AfG PLATELET fttig dL) (wg/di) RATIO BILIRUBIN COUNT
ims/dD (Thousands)
Group A 7.7 5.7 1 5.0 0.8 0.0 1235.3
Group B X X 1 X i .O X ! Ϊ Ί
Group D X X j X 1.1 0.0 1 163.8
Group E X X 1 X 1.0 V 1232.5
Group F X X i X 1.0 X 1 128.6
Example 26: Pharmacokinetics of 5-traos isomer of GGA's in rats.
[0361] Rats were administered a single dose of CNS-102 either intravenously (12mg/kg) or orally (24mg/kg), and blood samples were taken to measure the average plasma
concentrations of CNS-102 over tone.
10362] The formulation for this study includes the following:
Formulations: (Dose: 24 mg Kg; Study Date: 4»! 6-2012)
Gum Arabic 5%
Sodium Chloride 1%
Alpha-tocopherol 0.008%
CNS-102 0.4%
D. 1. H20 93.60%
[0363] The results for a 12mg/k g IV dose of CN S- 102 are shown in FIG. 4 and are tabulated below:
Figure imgf000122_0001
Figure imgf000123_0001
{0364} The results for a 24mg/kg oral dose of CNS-102 are shown in FIG. 5 and are tabulated below:
Figure imgf000123_0002
m n _ Example 27; Calculation of total brain to total plasma concentration ratio (Kp) of 5- irms hosier of GGA's in rats,
[0365] The ratio of total brain concentration to total plasma concentration (Kp) tor CNS- 102 was calculated. First, the total plasma concentration of CNS- 102 (in two different formulations) was calculated as described in Example 24. The total brain concentration of CMS- 102 in ng g for the same animals was also measured.
(0366) AUC(O-Iast) (min*ng ml}: area under the concentration-time curve from zero up to the last measurable time-point.
(036/] lz (1/mln): the first order rate constant associated with the terminal (log-linear) portion of the curve, estimated by linear regression of time vs. log concentration.
{0368] Terminal Half-fife (tl 12) - in{2)/lz: calculated using Lambda_z method to find best fit. If necessary, the concentration-time points were manually selected for use in the calculation, Bolded-italicized concentrations indicate points used for calculation.
[0369J AUC(O-inf) (mm*ng/tnl): Area under the concentration-time curve from zero up to infinity, based on the last observed concentration. Requires lz.
[0370] Ratio of Total Concentration of Drug in Brain relative to plasma (Kp):
,r AUCtot, brain
κρ -—
A UCiot, plasma
[0371 ] where AUCtot is the area under the concentration-time curve for total (bound and unbound) concentrations in brain or plasma.
[0372] The ratio data are summarized in the graphs in FIG. 6 and 7, as well as in the tabulations below (boided concentrations indicate points used for calculations).
Figure imgf000124_0001
Figure imgf000125_0001
Formulation 1 and Formulation 2 refer to the following: Formulation # 1 (Dose: 0.200 g/Kg)
Gum Arabic 5%
Sodium Chloride 1%
Alpha-tocopherol 0.008%
CNS-102 4%
D. I. H20 90% Formulation #2 (Dose:0.200 g Kg)
Gum Arabic: 5% Kydroxypropy] Cellulose (Mn= 100,000) 3%
Sodium Chloride 1 %
Alpha-tocopherol 0.008% CNS-102 4%
D. ί. H20 87% Example 28: C S-102 induces Heat Shock Protein Expression In Vitro
(03731 Murine neuro2A neuroblastoma cells were treated with various concentrations of CNS-102 (all tram-GGAl CNS-101 (mixture of GGA isomers), and CNS-103 (ail cis-GGA) for 48 hours. Differentiation was induced and cells incubated with geranyigeranyl transferase I inhibitor, GGTi-298. Cells were harvested and lysates were prepared and analyzed by western blot for HSP70 and 11SP90. Western signals in the absence of the compounds were normalized as 1.00. The results are tabulated below.
CNS-I IK-induced MSP Ex ression in euro2A Cells
Figure imgf000126_0001
(0374] Both CNS-102 and CNS-101 induce expression of HSP70 at various concentrations, and the GGA cis isomer (CNS-103) failed to induce expression of HSP70.
Example 29: Effect of CNS-102 on Neurite Outgrowth
(0375] Murine neuro2A neuroblastoma cells were treated with various concentrations of CNS-101, CNS-102. or CNS-103 for 48 h. Differentiation was induced and cells incubated with geranyigeranyl transferase I inhibitor, GGT1-298. After 24 h digital images were taken and neurite outgrowth was quantified. The comparisons of the neuroprotective effect of these three compounds are shown below.
Performance of CNS-102 and Comparison ιο CNS-101
[0376] The estimated mean iogio counts, from five replicate welis for each treatment group were calculated by P OC G1..M in SAS assuming a pooled estimate of variation. The anti-log values are given as the estimate of the median values and are tabulated below. Number of Cefis with Neurite Outgrowth when Treated with C!VS-SOI or CNS-102
Figure imgf000127_0001
[0377] The median count is estimated to be 95.5 in the absence of inhibitor and 23.5 when ceils are treated with inhibitor and PBS. This inhibited outcome is 0,225 of the uninhibited count (Relative Performance), and represents 0% protection from the adverse effects of the inhibitor (% Protection). The collected results are graphically plotted in FIG 8A. Increasing concentrations of CNS-102 (green) and CNS- 101 (red) results in increases in median counts with a maximum protection of -40% for CNS- 102 and 27% for CNS-101 -
[0378] The graph shows that both CNS-102 and the isomer mixture CNS-101 , give protection in a range from 10 nM to 10000 nM. The CNS-102 treatment consistently pro vides more protection than does CNS- 101 , even at the highest dose, which has reduced counts over the maximum seen at 1000 nM,
Comparison of Performance of CNS- J 03 and CNS-102
(0379] The estimated mean log 10 counts from five replicate weds for each treatment group were calculated by PROC GIM in SAS assuming a pooled estimate of variation. The anti-log values are given as the estimate of the median values and are tabulated below.
? Number of Cells with Neurite Outgrowth when Treated with CNS-182 orCWS-103
Figure imgf000128_0001
{0380| The median count is estimated to be 60,3 in the absence of inhibitor, and is 18,5 when cells are treated with inhibitor. This inhibited outcome is 0,307 of the uninhibited count (Relative Perfommnce). and represents 0% protection from the adverse effects of the inhibitor (% Protection), 'The collected results are graphically plotted in FIG 8B. The maximum protection is -36% for CNS-102 (green) and ~26% for CNS-103 (orange), .
