WO2017113268A1 - 用于治疗肌萎缩侧索硬化和额颞叶痴呆的化合物与应用 - Google Patents

用于治疗肌萎缩侧索硬化和额颞叶痴呆的化合物与应用 Download PDF

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WO2017113268A1
WO2017113268A1 PCT/CN2015/100061 CN2015100061W WO2017113268A1 WO 2017113268 A1 WO2017113268 A1 WO 2017113268A1 CN 2015100061 W CN2015100061 W CN 2015100061W WO 2017113268 A1 WO2017113268 A1 WO 2017113268A1
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compounds
compound
drug
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许顺良
金鹏
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Shandong University
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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
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4375—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having nitrogen as a ring heteroatom, e.g. quinolizines, naphthyridines, berberine, vincamine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/444—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring heteroatom, e.g. amrinone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5058—Neurological cells

Definitions

  • the present invention relates to compounds and applications for the treatment of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), and belongs to the field of neuropharmaceutical technology.
  • ALS amyotrophic lateral sclerosis
  • FTD frontotemporal dementia
  • GGGGCC repeat Amplification of the GGGGCC repeat between exons 1a and 1b in the C9ORF72 gene was found to be an important cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD).
  • ALS amyotrophic lateral sclerosis
  • FTD frontotemporal dementia
  • the amplified GGGGCC repeat not only accounts for 40% of the family of ALS or FTD families with dominant traits, but also accounts for 5-7% of all ALS and FTD cases in European and North American populations, making it both The most common genetic cause of one of the diseases.
  • ALS and FTD have phenotypic and pathological overlap.
  • ALS is a deadly degenerative disease that primarily affects motor neurons. The underlying feature is progressive weakness; however, cognitive impairment and behavioral changes (similar to those found in FTD) are increasingly recognized symptoms.
  • FTD is a progressive dementia disease that primarily affects neurons in the frontal cortex, insular cortex, and anterior temporal cortex, resulting in significant changes in personality, behavior, and/or language ability. A small number of individuals with FTD also develop ALS.
  • Pathologically, ALS and FTD are most commonly characterized by abnormal accumulation of TAR DNA binding protein (TDP-43). Genetically, clustering of families that isolate ALS or FTD (usually as autosomal dominant traits) supports the notion that each disease has a strong genetic basis.
  • TARDBP TAR DNA-binding proteins
  • FUS sarcoma fusion
  • GGGGCC amplification has since been shown to be the most common genetic cause of ALS and FTD.
  • GGGGCC amplification has an age-dependent penetrance that is almost completely apparent by the age of 80.
  • Biochemically, C9orf72 is largely uncharacterized, but appears to be universally transcribed in normal control brains.
  • RNA-mediated toxicity is to isolate normal RNA-binding proteins (RBPs) from amplified repeats of transcription, resulting in depletion of RBP useful for normal RNA metabolism.
  • RBPs normal RNA-binding proteins
  • the effects of depletion of these RBPs include loss of development-specific transcripts and significant splicing abnormalities.
  • RNA functional acquisition mechanism of ALS and FTD was proposed based on the increase of riboGGGGCC (rGGGGCC) amplification level.
  • This type of RNA function-acquisition mechanism has been suggested as a triplet-repeat-related ataxia in myotonic dystrophy (DM) and riboCGG (rCGG) repeats amplified in FXTAS such as SCA8, SCA10 And the mechanism of SCA12.
  • RNA-mediated functional acquired toxicity model of ALS and FTD Additional evidence supports the RNA-mediated functional acquired toxicity model of ALS and FTD.
  • Drosophila ALS and FTD models has been described and it has been demonstrated that the rGGGGCC repeat between exons 1a and 1b in C9orf72 is toxic and sufficient to cause neurodegeneration.
  • Drosophila has been the first model system for research on human neurodegenerative diseases in the past decade, recognizing that fruit flies share many structural and functional gene families with humans. It has been shown that genes associated with neurodegeneration can be expressed in Drosophila, causing a phenotype that is significantly similar to the phenotype of corresponding human diseases, including polyglutamine disease, Parkinson's disease, and Alzheimer's disease. These results indicate that the molecular mechanisms of neuronal toxicity and loss are conserved between humans and fruit flies. The development of such disease models in Drosophila allows the application of genetic methods to address specific hypotheses regarding disease progression and to test candidate modified genes or therapeutic drug compounds.
  • the present invention addresses the deficiencies of the prior art and provides compounds, compositions, and methods of use that can treat, prevent, and reduce the symptoms of ALS and FTD or delay the progression of ALS and FTD.
  • ALS amyotrophic lateral sclerosis
  • FTD frontotemporal dementia
  • the compound is selected from the group consisting of: OEM-34, OEM-62, OEM-77, OK-EM 026, OK-EM 027, OK-EM 028, OK-EM 029, OK-EM 033 and / Or OK-EM 036.
  • the compound is selected from the group consisting of: OEM-34, OEM-62, OEM-77, OK-EM 026, OK-EM 027, OK-EM 028, OK-EM 029, OK-EM 033 and / Or a salt, ester or prodrug of OK-EM 036.
  • a method of treating or preventing ALS and FTD comprising administering to a host having such an effect an effective amount of OEM-34, OEM-62, or OEM-77.
  • the host has 30 GGGGCC repeats in the C9orf72 gene of its genome. In certain embodiments, the host has amyotrophic lateral sclerosis. In certain embodiments, the host has frontotemporal dementia.
  • a pharmaceutical composition comprising OEM-34, OEM-62 or OEM-77 in combination with a pharmaceutically acceptable carrier is provided.
  • the compound is present at a dosage level effective to treat the condition, wherein the condition is amyotrophic lateral sclerosis or frontotemporal dementia.
  • Embodiments of the present disclosure also include methods of screening for compounds effective to treat or prevent ALS and FTD, comprising: 1) providing a library of compounds; 2) applying each of the compounds to a plurality of Drosophila embryos, wherein each fruit The fly embryo contains a GGGGCC 30 repeat that is linked to the hs-hid transgene, where hs-hid expression is activated after heat shock; 3) exposure of Drosophila embryos to heat shock; 4) measurement of sputum in each container formation and adult fly, which in the adult fly pupae formation and rescue container comprising a compound of Drosophila embryos GGGGCC 30 repeats the occurred; and 5) a compound selected to allow the formation of pupae or adult flies.
  • Embodiments of the present disclosure include a kit for screening a library of compounds, the kit comprising: a plurality of Drosophila embryos, wherein each Drosophila embryo comprises a GGGGCC 30 repeat fragment linked to a hs-hid transgene, said hs- The hid transgene is lethal to a Drosophila embryo carrying a GGGGCC 30 repeat after heat shock; and a set of instructions for screening a compound library.
  • the present disclosure relates to a method of treating amyotrophic lateral sclerosis comprising administering to a subject in need thereof an effective amount of a compound that inhibits toxicity caused by a GGGGCC repeat.
  • the subject is diagnosed with GGGGCC repeat amplification.
  • the subject is diagnosed with ALS.
  • the subject is diagnosed with FTD.
  • the compound is selected from the group consisting of: OEM-34, OEM-62, or OEM-77.
  • the compound is selected from the group consisting of: OEM-34, OEM-62, or OEM-77, or a salt, ester, or prodrug thereof.
  • the compound is selected from the group consisting of OEM-34, OEM-62, or OEM-77, or a salt, ester or prodrug thereof. In certain embodiments, the compound is OEM-34, OEM-62 or OEM-77. In certain embodiments, the compound is any compound provided herein substituted with one or more substituents.
  • the disclosure relates to genetically engineered fruit flies having increased expression of a GGGGCC-linked transgene.
  • Figure 1 Schematic diagram of the identification process of small molecules that inhibit the toxicity caused by rGGGGCC repeats by chemical screening for inhibition of rGGGGCC repeat-mediated lethality;
  • the Elav-Gal4 driver was hybridized with UAS-GGGGCC 30 -EGFP/Cyo on food supplemented with individual unique compounds from libraries of 2000 small molecules to generate progeny embryos.
  • the offspring embryos continue to develop on food supplemented with individual medications until the end of the offspring. Relative viability is obtained by comparing the number of adult offspring or ticks based on their genotypes after administration of heat shock;
  • Figure 2 Schematic diagram of the identification process of small molecules for improving motor defects caused by rGGGGCC repeats
  • the Nervana-GAL4 driver was hybridized with UAS-GGGGCC 30 -EGFP/Cyo on food supplemented with or without the selected compound to produce progeny embryos.
  • Adult progeny of genotype UAS-GGGGCC 30 -EGFP/nervana were selected and then 30 female uncrossed ligands at 48 hours of age were monitored for locomotor activity for several weeks. Simultaneous monitoring of the movement of 30 fruit flies fed with Drosophila food containing compounds;
  • Figure 4 Bar graph of the percentage results of the average locomotor activity for the selected drug at 7 days;
  • the term "subject” includes humans, mammals (eg, cats, dogs, horses, etc.) and other living species in need of treatment.
  • the living organism can be a human or a mammal.
  • a subject "susceptible" to a neuronal disorder and related condition may be defined as one that does not exhibit the apparent symptoms of one or more of these conditions but is genetically, physiologically, or otherwise in the presence of such conditions or Multiple subjects in the risk. Therefore, the present disclosure
  • the compositions and agents are prophylactically used in these conditions.
  • a “composition” or “agent” can include one or more of the chemical compounds and/or agents as described below.
  • An “active composition” can comprise one or more "active compounds.”
  • screening refers to the identification of one or more compounds from a library of compounds that meet criteria such as, but not limited to, the rescue of specially designed fruit flies from a lethal dose of glutamate.
  • the screening methods of the present disclosure can be used to identify compounds (e.g., drug candidates to be used in the active compositions) for treating disorders associated with rGGGGCC repeat-mediated neurodegeneration and other diseases and conditions described herein.
  • derivative refers to modifications of the disclosed compounds including, but not limited to, the hydrolysis, reduction or oxidation products of the disclosed compounds.
  • the term includes the opening of nitrogen-containing ring structures including, but not limited to, imidazoles of the disclosed compounds. Hydrolysis, reduction and oxidation reactions are known in the art.
  • terapéuticaally effective amount means that the amount of the compound administered will alleviate to some extent one or more symptoms of the condition being treated, and/or that amount will prevent the treatment to a certain extent. One or more symptoms of the condition of the subject at risk of attack.
  • “Pharmaceutically acceptable salt” means the bioavailability and properties of the free base, and by interaction with inorganic or organic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, Those salts obtained by the reaction of toluenesulfonic acid, salicylic acid, malic acid, maleic acid, succinic acid, tartaric acid, citric acid and the like.
  • inorganic or organic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, Those salts obtained by the reaction of toluenesulfonic acid, salicylic acid, malic acid, maleic acid, succinic acid, tartaric acid, citric acid and the like.
  • “Pharmaceutical composition” refers to a mixture of one or more of the compounds described herein, or a pharmaceutically acceptable salt thereof, with other chemical components, such as physiologically acceptable carriers and excipients.
  • One purpose of a pharmaceutical composition is to facilitate the administration of a compound to an organism.
  • pharmaceutically acceptable carrier refers to a carrier or diluent that does not cause significant irritation to the organism and does not abrogate the biological activity and properties of the administered compound.
  • Excipient means an inert substance that is added to a pharmaceutical composition to further facilitate administration of the compound.
  • excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars and various types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols.
  • Treatment or “therapy” of a disease includes preventing the disease from occurring in an animal that may be predisposed to the disease but has not experienced or manifested the symptoms of the disease (prophylactic treatment), inhibiting the disease (slowing or preventing its development), providing Relief of symptoms or side effects of the disease (including palliative care), as well as alleviation of the disease (resolved disease subsided).
  • prodrug refers to an agent that is converted in vivo to a biologically active form.
  • Prodrugs are often useful because, in some cases, they can be administered more readily than the parent compound. They can, for example, be taken orally It can be bioavailable for administration, but not for the parent compound. Prodrugs can also have improved solubility in the pharmaceutical composition relative to the parent drug. Prodrugs can be converted to the parent drug by a variety of mechanisms, including enzymatic processes and metabolic hydrolysis. Harper, NJ (1962). Drug Latentiation in Jucker, ed. Progress in Drug Research, 4:221-294; Morozowich et al. (1977).
  • topical active agent refers to a composition of the present disclosure that causes a pharmacological response at a site that is applied to a host (contacted in a locally applied manner).
  • topical refers to the application of the compositions of the present disclosure to mucosal cells and the surface of tissues and skin.
  • salts which are also within the scope of the invention.
  • References to compounds of any formula herein are understood to include references to salts thereof, unless otherwise indicated.
  • the term “salt” as used herein denotes an acidic and/or basic salt formed with inorganic and/or organic acids and bases.
  • a zwitterion can be formed and included within the term “salt” as used herein.
  • Pharmaceutically acceptable (e.g., non-toxic, physiologically acceptable) salts are preferred, although other salts may be employed, for example, in the separation or purification steps that may be employed in the preparation process.
  • Salts of the compounds of the active compounds can be formed, for example, by reaction with an active compound with an amount of, for example, an equivalent amount of an acid or a base, which is carried out, for example, in a medium in which the salt is precipitated or in an aqueous medium, followed by lyophilization.
  • the disclosed compounds containing a basic moiety can form salts with various organic and inorganic acids.
  • Exemplary acid addition salts include acetates (such as those formed with acetic acid or trihaloacetic acids such as trifluoroacetic acid), adipates, alginates, ascorbates, aspartates, benzoates , benzenesulfonate, hydrogen sulfate, borate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentane propionate, digluconate, dodecyl sulfate , ethanesulfonate, fumarate, Glucoheptanoates, glycerol phosphate, hemisulfate, heptanoate, hexanoate, hydrochloride (formed with hydrochloric acid), hydrobromide (with hydrogen) Bromoacid formation), hydroiodide, 2-hydroxyethanesulfonate, lactate, male
  • the disclosed acidic moiety-containing compounds can form salts with various organic and inorganic bases.
  • Exemplary basic salts include ammonium salts, alkali metal salts such as sodium salts, lithium salts and potassium salts, alkaline earth metal salts such as calcium salts and magnesium salts, and organic bases (for example, organic amines) such as benzathine, dicyclohexylamine, hydrabamine. (formed with N,N-di(dehydroindenyl)ethylenediamine), a salt of N-methyl-D-glucosamine, N-methyl-D-glucamide, tert-butylamine, and Amino acids such as arginine, salts of lysine, and the like.
  • the basic nitrogen-containing group may be, for example, a lower alkyl halide (for example, methyl, ethyl, propyl, and butyl chloride, bromide, and iodide), a dialkyl sulfate (for example, dimethyl group). , diethyl, dibutyl and dipentyl sulfate), long chain halides (eg, mercapto, lauryl, myristyl and stearyl chloride, bromide and iodide), aralkyl halides
  • the reagents such as benzyl and phenethyl bromide are quaternized.
  • Ra and Rb in this context may be the same or different and independently hydrogen, halohydroxy, alkyl, alkoxy, hydrocarbyl, amino, alkylamino, dialkylamino, carbocyclyl, carbocyclic Alkyl, heterocarbocyclyl, heterocarbocycloalkyl, aryl, arylalkyl, heteroaryl and heteroarylalkyl.
  • Solvates of the compounds of the present disclosure are also included herein.
  • the solvate of the compound is preferably a hydrate.
  • All stereoisomers of the compounds of the invention such as those which may exist due to asymmetric carbon atoms on various substituents, including enantiomeric forms (which may even exist in the absence of asymmetric carbon) And diastereomeric forms are within the scope of the present disclosure.
  • Individual stereoisomers of the compounds of the disclosure may, for example, be substantially free of other isomers, or may be admixed, for example, as a racemate or with all other or additional The selected stereoisomers are mixed.
  • the chiral centers of the compounds of the present disclosure may have the S or R configuration as defined by IUPAC 1974 Recommendation.
  • Embodiments of the present disclosure include screening methods, kits for screening libraries of compounds, active compositions comprising one or more active compounds, pharmaceutical compositions comprising one or more active compounds, therapeutic and/or A method for preventing amyotrophic lateral sclerosis and frontotemporal dementia, a kit for treating and/or preventing amyotrophic lateral sclerosis and frontotemporal dementia.
  • embodiments of the present disclosure include screening for high throughput methods of compounds (eg, drug candidates) that can be used to treat amyotrophic lateral sclerosis and frontotemporal dementia. Screening is based on compound screening of fruit flies (e.g., fruit fly), wherein the efficacy of the compounds is determined by scoring the number of sputum formed under certain culture conditions or the appearance of adult fruit flies. Compounds that meet the screening process can be selected as the active compound, which can be used in compositions and pharmaceutical compositions to treat a host in need of such treatment with an effective amount of the active composition.
  • compounds eg, drug candidates
  • Screening is based on compound screening of fruit flies (e.g., fruit fly), wherein the efficacy of the compounds is determined by scoring the number of sputum formed under certain culture conditions or the appearance of adult fruit flies.