[0381 ] The graph shows that both CNS-102 and the all-ay isomer, CNS-103, give protection in a ratige from 10 nM to 10000 nM, with the peak at about 100 tiM, and CNS-102 providing greater protection at the optimal, concentration of 100 nM
Comparison of the Effect of GG A isomers on Nmite Outenwth
[0382j Differences in mean 3og(counts) between treatments for each concentration were calculated using PROC GLM in SAS. The p-value given is for the comparison of the estimated difference to zero, the expected difference value for no treatment effect. The anti- log of the differences represent the estimated ratio of counts (relative effect on inhibition) and are tabulated below along with the lower and upper 95% confidence intervals.
Comparison of Ratio of Neurite Outgrowth
Figure imgf000128_0002
(0383) There is insufficient difference between the treatments at ail but one concentration for statistical significance at the 0,05 level. There exists, for CNS-1Q2 and CNS-101, a concentration dependent trend in the ratio estimate above a threshold concentration (100 nM, p = 0,06) as graphically shown in FIG 8.C, Rescue Ratio for CNS-102/CNS-I0I versus Log!O Concentration, It is notable that the ratio continues the trend at 4,0 log concentration even in the face of significant depression of the total number of cells exhibiting neurite outgrowth at this highest concentration, shown in the previous graphs in FIGS. 8A and 8B.
[0384J From this it can be concluded that CNS-102 is more effective at protecting neurite outgrowth than is either the GGA mixture or cis GGA,
Example 3D: Time Course of CNS-102 Induced HSP70 Expression in Vivo
0385| The time course of protein expression, as measured by western blot for HSP70, was determined in triplicate for hippocampus, and cortex tissue samples taken f om each of 5 animals per group at each of four time points (24, 8, 72, and 96 h) after treatment with either PBS or 12 mg/kg CNS-102, administered orally. The average expression for each treatment group is calculated at each time point for each tissue using PROC MLXED in SAS and are tabulated, along with the difference (delta) between treatment averages and a p-value comparing the difference to zero, below.
HSP70 Expression Fct!Io ing Administration of CNS-102 vs PBS
Figure imgf000130_0001
[0386] Expression of HSP70 was observed after CNS-102 administration and the difference between CNS-102 and PBS induced expression (delta, in the table) in both the cortex at 24 h a d the hippocampus at 72 h was statistically significant (bolded in the table).
1038?] These results demonstrate that CNS-102 induces expression of I-ISP7G as measured in the corte 24 h after administration while in the hippocampus the level of HSP70 was not significant until 72 h after administration. No significant levels of HSP70 were found in the cortex after 24 h, howe ver since no time points before 24 h were taken, it may be that HSP70 is expressed earlier, in the hippocampus the expression appears to peak after 48 h with significant levels measured at 72 hours.
Example 31; CNS-102 Induces Expression of Seleciiid Heat Shock Proteins Irs Vivo
[0388] CNS-102 was administered orally to Sprague-Dawie rats at ] 2 mg/kg and brain tissue was extracted at 12. 24, 48. and 96 h after dosing. Expression of selected HSPs was detected using q T-PCR with expression of the GAPDH gene used as the control. Results are shown in FIG. 9. To represent the effect of CNS-102 on HSP expression, mean fold change is calculated as CNS-102 treated/PBS treated for each HSP at each time point,
[0389) Gene expression of HSP60 (chaperonin), HSP70, and HSP 0 was increased in CNS- 102 treated aniraals compared to PBS treated animals for up to 96 h post dose with peak effects generally after 48 h post dose.
Example 32: Determination, of Time Course of Neuroprotection by CNS-102 is the Kainic Add Model
[0390] CNS-102 was administered orally to Sprague Dawk-}? rats at 12 mg/kg. After dosing, A was stereotactically injected into the hippocampus at 24, 48, 72, 96 and 168 hours to induce neuron damage. After 24 h, hippocampus tissue was collected, stained, and imaged. The scans were analyzed by Image J to calculate the fraction of ceils having KA-induced damage. Average fraction damaged in the hippocampus from ten ammals for each treatment (PBS vs. 12 mg/kg CNS- 102) by time (24, 48, 72, 96 arid 168 hours) combination was estimated with PROC MIXED in SAS, Comparisons of the CNS-102 treatment groups to the pooled PBS treatment at the different times can be made, giving the results tabulated below.
Ti it) e Course of Neuroprotection by CNS-i02
Figure imgf000131_0001
[0391 j The results of this study demonstrated a statistically significant (p- value of 0.004) efficacy of neuron protection by a single dose of 12 mg/kg of CNS-102. The maximal protection effect by CNS-102 was achieved in this model at the 72 h timepoint. This time frame was incorporated into subsequent concentration dependence studies.
Example 33: Concentration Dependence of Neuroprotection by CNS-102 in the Kainic
Acid Model
[0392] The fraction of cells damaged in the kainic acid model was determined on 130 rats, each receiving one dose of CNS-102 at either 0, 1 , 3, 6, 12 or 24 mg kg. PBS was used for
29 dosing at 0 mg kg. The linear model of the logic percent damage versus concentration is graphically shown FIG. 10,
[0393 j The ANOVA results for the model are tabulated below. The concentration dependence is statistically significant with a p-value of 0.007.
ANOVA Table for Concentration Dependence of Damage in Kaiaic Acid Rat Model
Analysis of Variance For L% D
No Seiector
1 30 total cases of which 1 1 are missing
Source d» burns of squares Mean Square F - rat io Pro b
Const 1 471 .706 ' 471.706 982.53 0.0001 cm 1 3.58419 3.5841 9 7,4556 0.0073
Error 1 17 56. 1 708 0.480092
Total 118 59.7550
[0 94 j These results demonstrate that neuroprotection by CNS-302 is concentration dependent with increasing protection up to at least. 24 mg kg. This study supports 12 mg/kg as the mini-rial effective dose.
Example 34: .Effect of CNS-102 on Survival in the SODl Mouse Model of ALS
(0395) CNS-102 was administered daily b oral gavage to male SOD l and wild type mice at 12 mg/kg beginning on postnatal day (P) 38, Groups of SOD i mice were also administered 8 mg/kg riiuzole.
|0396] Survival data showed no transgenic animals survived past age PI 53. Longer survival for subjects in group A (CNS-102) and B (riiuzole) was observed. In all testing, both wiidtype groups performed better than any transgenic group. There was negligible difference between wiidtype animals dosed with vehicle versus CNS-102, suggesting that treatment with CNS- 102 does not lead to obvious toxicity or behavioral abnormalities as tested and observed in the study,
{§397] The survival data was analyzed using PROC LIFE-TEST in SAS. The wild-type treatment groups were removed from the study since all of these animals, save one. survived to the end of the study. The remaining four groups were subjected to the standard survival analysis by the method of Kaplan-Meier, with median estimates tabulated below, and survival shown graphically in FIG. 1 1. Median Survival for Drug Treatment Groups in SOD? Mice
Figure imgf000133_0001
[03981 A survival analysis comparison of the two drugs and the vehicle was conducted using PROC IJFETEST, yielding a p-value of 0.04. This outcome indicated that the data contains evidence of a significant difference between at least two of the three single compound treatment groups A, B, and D.
[0399] Parametric regression with PROC LiFEKEG in SAS, using the gamma distribution as the model (0=0.0024), was conducted to further elucidate the nature of this difference. Separately comparing each of the two drag treatment groups A and B with the vehicle D, finds thai there is significant, evidence of differences for both groups. The magnitude of change in median time to death is +9% for the CNS-102 treatment group and +6% for the riluzoie treatment group, respectively, compared to that of the vehicle group as tabulated below. p- Values for Treatment Group Comparisons to Vehicle Grosp
Figure imgf000133_0002
Example 35: Effect of CNS-102 on CaiWalk
[0400] Cat Walk results showed a severe change in gait during onset and progression of disease. Prior to onset, there was little to no difference between the transgenic and wikitype groups. Transgenic animals tended to rely more on front paws for movement as weakness and paralysis in the hind paws progressed. This began to show around timepoint PI 00, coinciding with the expected onset of disease. This impairment was seen through changes in front and hind paw step cycle, print area, mean intensity, and base of support. [04011 In general, group A (CNS- 102) outperformed the riliizoie-tfansgenic treatment group in Cat Walk. This was seen in. multiple time points and through several metrics. Group A had a lower ran duration, lower % body, and higher regulatory index, suggesting a less- impaired gait. There is very littie difference between i!dtype groups E (vehicle) and F (CNS-102). See, FIG 12.
[0402] Three neurological function outcomes (stride length, run duration, and swing speed) were compared for CNS-102 and vehicle by the Bootstrap method. Prior to Pi 20, there were only small differences between treatment group averages. By PI 20, the progression of the disease had both increased the measured differences between CNS-102 and vehicle and removed significant but unequal fractions of each treatment group, 'Hie p-value for each difference in CNS- i 02-rninus- vehicle averages is tabulated beiow.
Bootstrap Comparison of Averages at P120
Figure imgf000134_0001
[0403] A minus-one jackknife procedure demonstrated that no single animal unduly influenced the construction of the reference distribution, thereby validating these p-values. Clearly, the fact that all three metrics show significantly better outcomes compared to PBS. indicates that CNS-102 not only prolongs survival but also slows the symptomatic progression of the disease.
Error! Not a valid link. CNS-102-vehicie/P124(+/-)3: P (bootstrap comparison method)^ 0.0062
Error! Not a valid link CNS - 102-vehicle/P 124(+/-)3 : P (bootstrap comparison method)= 0.0206
Example 36: Effect of CNS-1 2 si Blood Chemistry and Hematology
[0404] Blood and serum samples from days P70 and i 0 were collected on three animals from each of the six treatment groups. Hematology measures were white blood count, red biood count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, and platelets. Chemistry values were alanine aminotransferase, aspartate aminotransferase, albumin, total protein, gamma globulin, blood urea nitrogen, cholesterol, and glucose. No analyses were conducted on any samples collected after PI 00.
[0405] Each outcome was fit to a linear discrete model with the age of the animal included as a covariate. None of these outcomes in either hematology or chemistry showed statistical significant differences between any of the 6 treatment groups, except for glucose. In this case, the observed difference resides in the mouse strain (SOD1 vs. wild type), not in the treatment groups A-D, or E-F.
[0406] In conclusion, there are no statistically significant treatment differences. There is a genotype difference in the glucose outcome. Time is a significant factor in several of the observed outcomes (red blood count, platelets, and hematocrit) across all treatments in the SOD1 animals, attributable to the progression of the disease state.
Example 37; Determination of Neuroprotection by CNS-102 in the presence of GGA cis- isorner (CNS-103) in the Kainic Acid Model
|0407] GGA trans isomer (CNS-102) was administered orally to Sprague Dawley rats at 24 mg/kg. In parallel experiments, a mixture of GGA cis- and trans isomer (10:90) was administered orally to Sprague Dawley rats at 26.66 mg/kg. An amount of GGA trans isomer that contained in the mixture was equivalent to 24mg/Kg. After dosing, KA was
stereotacticaliy injected into the hippocampus ai 72 hours to induce neuron damage. After 24 h, hippocampus tissue was collected, stained, and imaged. The scans were analyzed by Image J to calculate the fraction of cells having KA -induced damage.
GGA Mean of SE
Hippocampus CA3
neurons damaged
(Arbitrary units)
CNS-102 (24 mg/Kg rat) 19.8182 1.9756
GG A els- and trans mixture 23.2589 2.2362
(10:90) [0408] In the presence of the GGA cis-isomer, neuroprotective effects of the GGA trans isomer were diminished demonstrating that the cis isomer inhibits the neuroprotective activity of trans isomer.
Example 38: Efficacy of compounds ia alleviating tieu rodegeneratkm induced by Kainic acid,
[0409] The indicated compounds or vehicle control were orally dosed to Sprague-Dawiey rats, and Kainic acid was injected. After dosing, KA was stereotaetieali injected into the hippocampus at 72 hours to induce neuron damage. After 24 h, hippocampus tissue was collected, stained, and imaged. The scans were analyzed by Image J to calculate the fraction of ceils having KA-induced damage. Neurons damaged by Kainic acid {mean of hippocampus CA3 neurons damaged) were quantified. These results are depicted in the followin tables:
Figure imgf000136_0001
e c e: rag g ose, = .
Figure imgf000136_0002
Figure imgf000137_0001
Figure imgf000137_0002
Vehicle 20.il
Figure imgf000137_0003
Vehicle 20.1 S
Figure imgf000137_0004
Figure imgf000138_0001
Vehicle 20.11
The results for the following compound were obtained employing a stereotactics! surgical injection, as is well known to the skilled artisan.
Figure imgf000138_0002
Vehicle 20.35
Example 39; The effect of CNS102 on pmn hiik.-n of Rap I in fVean>2A cells
[0410] Mouse neuroblastoma Neuro2A cells were cultured in DMEM supplemented with 10% FBS for 24 his. The cells were then either treated with CNS-102 only for 48 hrs, or treated with CNS-102 for 24 hrs first before GGTase inhibitor GGTI-298 was added to the culture medium for additional 24 hr incubation. At the end of 48 fir treatments, whole cell lysates were prepared from both control and the treated cell populations for western blotting analysis using antibodies specific for the un-prenylated or total level of RAPL Both un- prenylated and total Rap 1 protein bands were quantified using Image J arid, normalized to their respective loading control GAPDH. The data shown in the chart were averaged from two repeats of one experiment and presented as the ratio of normalized un-prenylated Ra l t total Ra l.
Figure imgf000139_0002
Compared to control, CNS 102 treatment at 100 nM appears to slightly increase the prenylation of Rapl in Neuro2A cells (student's t-test, P - 0.08).