  • Compounds that meet the screening process can be selected as the active compound, which can
  • Embodiments of screening methods are advantageous because they provide the only known method for screening compounds that reverse the clinical consequences of rGGGGCC repeat-mediated toxicity.
  • the use of fruit flies provides screening of complex organisms, which are relatively simple screenings, such as cell-based screening, are more likely to provide useful compounds.
  • transgenic flies containing the rGGGGCC repeat in C9orf72 showed neuronal cell death consistent with human phenotype.
  • a transgenic Drosophila model has been previously established that expresses amplified repeats upstream of the reporter gene EGFP and shows direct involvement of neuronal cell death in the pathogenesis of human ALS.
  • the GAL4-UAS system is used to drive expression of the transgene in different tissues, including Drosophila eyes. Drosophila shows neurodegeneration of the eye and motor neurons that mimic the human phenotype.
  • embodiments of the present disclosure provide active compositions comprising one or more active compounds useful for the treatment and/or prevention of ALS and FTD.
  • Embodiments of the present disclosure include high throughput methods for screening compounds (e.g., drug candidates) useful for treating ALS and FTD.
  • compounds e.g., drug candidates
  • embodiments of the present disclosure include methods for screening compounds to identify drug candidates for the treatment of ALS and FTD using a fruit fly based compound screening.
  • Drosophila carrying the GGGGCC 30 repeat between exons 1a and 1b in C9ord72 exhibited neuronal and behavioral defects similar to those reported in the mouse model and in human patients. It has been found that expression of the rGGGGCC repeats results in lethality in early embryonic development.
  • a Drosophila strain transgenic with UAS-GGGGCC30-EGFP equilibrated with the Cyo balancer carrying the hs-hid transgene was generated. After heat shock, the expression of hid is activated, which results in cell death and lethality in GGGGCC 30 Drosophila embryos. As shown in Figure 1, no live fruit flies or ticks were produced after heat shock during early embryonic development.
  • the mutated embryos are placed in a suitable container (eg, a 96-well container) containing food with or without a compound from a library of drug candidates. Screening of the library from 2,000 FDA-approved drugs and natural products (The Spectrum Collection TM) of 5 ⁇ M and 40 ⁇ M individual compounds. The vials were then kept at 25 ° C for 10 to 15 days to score for vigor. The efficacy of drug candidates is determined by scoring the number of sputum formed after a certain time frame or the appearance of adult fruit flies.
  • Embodiments of the present disclosure provide screening for compounds that directly and indirectly modulate rGGGGCC repeat-mediated toxicity and rescue mutant flies from avoiding heat shock-induced lethality. Further details regarding the active compositions and conditions are described in the examples below.
  • active compositions comprising one or more active compounds are useful for the treatment and/or prevention of ALS and FTD.
  • the compound is OEM-34, OEM-62 or OEM-77.
  • Dosage regimens for active compositions have been developed based on the discussion in the embodiments and examples of the present disclosure.
  • the initial dose for most Phase I clinical trials is based on preclinical trials and is generally quite conservative.
  • the standard measure of drug toxicity in preclinical testing is due to the percentage of animals (rodents) that are treated to die.
  • the dose at which 10% of animals die is called LD10, which used to be associated with the maximum tolerated dose (MTD) in humans (adjusted for body surface area). Adjustments to body surface area include host factors such as surface area, body weight, metabolism, tissue distribution, absorption rate, and excretion rate.
  • the standard conservative starting dose is one tenth of the mouse LD10, although other species (ie, dogs) may be lower if they are more sensitive to the drug. It is expected that the starting dose of the active composition in a Phase I clinical trial in humans will be determined in this manner. This dosing regimen is discussed in more detail in Freireich E J, et al., Cancer Chemother Rep 50: 219-244, 1966, which is incorporated herein by reference.
  • the therapeutically effective dose level will depend on a number of factors. Moreover, within the skill of the art The dosage of the active composition is initiated at a relatively low level and the dosage is increased until the desired effect is achieved.
  • Embodiments of the present disclosure provide compositions and pharmaceutical compositions comprising an effective amount of an active composition (e.g., one or more active compounds) to treat and/or prevent conditions such as those described herein.
  • an active composition e.g., one or more active compounds
  • compositions and dosage forms of the present disclosure include pharmaceutically acceptable salts of the disclosed compounds or pharmaceutically acceptable polymorphs, solvates, hydrates, dehydrates, co-crystals thereof, Water or amorphous form.
  • Particular salts of the disclosed compounds include, but are not limited to, the sodium, lithium, potassium, and hydrate thereof.
  • compositions and unit dosage forms of the present disclosure will generally also comprise one or more pharmaceutically acceptable excipients or diluents.
  • Advantages provided by particular compounds of the present disclosure such as, but not limited to, increased solubility and/or enhanced flow, purity or stability (e.g., hygroscopicity) characteristics may make them more suitable for pharmaceutical formulation than the prior art. And/or to the patient.
  • the pharmaceutical unit dosage form of the compounds of the present disclosure is suitable for oral, mucosal (eg, nasal, sublingual, vaginal, buccal or rectal), parenteral (eg, intramuscular, subcutaneous, intravenous, intraarterial or projectile) Injection, topical, or transdermal administration to a patient.
  • mucosal eg, nasal, sublingual, vaginal, buccal or rectal
  • parenteral eg, intramuscular, subcutaneous, intravenous, intraarterial or projectile
  • Injection e.g, topical, or transdermal administration to a patient.
  • dosage forms include, but are not limited to, tablets; caplets; capsules, such as hard gelatin capsules and soft elastic gelatin capsules; cachets; troches; lozenges; dispersing agents; suppositories; ointments; Mud; paste; powder; dressing; cream; plaster; solution; patch; aerosol (for example, nasal spray or inhaler); gel; liquid suitable for oral or mucosal administration to patients
  • dosage forms including suspensions (for example, aqueous or non-aqueous liquid suspensions, oil-in-water emulsions or water-in-oil liquid emulsions), solutions and elixirs; liquid dosage forms suitable for parenteral administration to patients; and sterile solids ( For example, a crystalline or amorphous solid) that can be reconstituted to provide a liquid dosage form suitable for parenteral administration to a patient.
  • compositions, shape and type of dosage forms of the compositions of the present disclosure will generally vary depending on their use.
  • a dosage form for use in the acute treatment of a disease or condition can comprise a greater amount of active ingredient, such as a disclosed compound or a combination thereof, in a dosage form for use in the chronic treatment of the same disease or condition.
  • parenteral dosage forms can contain smaller amounts of active ingredient than oral dosage forms used to treat the same disease or condition.
  • Typical pharmaceutical compositions and dosage forms comprise one or more excipients.
  • Suitable excipients are pharmaceutical or pharmaceutical
  • suitable excipients are known to those skilled in the art and are provided herein. Whether a particular excipient is suitable for incorporation into a pharmaceutical composition or dosage form depends on a variety of factors well known in the art including, but not limited to, the manner in which the dosage form will be administered to the patient.
  • oral dosage forms such as tablets or capsules may contain excipients which are not suitable for use in parenteral dosage forms.
  • the suitability of a particular excipient may also depend on the particular active ingredient in the dosage form. For example, the decomposition of some of the active ingredients can be accelerated by some excipients such as lactose or when exposed to water. Active ingredients containing primary or secondary amines are particularly susceptible to such accelerated decomposition.
  • compositions and dosage forms that contain one or more compounds that reduce the rate of decomposition of the active ingredient.
  • compounds which are referred to herein as “stabilizers”, include, but are not limited to, antioxidants such as ascorbic acid, pH buffers, or salt buffers.
  • the pharmaceutical compositions or dosage forms of the present disclosure may contain one or more solubility modifiers such as sodium chloride, sodium sulfate, sodium or potassium phosphate or organic acids. A particular solubility modifier is tartaric acid.
  • the amount and specific type of active ingredient in the dosage form can vary depending on factors such as, but not limited to, the route by which it is administered to the patient.
  • typical dosage forms of the compounds of the present disclosure include pharmaceutically acceptable salts, or pharmaceutically acceptable polymorphs, solvates, hydrates, dehydrates, co-crystals, anhydrous or amorphous forms thereof.
  • the amount is from about 10 mg to about 1000 mg, preferably from about 25 mg to about 750 mg, more preferably from 50 mg to 500 mg.
  • the compounds and/or compositions can be delivered using lipid or polymer based nanoparticles.
  • nanoparticles can be designed to improve the pharmacological and therapeutic properties of drugs for parenteral administration (Allen, TM, Cullis, PRDrug delivery systems:entering the mainstream. Science. 303 (5665): 1818-22 (2004)) .