Example 40: The effect of CNS102 on activity of Rho GTPase in Neuro2A cells
{0411] Mouse neurobl stoma Neuro2A cells were cuitured in DMEM suppiemented with 10% FBS for 24 hrs. The cells were then either treated with CNS-102 for 48 hrs, or treated with GGTase inhibitor GGT!-298 for 24 hrs before harvesting. Cell lysates were quantified and subjected to active Rho assay using G-LISA RhoA activation Assay Biochem it from Cytoskeleton Inc. The data were averaged from 4 repeats of one experiment with background subtraction.
Figure imgf000139_0001
Example 41: Stereotactic injections of Kasaie acid into rat hippocampus tissues led to increased damages in neurons.
( 0412J Stereotactic injections of Kainic acid into rat hippocampus tissues led to increased damages in neurons. However, when GGA trans isomer (CNS-102) was dosed before the A injection, neurons were protected. Under this condition, neurons and HSP70 expression was stained by imraunohistoebemistry techniques. HSP70 expression was induced in a very specific area only that KA was injected. The induced area was not stained by HSP70 antibody when KA was not injected, but CNS-102 was dosed, Therefore these data suggest that HSP70 induction by dosing CNS-102 which protects neurons really depends on neuron damages or stressors. Furthermore, interestingly, the induced area did not match with neurons. Therefore, the data suggest that glial cells play an important role in the HSP70 induction, but not neurons,
10413] HSP70 induction by dosing CNS-102 in the absence of the KA injection was limited in western blot experiments using hippocampus lysates. 120% or 130% of'HSP70 expression could be seen by comparing those of vehicle dosing control experiments. But by siereoiactically injecting KA in hippocampus tissues, a strong induction of HSP70 was seen very clearly, and the induction was very localized in the injection site only. The results are shown in FIG. 13.
Without being limited to a particular theory, it is believed that one or more of the following may be occurring:
1. CNS-102 will activate m NA stability of HSP70, and the stabilization will be involved in activities of micro RNAs (378 and/or 71 1),
2. The stabilization will raise a minimal amount of HSP70 induction. But when a stressor or toxin that include SOD1 mutants of ALS, tau aggregations of Alzheimer disease, A-b aggregations, etc attacks neurons or glia ceils, HSP70 is strongly induced in a specific area only,
3. CNS-102 will be a facilitator that optiraally induces HSP70 in cells or neurons damaged by those stressors only. HSF1 will be a key driver to strongly induce HSP70 transcription.
Example 42s Sereeisisg for compounds that post-iranseriptionally regulate HSP 70 activity,
Hsp70.3-hiciferase reporter constructs can be made using either a CMV promoter or the endogenous Hsp70,3 promoter (3974 bp upstream from the protein coding sequence including the 5 -UTR). The Hsp70.3 promoter can be PCR-puritied from genomic DNA of C57/B16 mice and cloned into the Promega pGL4.10 hiciferase reporter vector. The CMV promoter used can be isolated from the Promega pGL4.75 vector. The Hsp70.3 ,3'-UTR sequence (1253 bp from the end of the protein coding sequence) can also be PCRpurii!ed from genomic DNA of C57 B16 mice, All constructs may be amplified m Escherichia coli under the selection of 100 pg/m! ampicillin and sequence-verified prior to use. The test, control, 5-trans GGA or derivative compounds CM men be added to the cells in varying amounts. For luciftrase assays, cells can, be rinsed with sterile PBS, 40 ul of celt culture lysis buffer (Promega) can be added per well, and plates can be incubated at room temperature for 5 min. 100 μΐ of hiciferase assay reagent (Promega) can then be added per well, and luminescence can be read immediately. The data (liiciferase output) for each 3 -UTR construct can be normalized to the same promoter construct lacking the 3 -UTR,
}0414| It is contemplated that the addition of GGA trans isomer will increase lociferase activities to the level of CMV -Liiciferase, This will suggest that the addition of GGA trans isomer removes the suppressive effect of the HSP70 3' UTR, and that the addition increased HSP70 inRNA stability. Activities of analogues of GGA trans isomers and other test compounds can be evaluated in the experimental system,
{0415] The above assay may be further modified to include increasing doses ofmiR-378, miR-71 1 , or an Hsp70.3 3 -UTRspecific siRNA. It is contemplated that the addition of GGA trans isomer will increase reporter activities of CMV-I,uciferase-U3 suppressed by raiRNA- 378 and aiiRNA-71 1. This would suggest that the addition of GGA trans isomer removes the mENA suppression by miR A-378 and miRNA-711. Activities of analogues of GGA trans isomers can be evaluated in the experimental system. Other test compounds and GGA derivatives may he analyzed by the same assay.
Example 43: SOI) I Mice Tests
{0416] Four treatment groups of 16 animals each giving a total of 64 male transgenic SOD1 G93A and two treatment groups of 30 wild type mice each, organized into two cohorts,
[0417J Transgenic mice were randomized into the four experimental groups and were administered CNS 102, rihizole, CMS 102 and riluzoie or Vehicle {N=io7group). Wild type mice were randomized into two experimental groups and administered either
CNS 102 or vehicle (N-10),
|0418] Daily dosing of 12 rag/kg of CNS102 alone, 8 mg/kg of riiazole alone, 8 mg/kg of riluzole plus 12 mg/kg of CNS102, or Vehicle were administered beginning postnatal-day (P) 38 at a volume of 8.88 mL/kg body weight, via a single bolus oral gavage injection, (0419] The transgene number of the individual SOD1 mice used in this study was determined by qPCR. There was no difference in the means of the copy numbers in the different treatment groups (86 ± 18 and 91 ±16 for Vehicle treated and CNSI 02 treated, respectively; Student's t-test: p = 0,50). la addition, there was no evidence of correlation between transgene copy number and survival (Figure 16). The correlation coefficients were r - 0.49 (p - 0.12) for Vehicle treated mice and r = 0.24 (p = 0.46} for CNS 102 treated mice,
{'0420) Analysis of the 16 animals from each of the CNS 102 and Vehicle control treatment groups gives 7,5% and 6.4% increase in median arid mean life span, respectively, for those animals receiving CNS i 02 compared to Vehicle control,
[04211 Comparison by a parametric method under the assumption that a generalized (3 parameter) gamma distribution is an appropriate model for survival (PROC LIFEREG SAS) found a 9.5% increase in the estimated location parameter for the distribution of deaths in the group treated with CNS 102 over that for the group treated with Vehicle alone (p = 0.001 ), This difference translates to approximately 12 days. Cox proportional hazard analysis found a p- value of 0.021 (PROC PHREG, SAS).
[0422] Body Weight Body weights were recorded daily during the first two weeks of the study and 3 times a week thereafter until necropsy. Body weights were taken immediately prior to closing, Over time it wasobserved that C S 102 slowed the loss of body weight relative to Vehicle control Below is a plot of the mean weight of each group for all of the surviving animals.