  • compositions suitable for oral administration of the present disclosure may be presented as discrete dosage forms such as, but not limited to, tablets (including but not limited to scored or coated tablets), pills, sachets Tablets, capsules, chewable tablets, powder packs, cachets, troches, wafers, aerosol sprays, or liquids such as, but not limited to, syrups, elixirs, aqueous liquids, solutions in non-aqueous liquids Or a suspension, an oil-in-water emulsion or a water-in-oil emulsion.
  • Such compositions comprise a predetermined amount of a pharmaceutically acceptable salt of the disclosed compounds, and may be prepared by methods of pharmacy well known to those skilled in the art. See generally, Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing, Easton, Pa. (1990).
  • a typical oral dosage form of a composition of the present disclosure will be disclosed by conventional drug compounding techniques.
  • a pharmaceutically acceptable salt of the compound is prepared in combination with at least one excipient in an intimate admixture.
  • the excipient can take a variety of forms depending on the form of the composition desired for administration.
  • excipients suitable for oral liquid or aerosol dosage forms include, but are not limited to, water, glycols, oils, alcohols, flavoring agents, preservatives, and coloring agents.
  • excipients suitable for solid oral dosage forms such as powders, tablets, capsules and caplets include, but are not limited to, starch, sugar, microcrystalline cellulose, kaolin, diluents, granulating agents, lubricants, Binder and disintegrant.
  • tablets and capsules represent the most advantageous solid oral unit dosage form, in which case solid pharmaceutical excipients are employed. If desired, the tablets can be coated by standard aqueous or non-aqueous techniques.
  • These dosage forms can be prepared by any of the pharmaceutical methods. In general, the pharmaceutical compositions and dosage forms can be prepared by uniformly and intimately admixing the active ingredient with liquid carriers, finely divided solid carriers, or both, and, if necessary, shaping the product into the desired form.
  • tablets can be prepared by compression or molding.
  • the compressed tablet can be prepared by compressing the active ingredient in a suitable machine in a free flowing form such as a powder or granules, optionally with one or more excipients.
  • Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
  • Suitable binders for pharmaceutical compositions and dosage forms include, but are not limited to, corn starch, potato starch or other starches, gelatin, natural and synthetic gums such as acacia, sodium alginate, alginic acid, other alginates, Astragalus gum powder, guar gum, cellulose and its derivatives (for example, ethyl cellulose, cellulose acetate, calcium carboxymethyl cellulose, sodium carboxymethyl cellulose), polyvinylpyrrolidone, methyl cellulose , pregelatinized starch, hydroxypropyl methylcellulose, (for example, No. 2208, 2906, 2910), microcrystalline cellulose, and mixtures thereof.
  • Suitable forms of microcrystalline cellulose include, but are not limited to, materials sold as AVICEL-PH-101, AVICEL-PH-103 AVICEL RC-581 and AVICEL-PH-105 (available from FMC Corporation, American Viscose Division, Avicel Sales, Marcus Hook, Pa., USA), and mixtures thereof.
  • An exemplary suitable binder is a mixture of microcrystalline cellulose and sodium carboxymethylcellulose sold as AVICEL RC-581.
  • Suitable anhydrous or low moisture excipients or additives include And Starch 1500LM.
  • fillers suitable for use in the pharmaceutical compositions and dosage forms disclosed herein include, but are not limited to, talc, calcium carbonate (eg, granules or powders), microcrystalline cellulose, powdered cellulose, glucose binder, kaolin, nectar Alcohol, silicic acid, sorbitol, starch, pregelatinized starch, and mixtures thereof. Medicine in the present disclosure
  • the binder or filler in the composition is typically present at from about 50 to about 99 weight percent of the pharmaceutical composition or dosage form.
  • Disintegrants are used in the compositions of the present disclosure to provide tablets that disintegrate when exposed to an aqueous environment. Tablets containing too much disintegrant may swell, crack, or disintegrate upon storage, while tablets containing too little disintegrant may not be sufficient to disintegrate and thus alter the rate at which the active ingredient is released from the dosage form and degree. Thus, a solid oral dosage form of the present disclosure should be formed using a sufficient amount of disintegrant that is neither too little nor too much (not adversely altering the release of the active ingredient). The amount of disintegrant used varies based on the type of formulation and mode of administration, and is readily discernible to those of ordinary skill in the art.
  • a typical pharmaceutical composition comprises from about 0.5 to about 15 weight percent of a disintegrant, preferably from about 1 to about 5 weight percent of a disintegrant.
  • Disintegrants which may be used in forming the pharmaceutical compositions and dosage forms of the present disclosure include, but are not limited to, agar, alginic acid, calcium carbonate, microcrystalline cellulose, croscarmellose sodium, crospovidone, wave Laclin potassium, sodium starch glycolate, potato or tapioca starch, other starches, pregelatinized starch, clay, other alginates, other celluloses, gums, and mixtures thereof.
  • Lubricants that can be used to form the pharmaceutical compositions and dosage forms of the present disclosure include, but are not limited to, calcium stearate, magnesium stearate, mineral oil, light mineral oil, glycerin, sorbitol, mannitol, poly Ethylene glycol, other glycols, stearic acid, sodium lauryl sulfate, talc, hydrogenated vegetable oils (eg, peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil and soybean oil), zinc stearate , ethyl oleate, ethyl laurate, agar, and mixtures thereof.
  • lubricants include, for example, syloid silica gel ( 200, manufactured by WR Grace Co. of Baltimore, Md.), a synthetic aerosol of silica (sold by Degussa Co. of Plano, Tex.), (Sintered silica product sold by Cabot Co. of Boston, Mass.), and mixtures thereof. If used, the lubricant is typically employed in an amount of less than about 1 weight percent of the pharmaceutical composition or dosage form to which it is incorporated.
  • lactose-free pharmaceutical compositions and dosage forms wherein such compositions preferably contain little, if any, lactose or other monosaccharides or disaccharides.
  • lactose free means that the amount of lactose present, if any, is insufficient to substantially increase the rate of degradation of the active ingredient.
  • lactose-free compositions of the present disclosure may comprise excipients that are well known in the art and are listed in USP (XXI) / NF (XVI), which is incorporated herein by reference.
  • the lactose-free composition comprises a pharmaceutically acceptable salt of the compound in the active composition, a binder/filler, and a pharmaceutically compatible and pharmaceutically acceptable amount of a lubricant.
  • Preferred lactose-free dosage forms comprise pharmaceutically acceptable salts, microcrystals of the disclosed compounds Cellulose, pregelatinized starch and magnesium stearate.
  • the present disclosure also encompasses anhydrous pharmaceutical compositions and dosage forms comprising the disclosed compounds as active ingredients, as water can promote degradation of some of the compounds.
  • water e.g., 5%
  • water is widely accepted in the pharmaceutical arts as a means of simulating long-term storage to determine the properties of the formulation over time such as shelf life or stability. Participation, for example, Jens T. Carstensen, Drug Stability: Principles & Practice, 379-80 (2nd ed., Marcel Dekker, NY, N.Y.: 1995). Water and heat accelerate the decomposition of some compounds.
  • the effect of water on the formulation can be important because moisture and/or humidity are often encountered during the manufacture, handling, packaging, storage, transportation and use of the formulation.
  • Anhydrous pharmaceutical compositions and dosage forms of the present disclosure can be prepared using anhydrous or low moisture containing ingredients and low moisture or low humidity conditions.
  • Pharmaceutical compositions and dosage forms comprising lactose and at least one active ingredient comprising a primary or secondary amine are preferably anhydrous if substantial contact with moisture and/or moisture is expected during manufacture, packaging and/or storage. .
  • anhydrous pharmaceutical compositions should be prepared and stored such that their anhydrous nature is maintained. Accordingly, anhydrous compositions are preferably packaged using materials known to prevent exposure to water such that they can be included in suitable pharmaceutical kits. Examples of suitable packaging include, but are not limited to, sealed foils, plastics, unit dose containers (eg, vials) with or without a desiccant, blister packs, and strip packs.
  • the pharmaceutically acceptable salts of the disclosed active compounds can be administered by controlled or sustained release devices.
  • Controlled release drug products have a common goal of improving drug therapy relative to their non-controlled release counterparts.
  • the use of an optimally designed controlled release formulation in medical therapy is characterized by minimizing the drug substance used to cure or control the condition in a minimal amount of time.