{0423] Statistical analysis (tabulated below; Anova, DataDesk, Data Description) found that the treatment x day interaction term is significant, indicating that this --50 day range of data from the two treatment groups have different slopes. The Vehicle (blue) had a slope of -0, 135 and the CNS 102 (red) treated group had a slope of -0.081. This treatment difference corresponds to a reduction of over 40% in the rate of weight loss compared to vehicle treated animals,
Anova for Body Weight Data
So u rce df Sums of Squares Mean Square F - r a t i o P ro b Const 1 1 6899.4 1 8899.4 1 09403 < 0.0001
1.54021 1 ,54021 9,971 2 0.0037
36.7620 36.7620 237,99 < 0,0001
2.36223 2.36223 1 5.293 0.0005 4.47951 0.1 54466
48.6503 trt 1
day ^
trt*dsy
Error ^9
Total 32
{0424J Analysis of body weight at P127 showed that CNS102 has slowed the progressive loss of weight relative to the Vehicle (Figure 18; Average weight 8.1% greater than that of the control, p = 0.0325)
{04251 Neuroscore. Neuroscores were assigned to subjects based on a 5-point scale [Bruestle, 2009J with 0 representing normal gait and 5.0 representing advanced paralysis and lack of righting reflex. Average neuroscore for all animals at each time point is plotted below (Figure 520) for the CNS 102 and Vehicle control treatments for both SOD! and Wild type groups. Mote that this metric is set to the highest score (5.0) when an animal died or was euthanized, and remained at 5,0 for the duration of the study.
Fitting a linear model to the average neuroscore data from 1.0 to 3.5 gives slope estimates for each of the two treatments. These treatments are indicative of the average rate of change of the score over time and represent days 110 to 135 for Vehicle and 1 30 to 150 for C S102.
{04261 The slope estimate for Vehicle (red) is 0.124 and that for CNS 102 (blue) is 0.093, and are statistically significantly different (p <= 0.0001 ). The rate of increase of the neuroscore is 33% greater for the vehicle, which is taken as a measure of the ability of CNS102 to lower the rate of loss of motor function. [0427} Grip Strength. Comparison of the average strength at day 325 using a standard t- test indicates a significant difference with a p- alue of 0.006. Converting the difference in average strength (vertical axis) to a difference in time (horizontal axis) using the slope estimate predicts that the redaction of motor strength to 1 Newton occurred 8 days later with CNS 102 (125 days) compared to the Vehicle (1 17 days) (6.8% difference). This is contemplated to indicate an 8 delay in ihe time to reach the 1 Newton level of motor dysfunction with CNS 102 treatment.
As stated previously, CNS 102 is contemplated to target multiple mechanisms relevant to familial and sporadic ALS. Example 44: Kainic Acid Model Results
[0428) HSP Induction. Applicant has demonstrated that CNS102 induces HSP expression in various model systems and tissues, including the CNS, Previous studies using high doses of ieprenone showed induction of HSP70 accompanied by
neuroprotection in in vitro models: one mimicking Parkinson's disease-related degeneration, and a second, using proteasome impairment in cultured motor neurons, simulating conditions that may be found in ALS. To evaluate a role for CNS 102 in HSP induction, a series of experiments using in vivo and in vitro models was performed.
[0429] The kainic acid (KA) rat model to test this mechanism in vivo was utilized.
Preliminary studies using a single CNS 102 dose revealed reduced damage in the CA3 region of the hippocampus vs. the vehicle-treated cohort. In a follow-up experiment, CNS 102 (100 mg/kg) was administered daily by sublingual administration five days before the stereotactic surgery when KA or vehicle was injected bilaterally to the CA3 region of the hippocampus (Figure 23).
[1)430] In this experiment, on the surgery day, CNS102 was administered 1 hour before kainic acid or vehicle injection, and on the perfusion day, CNS 102 was administered 4 hours before perfusion. The toxin KA or vehicle was bilaterally injected by stereotactic surgery into the C A3 regions of both hippocampi of each rat (Figure 23). Upon tissue harvesting, each brain was removed from the rat skull, one half of which was subjected to snap freezing in liquid nitrogen for protein and total RNA extraction, and the other half was subjected to immersion fixation in 4% PF A followed by cryosectioning for histological examination of neuron loss, HSP70 expression, apoptosis and inflammation.
Tissues samples from hippocampus were analyzed for GRP78 using Western blot and HSP70 using ELJSA. and the p-value was calculated by Student's t-tes
[0431 ] The results (Figure 24) provides evidence that CNS 102 is able to increase both HSP'70 and ER-residerit GRP78 chaperone protein expression under basal conditions in the rat hippoeampal tissues. The effect is small but statistically significant. CNS 102 administration markedly increases both chapcrones HSP70 and GRP78 following the stress conditions caused by KA challenge.
j0432j These outcomes indicate that CNS102 may not only help to improve the protein folding through HSP70 and GRP78 chaperoning function, but also help the regulation of unfolded protein response (UPR) through GRP78. The latter is a key regulator of E stress. As ALS is characterized by activation of the UPR signaling and the accumulation of the intracellular or extracell ular aggregates of misfoided proteins or mutated gene products, CNS I 02 may function as an effective therapeutic strategy to augment the expression of 5 ISP70 and G P78. and curtail the progression of neurodegenerasiors through HSP 70- and GPR.78- mediated neuroprotective functions,
(04331 CNS 102-mediated HSP70 induction was also revealed in cerebral cortex (relevant to upper motor neurons) and hippocampus following a single oral dose in naive i.e., non- stressed animals. In this experiment, the time course of protein expression was determined in triplicate for these regions taken from 5 animals per group at four time points (24, 48, 72, and 96 h) after treatment with PBS or 12 mg kg CNS 102, administered orally. The average expression for each treatment group is calculated at each time point for each tissue using PROC MIXED in SAS and is shown, along with the difference (delta) between treatment averages and a p- value comparing the difference to zero. At 24 fa following CNS 102 administration, HSP70 was significantly elevated in cerebral cortex, and at 48 h, in the hippocampus.
Time Course of Brain HSP70 Measured by Western Blot.
Figure imgf000146_0001
[0434] It is demonstrated that CNS-102 upreguiaies HSP70 in vitro, in the murine neuroblastoma cell neuro2A (N2A) system (Figure 25). Furthermore, G P78, HSP90 and the HSP70 co-chaperone HSP40 also are induced in dose-response experiments. Of interest, the HSFl, a critical HSP70 transcription factor, is also subtantiali upregulated. This effect and its implications are discussed in greater detail below.
Example 45: Cell based results j 0435 J induction of HSPs is regulated transcriptionally by interaction of a consensus cis- elemenl (heat shock element; HSE) with a heat shock transcription factor ! (HSFl ). HSFl , in a trimer conformation, binds specifically to HSE located in the FiSP promoter region. Under normal conditions, HSFl resides as a monomer in the cytosol. Interaction with HSP70 prevems formation of the transcriptionally active HSF 1 trimer. Under stress conditions, increased protein misfolding occurs, and the binding is reduced,
[0436 [ Interestingly, a OGA-bindmg domain has been identified at the C terminus of HSP70, OGA interacts with this sequence effectively inhibiting its binding to HSFl . This raises the possibility thai CMS 102 may act at the GGA -binding site and prevent HSP70- mediated inhibition of HSF1
Figure imgf000147_0001
activity at the HSE site.
N2A ceils were iransfected with a construct containing the 5'UTR of Hspala, the primary HSP70 subtype, with a iuciferase reporter. Cells were then treated with
CNS 102 or vehicle. CNS 102 is able to induce HSP70 promoter activity even in the absence of stressor (Figure 26).
[0437] Prenylation and Ncurite Outgrowth. Additional experiments were conducted in vitro using the N2A celi line to test the idea that CMS 102 acts by modulating prenylation based on its structural similarity to geran lgeranyl pyrophosphate (GGPP).
N2A cells were incubated with vehicle or varying doses of CNS 102 and the level of Ra lA prenylation was measured. As shown in Figure 27 (top panels), CNS 102 gives approximately 60% increase in Rapl A prenylation, while geranylgeranyltransierase I inhibitor, GGl'h decreases RaplA prenylation. This indicates that CNS 102 can potentially regulate RaplA activity. It has also been shown thai CNS i 02 can increase the active RhoA level in N2A cells, compared to the inhibitory effect of GOTI on RhoA activity. In addition, increasing concentrations of CNS 102 promotes neurtte outgrowth of N 2A eel Is (Figure 27, bottom panels).
[0438] This data provides evidence that CNS102 may function through prenylation of small G protein to regulate neuronal morphogenesis and synaptic plasticity during neuronal regeneration and contribute to regenerative processes in neurodegenerative diseases. Example 46: Beneficial inflammation and other beneficial sreaimeni outcomes
[0439] The effect of CNS 302 on neuroinfiammation using a microglial marker was also examined. Expression in rat hippocampus 24 hours after A treatment was evaluated by Western blot analysis and a statistically significant increase in this microglial activation marker was observed (Figure 28). Thus, these results indicate a novel and additional mechanism by which CNS 302 contributes to neuroprotection.
[0440] Based on these data and other findings shown herein, it is contemplated thai treatment with CNS 102 can improve several mechanistic outcome measures (motor neuron denervation' europathoiogy in brain and spinal cord) in the SOD1 model.
[0441] A notable metric to demonstrate the delay of disease onset is shown in the survival curve itself, Figure 17, There is no statistical evidence that the slopes of the two curves are different, but the median is delayed by 8 days in the CNS 102 treated group. Additionally, the loss of motor function as measured by the mouse tail hang test discussed below in Question 14, shows a marked delay.
(0442] While there was no change in the slopes of the K/M survival curves, it was found that several different metrics demonstrate that CNS 102 slows disease progression, which results in the 8-day difference in the median survival time. Those metrics thai indicate a reduction of the rate of disease progression include body weight loss (Figure 19) and the neuroscore data (Figure 21). Additionally, a reduction in progression of motor deficits was observed in Run Time on CatWalk (Figure 30) and the Tail Hang Test (Figure 31 ).
[0443] CatWalk, Mice were tested in CatWalk every two weeks for three months, and thereafter on a weekly basis xmtil end of study. CatWalk (Moldus Information Technology) is a quantitative gait analysis system measuring pressure from the subject's paws illuminated on a glass walkway. The behavior experimenter, who was blind to the experimental group, labeled each paw on the recorded video after the trials. After classification was completed, gait parameters were analyzed.
[0444 j CatWalk analysis provides evidence that disease onset was significantly delayed in CNS 102 treated mice, A significant difference in stride length between control and the treated animals was observed al PI 27 in SOD1 mice (Figure 29) as determined from foot print maps from the Catwalk behavioral test
{0445] The gait impairments of PI 27 SO 1 mice were quite obvious when observing the mice traveling on the CatWalk (Video 1 , Video 2). CNS102 treatment resulted in marked better running, as evidenced by a significantly faster run speed compared to Vehicle treated mice (Figure 30).
[0446 J Analysis of swing speed from the CatWalk at P 120 showed thai CNS 102 had slowed the progressive loss of mobility relative to the vehicle (see tables below; Average speed 50% greater than that of control, p = 0.01 ).
Table of Mean Swing Speed
Figure imgf000148_0001
p- Values for Comparisons between Means
Figure imgf000149_0001
[0447] Tail Hang Test. For the Hang Test, scores were assigned on a scale ranging from
0 with full extension of hind legs away from iateral midline when suspended by tail to 0.5 exhibiting collapse or partial collapse of leg extension toward midline (-weakness).
Animals were suspended by the tail for several seconds while a trained observer monitored limb movement. The hang test scores were taken in conjunction with neuroseores.
[0448| CNS 102 delays onset of test score rise by approximately 15 days at day 1 15, and suppresses the slope of that rise by >20% over the time period of day 100- 140, with p- vaiues of -0,001 and—0.03, respectively, as determined by a general linear model using DataDesk from Data Description.
[0449] The neuroprotective effect of CNS 102 was tested in KA-challenged rats, a model for excitatory neuronal damage. The toxin KA was injected into the hippocampus 1 ,2,3,4 oi 7 days after a single oral dose of 12mg/kg CNS 102. The brains were harvested 24 hours later and prepared for cresyl violet staining and digital imaging. The damaged area in the CA3 and CA2 areas of the hippocampus were quantified as a percentage of the total area (Figure 32). 72 hours after CNS 102 administration a significant reduction of KA induced damage compared to vehicle treated rats (p- values were determined by Student's t-iest) was found.
Example 47: PK Results
[0450] A PK study was conducted in rats with 12mg kg CNS 102 administered intravenously and by oral gavage. Bioavailability (f) was determined by dividing AUC of PO dosed rats by the AUC of IV dosed rats (Figure 33).
Although oral administration of CNS 102 shows very good absorption, it was found thai CNS 102 absorption by sublingual administration is about five times more efficient than oral gavage (Figure 34),
[0451 ] To better understand the pharmacokinetics of C S102 and its potential for brain penetration, CNS 102 was administered sublinguall in a daily dosing regimen comparable to an SODl experiment. CNS 102 (lOOmg/kg) was given either in a single dose or repeatedly for 6 consecutive days; plasma and brains were harvested at 0.5h, 1 h, 2h, 4h, Sh, and 24h after the last dose (Figure 35), No significant differences were found in the PK profiles of single versus repeat dose administration. The p(brain) values were found to be 7% and 4%. respectively.
[0452] To obtain data on the safety of chronically administered CNS 102, blood and serum samples were collected in the SOD! study. Samples were collected from days P70 and PI 00 on three animals from each of the six treatment groups.