  • Advantages of controlled release formulations include: 1) prolonged drug activity; 2) reduced dosing frequency; 3) improved patient compliance; 4) use of less total drug; 5) reduced local or systemic side effects; Accumulation of chemical drugs; 7) reduce blood level fluctuations; 8) improve treatment efficacy; 9) reduce the increase or loss of drug activity; and 10) improve the speed of controlling diseases or conditions. Kim, Chemg-ju, Controlled Release Dosage Form Design, 2 (Technomic Publishing, Lancaster, Pa.: 2000).
  • Conventional dosage forms typically provide for rapid or immediate release of the drug from the formulation.
  • the use of conventional dosage forms can result in a wide range of fluctuations in the concentration of the drug in the blood and other tissues of the patient. These fluctuations can affect many parameters such as frequency of administration, initiation of action, duration of efficacy, maintenance of blood levels of treatment, toxicity, side effects, and the like.
  • the controlled release formulation can be used to control the onset of action and persistence of the drug Time, plasma levels within the treatment window, and peak blood levels.
  • controlled or sustained release dosage forms or formulations can be used to ensure maximum drug performance while minimizing potential adverse effects and safety issues, which may be due to insufficient drug dose (ie, below the minimum therapeutic level) and beyond.
  • the toxicity level of the drug occurs.
  • Controlled release of the active ingredient can be stimulated by a variety of conditions including, but not limited to, pH, ionic strength, osmotic pressure, temperature, enzymes, water, and other physiological conditions or compounds.
  • dosage forms can be used to provide slow or controlled release of one or more active ingredients using, for example, hydroxypropyl methylcellulose, other polymeric matrices, gels, permeable membranes, osmotic systems (eg, (Alza Corporation, Mountain View, Calif. USA)), multilayer coatings, microparticles, liposomes, or microspheres, or combinations thereof, to provide the desired release profile in varying ratios.
  • ion exchange materials can be used to prepare fixed, adsorbed salt forms of the disclosed compounds to achieve controlled delivery of the drug. Examples of specific anion exchangers include, but are not limited to, A568 and AP143 (Rohm & Haas, Spring House, Pa. USA).
  • One embodiment of the present disclosure includes a unit dosage form comprising a pharmaceutically acceptable salt (eg, a sodium salt, a potassium salt or a lithium salt) of the disclosed compounds, or a polymorph, solvate thereof a hydrate, dehydrate, co-crystal, anhydrous or amorphous form, and one or more pharmaceutically acceptable excipients or diluents, wherein the pharmaceutical composition or dosage form is formulated for controlled use freed.
  • Particular dosage forms utilize an osmotic drug delivery system.
  • the active compositions of the present disclosure and the salts of the compounds of the complex can be much more water soluble than the active compound itself, they are well suited for delivery to patients based on osmosis.
  • the present disclosure does include the incorporation of active compounds and their non-salt isomers and isomer mixtures Dosage form.
  • a particular dosage form of the active composition of the present disclosure includes a wall defining a cavity having an exit aperture formed therein or formed therein and at least a portion of the wall being semi-permeable; being located within the cavity remote from the exit aperture and An expandable layer in fluid communication with the semi-permeable portion of the wall; a dry or substantially dry drug layer located in a cavity adjacent the exit aperture and in direct or indirect contact with the expandable layer; and an inner surface of the wall a flow promoting layer between at least an outer surface of the drug layer located within the cavity, wherein the drug layer comprises a salt of a compound of the active composition, or a polymorph, solvate, hydrate, dehydrate thereof, Co-crystal, anhydrous or amorphous form. See U.S. Patent No. 6,368,626, the disclosure of which is incorporated herein in its entirety.
  • Another particular dosage form of the present disclosure includes: a wall defining a cavity having an exit aperture formed therein or formed therein and at least a portion of the wall being semi-permeable; being located within the cavity remote from the exit aperture and with the wall An expandable layer in which the semi-permeable portion is in fluid communication; a drug layer located in a cavity adjacent the exit orifice and in direct or indirect contact with the expandable layer; the drug layer comprising a liquid, an active agent formulation adsorbed in the porous particles,
  • the porous particles are adapted to withstand a compressive force sufficient to form a layer of compacted drug that exhibits significant exudation without a liquid, the active agent optionally having a placebo layer between the exit orifice and the drug layer, wherein the active layer Formulation A salt of a compound comprising the active compound, or a polymorph, solvate, hydrate, dehydrate, co-crystal, anhydrous or none Shaped form.
  • Parenteral dosage forms can be administered to a patient by a variety of routes including, but not limited to, subcutaneous, intravenous (including bolus injection), intramuscular, and intraarterial routes. Because the administration of the parenteral dosage form generally circumvents the patient's natural defense against contaminants, the parenteral dosage form is preferably sterile or can be sterilized prior to administration to the patient. Examples of parenteral dosage forms include, but are not limited to, solutions prepared for injection, dry products ready to be dissolved or suspended in a pharmaceutically acceptable injectable vehicle, suspensions ready for injection, and emulsions. In addition, controlled release parenteral dosage forms can be prepared for administration to a patient, including, but not limited to, administration. Formulations, as well as dosage drops.
  • Suitable vehicles that can be used to provide parenteral dosage forms of the present disclosure are well known to those skilled in the art. Examples include, but are not limited to, sterile water; USP water for injection; saline solution; glucose solution; aqueous vehicle such as, but not limited to, sodium chloride injection, Ringer's injection, dextrose injection, right Spirulina and sodium chloride injections and lactated Ringer's injection; water-miscible vehicles such as, but not limited to, ethanol, polyethylene glycol, and propylene glycol; and non-aqueous vehicles such as, but not limited to, corn oil, cottonseed Oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate and benzyl benzoate.
  • Topical dosage forms of the present disclosure include, but are not limited to, creams, lotions, ointments, gels, shampoos, sprays, aerosols, solutions, emulsions, and other forms known to those skilled in the art. See, for example, Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing, Easton, Pa. (1990); and Introduction to Pharmaceutical Dosage Forms, 4th ed., Lea & Febiger, Philadelphia, Pa. (1985).
  • a viscous to semi-solid or solid form comprising a carrier or one or more excipients compatible with topical application and having a dynamic viscosity preferably greater than water is typically employed.
  • Suitable formulations include, but are not limited to, solutions, suspensions, emulsions, creams, ointments, powders, elixirs, salves, and the like, if necessary, sterilized or auxiliaries (eg, preservatives, stabilizing) Mixtures, wetting agents, buffers or salts) are used to affect various properties, such as osmotic pressure.
  • suitable topical dosage forms include sprayable aerosol formulations in which the active ingredient (preferably in combination with a solid or liquid inert carrier) is packaged in a mixture with pressurized volatiles (for example, a gas propellant such as Freon) or extruded. In the bottle.
  • Moisturizers or humectants can also be added to the pharmaceutical compositions and dosage forms if desired.
  • An example of such an additional component is Ben The field is well known. See, for example, Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing, Easton, Pa. (1990).
  • Transdermal and mucosal dosage forms of the active compositions of the present disclosure include, but are not limited to, ophthalmic solutions, patches, sprays, aerosols, creams, lotions, suppositories, ointments, gels, solutions, emulsions, Suspensions, or other forms known to those skilled in the art. See, for example, Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing, Easton, Pa. (1990); and Introduction to Pharmaceutical Dosage Forms, 4th Ed., Lea & Febiger, Philadelphia, Pa. (1985).
  • Dosage forms suitable for treating mucosal tissues in the oral cavity can be formulated as mouthwashes, oral gels, or oral patches.
  • Additional transdermal dosage forms include "depot type” or "matrix type” patches that can be applied to the skin and worn for a specific period of time to allow penetration of the desired amount of active ingredient.
  • transdermal dosage forms and methods of administration that can be used to administer the active ingredients of the present disclosure include, but are not limited to, those disclosed in U.S. Patent Nos. 4,624,665; 4,655,767; 4,687,481; 4,797,284; 4,810,499; 4,834,978; 4,877,618; 4, 880, 633; 4, 917, 895; 4, 927, 687; 4, 956, 171; 5, 035, 894; 5, 091, 186; 5, 163, 899; 5, 232, 702; 5, 234, 690; 5, 273, 755; 5, 273, 756; 5, 308, 625; 5, 356, 632; 5, 358, 715; 5, 372, 579; 5, 421, 816; 5, 466; 465; 5, 494, 680; 5, 505, 958; 5, 554, 381; 5, 560, 922; 5, 585, 111; 5, 656, 285; 5, 698, 217; 5, 741, 511; 5, 747, 783;
  • Suitable excipients e.g., carriers and diluents
  • other materials that can be used to provide transdermal and mucosal dosage forms for inclusion in the present disclosure are well known to those of ordinary skill in the pharmaceutical arts and depend on a given pharmaceutical composition. Or the dosage form will be applied to a particular tissue or organ.
  • typical excipients include, but are not limited to, water, acetone, ethanol, ethylene glycol, propylene glycol, butane-1,3-diol, isopropyl myristate, isopropyl palmitate, Mineral oils, and mixtures thereof, to form non-toxic and pharmaceutically acceptable dosage forms.
  • the treatment or treatment with a pharmaceutically acceptable salt of a compound of the active composition of the present disclosure Use additional components later.
  • penetration enhancers can be used to help deliver the active ingredient to or through the tissue.
  • Suitable permeation enhancers include, but are not limited to, acetone; various alcohols such as ethanol, oleyl alcohol, tetrahydrofuran; alkyl sulfoxides such as dimethyl sulfoxide; dimethyl acetamide; dimethylformamide; Alcohol; pyrrolidone, such as polyvinylpyrrolidone; polyvinylpyrrolidone grade (polyvinylpyrrolidone, povidone); urea; and various water soluble Sexual or insoluble sugar esters such as TWEEN 80 (polysorbate 80) and SPAN 60 (sorbitan monostearate).
  • the pH of the pharmaceutical composition or dosage form or the pH of the tissue to which the pharmaceutical composition or dosage form is applied may also be adjusted to improve delivery of the active ingredient.
  • the polarity of the solvent carrier, its ionic strength or tonicity can be adjusted to improve delivery.
  • Compounds such as stearates can also be added to the pharmaceutical compositions or dosage forms to advantageously alter the hydrophilicity or lipophilicity of the active ingredients, thereby improving delivery.
  • stearate can be used as a lipid vehicle for the formulation, as an emulsifier or surfactant, and as a delivery enhancer or penetration enhancer.
  • Different hydrates, dehydrates, co-crystals, solvates, polymorphs, anhydrous or amorphous forms of the pharmaceutically acceptable salts of the compounds of the active compositions can be used to further adjust the properties of the resulting compositions.
  • the active ingredients of the pharmaceutical compositions of the present disclosure may not be administered to a patient at the same time or by the same route of administration. Accordingly, the present disclosure includes kits that, when used by a physician, can simplify the administration of an appropriate amount of active ingredient to a patient.
  • a typical kit comprises a unit dosage form of a pharmaceutically acceptable salt of the active compound of the active composition.
  • the pharmaceutically acceptable salt of the active compound of the active composition is a sodium, lithium or potassium salt, or a polymorph, solvate, hydrate, dehydrate, co-crystal, anhydrous or Amorphous form.
  • the kit may further comprise means for administering the active ingredient. Examples of such devices include, but are not limited to, syringes, drip bags, patches, and inhalants.
  • the kit can contain instructions for using the kit.
  • Kits of the present disclosure may further comprise a pharmaceutically acceptable vehicle that can be used to administer one or more active ingredients (e.g., active compounds).
  • active ingredients e.g., active compounds
  • the kit may contain a sealed container of suitable vehicle in which the active ingredient can be dissolved to form a suitable preparation for parenteral administration.
  • Examples of pharmaceutically acceptable vehicles include, but are not limited to, USP water for injection; aqueous vehicles such as, but not limited to, sodium chloride injection, Ringer's injection, dextrose injection, dextrose And sodium chloride injection, and lactated Ringer's injection; water miscible vehicles such as, but not limited to, ethanol, polyethylene glycol, and propylene glycol; and non-aqueous vehicles such as, but not limited to, corn oil, cottonseed oil, Peanut oil, sesame oil, ethyl oleate, isopropyl myristate and benzyl benzoate.
  • aqueous vehicles such as, but not limited to, sodium chloride injection, Ringer's injection, dextrose injection, dextrose And sodium chloride injection, and lactated Ringer's injection
  • water miscible vehicles such as, but not limited to, ethanol, polyethylene glycol, and propylene glycol
  • non-aqueous vehicles such as, but not limited to, corn
  • kits for screening a compound of a candidate drug comprises a plurality of Drosophila embryos.
  • Each Drosophila embryo contains a GGGGCC 30 repeat in C9orf72 linked to the hs-hid transgene (where heat shock is lethal to the GGGGCC 30 transgenic Drosophila embryo) and a set of instructions for screening the compound library.
  • the Drosophila disease model of ALS and FTD provides experimental evidence that the rGGGGCC repeat in C9orf72 alone is sufficient to cause neurodegeneration in a repeated dose. See Xu et al., PNAS, 2013, 110: 7778-7783. Drosophila with expression of the rGGGGCC30 repeat in C9orf72 did not reach adulthood. Lethality occurs primarily during embryonic development prior to larval formation. Using this lethality, a high-throughput strategy was designed to screen for chemical libraries of small molecules associated with ALS and FTD.
  • the UAS-GGGGCC30-EGFP transgene was chromosomally balanced (w1118, UAS-GGGGCC30-EGFP/TM2Cyo:hs-hid) carrying a CFS (Cyo) marker recombined with heat shock-induced apoptosis hid-hed. Then, the Drosophila GAL4/UAS system was used to test the viability of adult offspring. Hybridization of UAS-GGGGCC30-EGFP/TM2Cyo:hs-hid was carried out using a homozygous pan-neuron elav-Gal4 driver.
  • this assay can be used for chemical screening to identify small molecules that can improve/restore the viability of Drosophila expressing the rGGGGCC repeat.
  • Figure 1 As described in the schematic ( Figure 1), supplemented with 40 ⁇ m individual unique compounds from 2000 US Food and Drug Administration (FDA) approved drugs and natural products (The Spectrum Collection, MicroSource Discovery Systems, Inc.) The food was fed a hybrid control flies between elav-GAL4 and UAS-GGGG30-EGFP/TM2Cyo:hs-hid.
  • FDA US Food and Drug Administration
  • the hybrids were heat shocked to clear (elav-GAL4/+; +/TM2Cyo:hs-hid) progeny, then the vials were kept at 25 ° C for 10-15 days to target any live Drosophila (elav-GAL4/+; UAS-GGGGCC30-EGFP/+), which is otherwise lethal, was scored.
  • a secondary behavioral assay was developed.
  • the UAS-GGGGCC30-EGFP transgenic Drosophila is crossed with the motor neuron-specific driver Ok371-GAL4, which drives the expression of rGGGGC repeats in motor neurons, allowing us to Analysis of motor defects in adult fruit flies.
  • Expression of the rGGGGCC repeat fragment resulted in dyskinesia ( Figure 2).
  • Figure 2A hybridization between UAS-GGGGCC30-EGFP/TM2Cyo and Ok371-GAL4 was established in food supplemented with various compounds.
  • the progeny Drosophila (UAS-GGGGCC30-EGFP/Ok371-GAL4) was collected to monitor exercise.