[0453] Hematology measures include white blood count, red blood count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, and platelets. Chemistry values were alanine aminotransferase, aspartate aminotransferase, albumin, total protein, gamma globulin, blood urea nitrogen, cholesterol, and glucose. No analyses were conducted on any samples collected after P I 00,
[0454] Each outcome was fit to a linear discrete model with the age of the animal included as a covariate. None of these outcomes in either hematology or chemistry showed statistical significant differences between any of the 6 treatment groups, except for glucose. In this case, the observed difference resides in the mouse strain (SOD 1 vs. Wild type), not in the treatment groups.
[0455] In conclusion, there are no statistically significant treatment differences in hematology or blood chemistry outcomes. There is a genotype difference in the glucose outcome, Time is a factor in several of the observed outcomes (red blood count, platelets, and hematocrit) across afl treatments in the SODl animals, attributable to the progression of the disease state,
[0456] Lastly, long term daily dosing with CNS 102 had no adverse effect on Wild type mice compared to animals dosed with Vehicle,
Example 48: mR A and Protein Expression analyses of HSPs in multi-dosing ainic Acid rat model
348 [0457J To study the in vivo efficacy of CNS 102, CNS102 was tested with consecutively multiple dosing paradigm (see Figure 36), in the rat models acutely subjected to kaitiic acid challenge
[0458] Specifically, each individual rat received 100 mg/kg CNS102 daily by sublingual administration, started five days before the surgery day with kainic acid or vehicle treatment and until the day of tissue harvesting. On the surgery day, CNS102 was administered 1 hour before kainic acid or vehicle injection, and on the perfusion day, CNS102 was administered 4 hours before perfusion, Kainic acid or vehicle was bilaterally injected by stereotactic surgery into the CA3 regions of both hippocampi of each rat, Upon tissue harvesting (24 hours or 72 hours after KA or vehicle injection), each brain was removed from the rat skull, one half of which was subjected to snap-freezing in liquid nitrogen for protein and total RNA extraction, and the other half was subjected to immersion fixation in 4% PFA followed by cryosectio ng for histological examination of neuron loss, HSP expression, apoptosts and inflammation. [0459] The above snap-frozen hippocampi was ground in liquid nitrogen and split the powder into two pails, one part was used for total protein extraction with a sonication method, and the other part was for the total RNA preparation.
[0460] With the total protein extracts from hippocampi, the expression level of multiple heat shock proteins was analyzed by either western blotting (HSP 90, 60, 40, 27 and GRP78), or by EL1SA (HSP70). Meanwhile, the q-RT-PCR analysis of the aforementioned genes for their mRNA expression was also conducted. The results are tabulated below.
349
Figure imgf000152_0001
[0461] The results indicate that 24 hr after Kainic acid injury, CNS102 selectively induces HSP70, GRP78 and HSP 7 expression, while it has no effects on HSP90, HSP60 and HSP40 at either the mRNA or protein expression level. There is no significant protein or mRNA induction on all these six HSPs by CNS102 72 hr after Kanic acid injury.
[04621 The effects of CNS102 on HSP70, GRP78 and HSP27 is compared in more detail in the table below (SS: Saline drag control with Saline injection, CS: CNS 102 with saline injection, S ; Saline drug control with Kainic Acid injection. C : CNS102 with Kainic Acid injection). The significant effects of CNS102 on above gene expressions were highlighted in bold.
[0463] The results showed that HSP70 is up-regulated by CNS 102 at both mRNA and protein level 24 hr after KA treatment, while there is only down-regulation of HSP70 mRNA without corresponding protein change 72 hr after KA treatment. These results provides evidence there might be different regulation mechanisms for I-ISP70 mRNA and protein expression in responding to CNS102 over time. As protein expression is a consequence of gene transcription, mRNA translation and post-translation modification, it is possible that additional mechanisms (such as miRNA regulation) are involved in regulation of the final protein level readout.
Figure imgf000153_0001
-1 protein expression analysed by westers blotting
Figure imgf000153_0002
[0464] Iba-3 data shown in the file of Kainic acid experiment histology specify an example of how neuro inflammation pathways induced by CNS- 102 work in protecting neurons.
Example 48: Histology of treated Kainic acid rat model [0465J To study the in vivo efficacy of CMS 102. we conducted a test of CNS 102 with consecutively multiple dosing paradigm in the rat models acutely subjected to kainic acid challenge.
[0466] Specifically, each individual rat received 100 mg/kg CMS 102 daily by sublingual administration, started five days before the surgery day, with kainic acid or vehicle treatment and until the day of tissue harvesting. On the surgery day, CNS102 was administered 1 hour before kainic acid or vehicle injection, and on the perfusion day, CNS 102 was administered 4 hours before perfusion. Kainic acid or vehicle was bilaterally injected by stereotactic surgery into the CA3 regions of both hippocampi of each rat. Upon tissue harvesting (24 hours or 72 hours after KA or vehicle injection), each brain was removed from the rat skull, one half of which was subjected to snap freezing in liquid nitrogen for protein and total RNA extraction, and the other half was subjected to immersion fixation in 4% PFA followed by cryosectioning for histological examination of neuron loss, HSP70 expression, apoptosis and inflammation..
[0467] Snap frozen hippocampi were ground in liquid nitrogen and the powder was split into two parts, one part was used for total protein extraction with sonication method, and the other part was for the total RNA preparation,
[0468] For histological examination hemispheres were embedded in optimum cutting temperature (OCT) compound and serially cut at 20 um on a cryostat.
Histological quantification of neuron loss
[0469] To quantify the effect of CNS102 on neuron loss, every I 0"1 section of KA treated animals was stained with cresyl violet and the damaged area of the CA3/CA2 pyramidal layer of the hippocampus was quantified with Imaged software.
Figure imgf000154_0001
This finding provides evidence a neuroprotective effect of CNS102 72h after kainic acid surgery. Histological quantification of I ha I expression
[0470] 5 sections bracketing the KA injection site by 200um were analyzed for Ibai expression by .iramuaoiiistoiogy, A trained observer analyzed the number of IBAI stained ceils per area on digitized nucrascope images. Three different activation states were analyzed: "activated microglia" have a thick bushy appearance, "resting microglia" have a strongly branched appearance with thin processes, and "macrophages are round with no processes.
The average number/are* is shown in tSie table.
Figure imgf000155_0001
CNS102 reduces the number of activated microglia and macrophages in the injured (KA treated) brain. Microglia are activated and macrophages are attracted by dying neurons. In addition activated microglia can add secondary cell stress to neurons and induce ceil death. This resul provides evidence that CNS102 is neuroprotective.
[0471 j From the foregoing i will be appreciated that, although specific embodiments of the invention have been described herein, for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention,
[0472] Throughout the description of this invention, reference is made to various patent applications arid pablications, each of which are herein incoiporated by reference in their entirety.

Claims

What is claimed is:
1. Λ method for treating a disease in a subject mediated in part by raicroRNA (miRNAV induced regulation of mRNA translation (e.g., miRNA-378 or miRNA-71 1 increased activity) comprising administering to the subject a therapeutically effective amount of 5- trans- geroaylgeranyl acetone (GGA) or a derivative thereof.