  • DAM Drosophila Activity Monitoring

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Abstract

本发明涉及用于治疗肌萎缩侧索硬化(ALS)和额颞叶痴呆(FTD)的化合物与应用，其公开了抑制由C9orf72的第一内含子中的rGGGGCC重复片段引起的毒性的化合物在制备用于治疗肌萎缩侧索硬化(ALS)和额颞叶痴呆(FTD)的药物中的用途。本发明公开的抑制由C9orf72的第一内含子中的rGGGGCC重复片段引起的毒性的化合物可显著改善由rGGGGCC重复片段诱导的运动障碍。

Description

用于治疗肌萎缩侧索硬化和额颞叶痴呆的化合物与应用 技术领域
本发明涉及用于治疗肌萎缩侧索硬化(ALS)和额颞叶痴呆(FTD)的化合物与应用，属于神经药物技术领域。
背景技术
C9ORF72基因中外显子1a和1b之间的GGGGCC重复片段扩增被发现是肌萎缩侧索硬化(ALS)和额颞叶痴呆(FTD)的重要病因。扩增的GGGGCC重复片段不仅解释了以显性性状遗传ALS或FTD的家族的40％的病因，还占了欧洲和北美人群中所有ALS和FTD病例的5-7％，从而使得其成为这两种疾病之一的最常见的遗传病因。
ALS和FTD具有表型和病理重叠。ALS是一种致命的退行性疾病，其主要影响运动神经元。基本特征是进行性乏力；然而，认知损害和行为改变(类似于见于FTD中的那些)是愈发得到认可的症状。FTD是进行性痴呆疾病，其主要影响额皮层、岛叶皮层和前颞叶皮层的神经元，从而导致人格、行为和/或语言能力的显著变化。少数患有FTD的个体也发生ALS。在病理学上，ALS和FTD最常见地由TAR DNA结合蛋白(TDP-43)的异常积累表征。在遗传学上，分离ALS或FTD(通常作为常染色体显性性状)的家庭的聚类支持了每种疾病具有强遗传基础的观点。连锁分析和基于家族的遗传研究已发现，几种基因(特别是TAR DNA结合蛋白(TARDBP)和肉瘤融合(FUS)蛋白)中的突变是ALS和FTD的罕见病因。还发现许多家族共分离ALS、FTD或两者作为常染色体显性性状并显示与9p21上的基因座连锁的证据。
最近，发现与9p21基因座连锁的原因是C9orf72中外显子1a和1b之间的异常扩增的GGGGCC重复片段。引起疾病所需的扩增的GGGGCC重复片段的确切大小不是精确已知的。GGGGCC重复片段的扩增已从此被显示为ALS和FTD的最常见遗传病因。GGGGCC扩增具有年龄依赖性外显率，到80岁时几乎完全外显。在生化学上，C9orf72是很大程度上未表征的，但似乎在正常对照的脑中是普遍转录的。
ALS和FTD的缓慢进展性质和GGGGCC扩增的非编码性质与其它非编码核苷酸重复片段病症(例如强直性肌营养不良、脆性X相关震颤/共济失调综合征(FXTAS)和脊髓小脑性共济失调)具有惊人的相似性。非编码重复片段如何导致疾病是未知的。一 种可能性是非编码重复片段通过破坏核和细胞质RNA加工来产生其毒性作用。据最近报道，非编码重复片段还可以被翻译成推测具有毒性的同聚物蛋白质。需要进一步的工作来阐明这些过程对疾病发病机制的相对贡献。RNA介导的毒性的一种可能的机制是通过由转录的扩增重复片段隔离正常的RNA结合蛋白(RBP)，从而导致可用于正常RNA代谢的RBP的耗尽。这些RBP的耗尽的效应包括：发育特异性转录物的丢失和明显的剪接异常。
以基于riboGGGGCC(rGGGGCC)扩增水平的增加，提出了ALS和FTD的RNA功能获得性机制。这种类型的RNA功能获得性机制已被建议作为在强直性肌营养不良(DM)和在FXTAS中扩增的riboCGG(rCGG)重复片段中的三联体重复片段相关性共济失调如SCA8、SCA10和SCA12的机制。
一些另外的证据还支持ALS和FTD的RNA介导的功能获得性毒性模型。已描述了使用果蝇(Drosophila)的ALS和FTD模型，并已证实C9orf72中外显子1a和1b之间的rGGGGCC重复片段是具有毒性的并且足以引起神经变性。
由于认识到果蝇与人共有许多结构和功能相关的基因家族，果蝇在过去十年中已经成为了用于人神经变性疾病的研究的首个模型系统。已显示，与神经变性相关的基因可以在果蝇中表达，引起与对应的人疾病(包括多聚谷氨酰胺疾病、帕金森病和阿尔茨海默病)的表型显著相似的表型。这些结果表明，神经元毒性和损失的分子机制在人和果蝇之间是保守的。在果蝇中这样的疾病模型的开发允许应用遗传方法来解决关于疾病进展的特定假说和测试候选修饰基因或治疗性药物化合物。
发明内容
本发明针对现有技术的不足，提供了可以治疗、预防ALS和FTD或减少ALS和FTD的症状或延迟ALS和FTD的进展的化合物、组合物和使用方法。
本发明技术方案如下：
抑制由C9orf72的第一内含子中的rGGGGCC重复片段引起的毒性的化合物在制备用于治疗肌萎缩侧索硬化(ALS)和额颞叶痴呆(FTD)的药物中的用途。
根据本发明优选的，所述化合物选自：OEM-34、OEM-62、OEM-77、OK-EM 026、OK-EM 027、OK-EM 028、OK-EM 029、OK-EM 033和/或OK-EM 036。
根据本发明优选的，所述化合物选自：OEM-34、OEM-62、OEM-77、OK-EM 026、OK-EM 027、OK-EM 028、OK-EM 029、OK-EM 033和/或OK-EM 036的盐、酯或前药。
在一些实施方案中，提供了治疗或预防ALS和FTD的方法，包括给有此症状的宿主施用有效量的OEM-34、OEM-62或OEM-77。
在某些实施方案中，宿主在其基因组的C9orf72基因中具有30个GGGGCC重复片段。在某些实施方案中，宿主患有肌萎缩侧索硬化。在某些实施方案中，宿主患有额颞叶痴呆。
在某些实施方案中，提供了药物组合物，其包含与药学上可接受的载体组合的OEM-34、OEM-62或OEM-77。在某些实施方案中，化合物以有效治疗病症的剂量水平存在，其中所述病症是肌萎缩侧索硬化或额颞叶痴呆。
本发明公开内容的实施方案还包括筛选有效治疗或预防ALS和FTD的化合物的方法，其包括：1)提供化合物文库；2)将化合物的每一种施用至多个果蝇胚胎，其中每个果蝇胚胎包含与hs-hid转基因连锁的GGGGCC30重复片段，其中在热休克后，hs-hid的表达将被激活；3)使果蝇胚胎暴露于热休克；4)测量每个容器中的蛹形成和成年果蝇，其中蛹形成和成年果蝇在包含挽救GGGGCC30重复片段的果蝇胚胎的化合物的容器中出现；和5)选择允许蛹或成年果蝇形成的化合物。
本发明公开内容的实施方案包括试剂盒以筛选化合物文库，所述试剂盒包含：多个果蝇胚胎，其中每个果蝇胚胎包含与hs-hid转基因连锁的GGGGCC30重复片段，所述hs-hid转基因在热休克后对于携带GGGGCC30重复片段的果蝇胚胎是致死的；和用于筛选化合物文库的一组指令。
在某些实施方案中，本发明公开内容涉及治疗肌萎缩侧索硬化的方法，其包括给有此需要的受试者施用有效量的化合物，所述化合物抑制由GGGGCC重复片段引起的毒性。在某些实施方案中，受试者被诊断具有GGGGCC重复片段扩增。在某些实施方案中，受试者被诊断患有ALS。在某些实施方案中，受试者被诊断患有FTD。在某些实施方案中，化合物选自：OEM-34、OEM-62或OEM-77。在某些实施方案中，化合物选自：OEM-34、OEM-62或OEM-77，或其盐、酯或前药。在某些实施方案中，化合物选自OEM-34、OEM-62或OEM-77，或其盐、酯或前药。在某些实施方案中，化合物是OEM-34、OEM-62或OEM-77。在某些实施方案中，化合物是用一种或多种取代基取代的本文提供的任何化合物。
在某些实施方案中，本公开内容涉及具有GGGGCC连锁的转基因的表达增加的遗传工程化果蝇。
本发明公开内容的其它组合物、方法、特征和优势在结合下文的附图和详细描述 后对于本领域技术人员而言将是或变得显而易见。意欲所有这样的另外的组合物、方法、特征和优势包括在本发明说明书内、包括在本发明公开内容的范围内、并受到所附权利要求书保护。
附图说明
图1、抑制rGGGGCC重复片段介导的致死性的化学筛选对抑制由rGGGGCC重复片段引起的毒性的小分子的鉴定过程示意图；
在补充有或没有来自2000个小分子的文库的个体独特化合物的食物上将Elav-Gal4驱动子与UAS-GGGGCC30-EGFP/Cyo杂交以产生子代胚胎。子代胚胎继续在补充有个体药物的食物上发育直到子代结束。在施用热休克后通过基于其基因型比较成年子代或蛹的数目获得相对活力；
图2、改善由rGGGGCC重复片段引起的运动缺陷的小分子的鉴定过程示意图；
在补充有或没有所示的所选化合物的食物上将Nervana-GAL4驱动子与UAS-GGGGCC30-EGFP/Cyo杂交以产生子代胚胎。选择基因型UAS-GGGGCC30-EGFP/nervana的成年子代并随后就运动活性监测48小时龄的30只雌性未交配体，连续进行数周。每次同时监测用含有化合物的果蝇食物喂养的30只果蝇的运动；
图3、可抑制由rGGGGCC重复片段引起的毒性的3种小分子的化学结构图；
图4、在7天时针对所选药物的平均运动活性的百分比结果柱状图；
误差线表示SEM；**P<0.001，*P<0.01。
图5、可抑制由rGGGGCC重复片段引起的毒性的6种小分子的化学结构图；
具体实施方式
本发明公开内容通过参考下面的详细描述和包括在其中的实施例可以更容易地理解。在公开和描述本发明的化合物、组合物和方法之前，应理解，本发明公开内容并不局限于特定的药物载体或特定的药物制剂或给药方案，因为这些当然可以变化。还应理解，本文所使用的术语仅是用于描述具体实施方案的目的，而不意欲是限制性的。
定义