2. A method for treating damaged neurons exhibiting increased miRNA-378 and/or miRNA-711 activity, said method comprising administering 5-trans-GGA or a derivative thereof in art amount effective to decrease miRNA-378 or miRNA-71 1 activity specifically in damaged neurons and/or neuron support cells proximate to damaged neurons.
3. A method for treating damaged neurons in a subject in need thereof, wherein the damage to the neurons is associated with increased miRNA-378 and/or miRNA-l l activity, said method comprising decreasing miRNA-378 or miRNA-71 ί activity specifically in damaged neurons and/or neuron support cells proximate to damaged neurons said method comprising administering a therapeutically effective amount of 5-trans-GGA or a derivative thereof to the subject.
4. The method of claim 3, wherein the subject has a disease mediated in part by miRN A- 378 or miRNA-71 1 increased activity.
5. The method of claim 2, wherein the damaged neurons have increased miRNA-378 and/or miRNA-711 activity.
6. The method of arty one of claims 2-5, wherein the method comprises decreasing the miRNA-378 or miRNA-711 activity,
7. The method of claim 6, wherein the miRNA-378 or miRNA-71 1 activity is decreased in neuron support cei!s.
8. The method of claim 7, wherein the neuron support cells are glial cells,
9. The method of any one of the previous claims wherein the subject has a neurodegenerative disease.
10. The method of claim 9, wherein the neurodegenerative disease is ALS.
1 1 . The method of claim 9, wherein the neurodegenerative disease is Alzheimer's disease.
12. The method of claim 2, wherein the method further comprises contacting undamaged neurons with an effective amount of 5-trans-GGA or a derivative thereof.
13. An assay for identifying compounds that regulate HSP 70, said assay comprising: expressing, in a pool of cells, a nucleotide construct comprising the 3'UTR of HSP 70 and a reporter;
adding a test compound to a first portion of the pool of cells; and
determining the output level of the reporter;
wherein an increase in the output level of the reporter in the first portion of ceils indicates a compound that increases HSP 70 protein levels and a decrease in the output level of the reporter in the first portion of ceils indicates a compound that decreases HSP 70 protein levels.
14. An assa for identifying compounds that regulate HSP 70, said assay comprising: expressing, in a pool of cells, a nucleotide construct comprising the 3'UTR of HSP 70 and a reporter;
adding a test compound to a first portion of the pool of ceils;
adding a control to a second portion of the pool of cells; and
determining the output level of the reporter;
wherein a higher output level of the reporter in the first portion of cells compaxed to the second portion of cells indicates a compound that increases HSP 70 protein levels and a lower output level of the reporter in the first portion of cells compared to the second portion of cells indicates a compound that decreases HSP 70 protein levels.
15. The assay of claim 13 or 14, wherein the ceils are neurons.
16. The assay of any one of claims 13-15, wherein the reporter is selected from the group consisting of green fluorescent protein, red fluorescent protein, dsRed, yellow fluorescent protein, luciferin, lueiferase, and aequorin.
1 7. The assay of an one of claims 13-16, wherein the cells were exposed to damage prior to addition of the test compound or control.
18. A compound identified by the assay of any one of claims 13-17,
19. A kit comprising
a pool of cells expressing a nucleotide construct comprising the 3'UTR of HSP 70 and a reporter;
355 a means for delecting the output of the reporter: and
instructions for use,
20. The kit of claim 19, wherein the cells are neurons.
21 . The kit of claim 19 or claim 20, wherein the leporter is selected from the group consisting of green fluorescent protein, red fluorescent protein, dsRed, yellow fluorescent protein, iueiferm, luciferase, and aequorin,
22. A method of treating a damaged neuron having increased raiRNA-378 and/or miRNA-71 1 activity, the method comprising:
contacting the damaged neuron with a therapeutically effective amount of 5-trans- GGA or a derivative thereof, and
if miRNA-378 and/or miRNA-71 1 activity decreases in the contacted neuron, continuing to contact the damaged neurons with a therapeutically effective amount of 5-trans- GGA or a derivative thereof.
23. The method of claim 22, wherein the damaged neuron are in a subject suffering from a neurodegenerative disease.
24. The method of claim 23, wherein the neurodegenerative disease is ALS, Alzheimer's Disease (AD), or Parkinson's Disease (PD).
25. The method of claim 23, wherein the 5-trans-GGA or a derivative thereof is administered sublingua!ly.
26. The method of claim 22, wherein the damaged neuron has reduced HSP activity
27 A method of treating a damaged neuron having reduced HSP activity, the method comprising:
contacting the damaged neuron with a therapeutically effective amount of 5-trans- GGA or a derivative thereof, and
if HSP activity increases in the contacted neuron, continuing to contact the damaged neurons with a therapeutically effective amount of 5-trans-GGA or a derivative thereof.
28. The method of claim 27, wherein the damaged neurons are in a subject suffering from a neurodegenerative disease.
29. The method of claim 28, wherein the neurodegenerative disease is AL8, Akhekaers Disease (AD), or Parkinson's Disease (PD).
30. The method of claim 29, wherein the 5-rrans-GGA or a derivative thereof is administered sublingual!}'.
31. The method of any one of claims 22-30, wherein the amount is administered as a daily amount.
32. The method of claim i, wherein the amount is administered once daily ,
33. The method of claim 1 , wherein the OGA or the derivative thereof is GO A.
34. The method of claim 5, wherein the OGA is trans GOA free of or substantially free of cis GGA.
.
35. A method of modulating i RNA-378 or ndRNA-711 activity in a damaged neuron comprising contacting the damaged neuron with an effective amount of 5-trans-GGA or a derivative thereof
36. The method of claim 35. wherein the damaged neron is damaged at least its part by ALS, Alzheimer's Disease (AD), or Parkinson's Disease (PD).
37. A method of treating a damaged neuron comprising causing neuro- flammation in the damaged neuron, wherein the neuroinfiarnmation results at least in part from contacting the damaged cell with an effective amount of 5-trans-GGA or a derivative thereof.
38. A method of treating a damaged neuron comprising increasing HSP activity in the damaged neuron, wherein the increasing HSP activity results at least in part from contacting the damaged cell with an effective amount of 5-trans-GGA or a derivative thereof.
39. A method of treating a damaged neuron having reduced GRF7S activity, the method comprising:
contacting the damaged neuron with a therapeutically effective amount of 5-trans- GGA or a derivative thereof, and
if GRP78 activity increases in the contacted neuron, continuing to contact the damaged neurons with a therapeutically effective amount of 5-trans-GGA or a derivative thereof. 40, A method of treating a damaged neuron comprising increasing GRP78 acti vity in the damaged neuron, wherein the increasing GRP78activity results at least in part from contacting the damaged cell with an effective amount of 5-trans-GGA or a derivative thereof,
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