如本文所用，术语“受试者”包括人、哺乳动物(例如，猫、狗、马等)和需要治疗的其它活物种。活生物体可以是人或哺乳动物。“易患”神经元病症和相关病状的受试者可以被定义为不显示这些病状的一种或多种的明显症状但在遗传上、生理上或以其它方式处于发生这些病状的一种或多种的风险中的受试者。因此，本公开内容 的组合物和药剂可预防性地用于这些病状。此外，“组合物”或“药剂”可以包含如下文所述的一种或多种化学化合物和/或试剂。“活性组合物”可以包含一种或多种“活性化合物”。
术语“筛选”是指从化合物的文库鉴定满足标准(例如，但不限于，从致死剂量的谷氨酸挽救特别设计的果蝇)的一种或多种化合物。本公开内容的筛选方法可用于鉴定用于治疗与rGGGGCC重复片段介导的神经变性和本文所述的其它疾病和病症相关的病症的化合物(例如，待用于活性组合物的药物候选物)。
术语“衍生物”是指对所公开的化合物的修饰，包括但不限于所公开的化合物的水解、还原或氧化产物。特别地，该术语包括含氮环结构(包括但不限于，所公开的化合物的咪唑)的打开。水解、还原和氧化反应是本领域已知的。
如本文所用的术语“治疗有效量”是指所施用的化合物的量将在一定程度上减轻所治疗的病症的一种或多种症状，和/或该量将在一定程度上预防所治疗的处于发作风险的受试者的病症的一种或多种症状。
“药学上可接受的盐”是指保留游离碱的生物有效性和性质，并且通过与无机酸或有机酸如盐酸、氢溴酸、硫酸、硝酸、磷酸、甲磺酸、乙磺酸、对甲苯磺酸、水杨酸、苹果酸、马来酸、琥珀酸、酒石酸、柠檬酸和类似物反应而得到的那些盐。
“药物组合物”是指本文所述的一种或多种化合物或其药学上可接受的盐与其它化学组分例如生理上可接受的载体和赋形剂的混合物。药物组合物的一个目的是促进化合物至生物体的施用。
如本文所使用的，“药学上可接受的载体”是指不会对生物体引起显著刺激并且不会消除所施用化合物的生物活性和性质的载体或稀释剂。
“赋形剂”是指加入到药物组合物中以进一步促进化合物的施用的惰性物质。赋形剂的实例包括但不限于碳酸钙、磷酸钙、各种糖和各种类型的淀粉、纤维素衍生物、明胶、植物油和聚乙二醇。
疾病的“治疗”或“疗法”包括预防疾病在可能易患该疾病但还没有经历或表现出该疾病的症状的动物中发生(预防性治疗)，抑制疾病(减缓或阻止其发展)，提供疾病症状或副作用的缓解(包含姑息治疗)，以及减轻疾病(引起的疾病消退)。
术语“前药”是指在体内被转化成生物活性形式的药剂。前药通常是有用的，因为在一些情况下，它们可以比母体化合物更容易地施用。它们可以，例如，通过口服 给药而可生物利用，而母体化合物则不能。前药还可在药物组合物中具有相对于母体药物的改善的溶解度。前药可以通过各种机制转化为母体药物，包括酶促过程和代谢水解。Harper,N.J.(1962).Drug Latentiation in Jucker,ed.Progress in Drug Research,4:221-294；Morozowich等人(1977).Application of Physical Organic Principles to Prodrug Design in E.B.Roche ed.Design of Biopharmaceutical Properties through Prodrugs and Analogs,APhA；Acad.Pharm.Sci.；E.B.Roche编辑(1977).Bioreversible Carriers in Drug in Drug Design,Theory and Appl ication,APhA；H.Bundgaard编辑(1985)Design of Prodrugs,Elsevier；Wang等人(1999)Prodrug approaches to the improved delivery of peptide drug,Curr.Pharm.Design.5(4):265-287；Pauletti等人(1997).Improvement in peptide bioavailability:Peptidomimetics and Prodrug Strategies,Adv.Drug.Delivery Rev.27:235-256；Mizen等人(1998).The Use of Esters as Prodrugs for Oral Delivery ofβ-Lactam antibiotics,Pharm.Biotech.11,:345-365；Gaignault 等人(1996).Designing Prodrugs and Bioprecursors I.Carrier Prodrugs,Pract.Med.Chem.671-696；M.Asgharnejad(2000).Improving Oral Drug Transport Via Prodrugs,in G.L.Amidon,P.I.Lee and E.M.Topp,Eds.,Transport Processes in Pharmaceutical Systems,Marcell Dekker,p.185-218；Balant等人(1990)Prodrugs for the improvement of drug absorption via different routes of administration,Eur.J.Drug Metab.Pharmacokinet.,15(2):143-53；Balimane and Sinko(1999).Involvement of multiple transporters in the oral absorption of nucleoside analogues,Adv.Drug Del ivery Rev.,39(1-3):183-209；Browne(1997).Fosphenyloin(Cerebyx),Clin.Neuropharmacol.20(1):1-12；Bundgaard(1979).Bioreversible derivatization of drugs--principle and applicability to improve the therapeutic effects of drugs,Arch.Pharm.Chemi.86(1):1-39；H.Bundgaard编辑(1985)Design of Prodrugs,New York:Elsevier；Flei sher等人(1996).Improved oral drug delivery:solubility limitations overcome by the use of prodrugs,Adv.Drug Del ivery Rev.19(2):115-130；Fleisher等人(1985).Design of prodrugs for improved gastrointestinal absorption by intestinal enzyme targeting,Methods Enzymol.112:360-81；Farquhar D,等人(1983). Biologically Reversible Phosphate-Protective Groups,J.Pharm.Sci.,72(3):324-325；Han,H.K.等人(2000).Targeted prodrug design to optimize drug del ivery,AAPS PharmSci.,2(1):E6；Sadzuka Y.(2000).Effective prodrug liposome and conversion to active metabolite,Curr.Drug Metab.,1(1):31-48；D.M.Lambert(2000)Rationale and applications of lipids as prodrug carriers,Eur.J.Pharm.Sci.,11Suppl 2:S15-27；Wang,W.等人(1999)Prodrug approaches to the improved delivery of peptide drugs.Curr.Pharm.Des.,5(4):265-87。
如本文所用，术语“局部活性剂”是指在施加至宿主(以局部施加的方式接触)的部位引起药理学应答的本公开内容的组合物。
如本文所用，术语“局部”是指将本公开内容的组合物施加至粘膜细胞和组织及皮肤的表面。
在整个说明书中，可选择基团及其取代基以提供稳定的部分和化合物。
所公开的化合物形成也在本发明的范围内的盐。对本文中任何式的化合物的提及被理解为包括对其盐的提及，除非另有说明。如本文所用的术语“盐”表示与无机和/或有机酸和碱形成的酸性和/或碱性盐。此外，当式I的活性化合物包含碱性部分和酸性部分两者时，可形成两性离子(“内盐”)并且其包括在如本文所用的术语“盐”内。药学上可接受的(例如，无毒性的，生理学可接受的)盐是优选的，尽管例如在制备过程中可使用的分离或纯化步骤中，也可使用其它盐。活性化合物的化合物的盐可以例如通过用一定量(例如等量)的酸或碱与活性化合物反应来形成，这在诸如其中盐沉淀的介质中进行或在水性介质中进行随后冻干。
含有碱性部分的所公开的化合物可以与各种有机酸和无机酸形成盐。示例性的酸加成盐包括乙酸盐(诸如与乙酸或三卤乙酸例如三氟乙酸形成的盐)，己二酸盐，藻酸盐，抗坏血酸盐，天冬氨酸盐，苯甲酸盐，苯磺酸盐，硫酸氢盐，硼酸盐，丁酸盐，柠檬酸盐，樟脑酸盐，樟脑磺酸盐，环戊烷丙酸盐，二葡糖酸盐，十二烷基硫酸盐，乙磺酸盐，延胡索酸盐，葡糖庚酸盐(Glucoheptanoates)，甘油磷酸盐，半硫酸盐，庚酸盐，己酸盐，盐酸盐(与盐酸形成)，氢溴酸盐(与氢溴酸形成)，氢碘酸盐，2-羟基乙磺酸盐，乳酸盐，马来酸盐(与马来酸形成)，甲磺酸盐(与甲磺酸形成)，2-萘磺酸盐，烟酸盐，硝酸盐，草酸盐，果胶酸盐，过硫酸盐，3-苯基丙酸盐，磷酸盐，苦味酸盐，新戊酸盐，丙酸盐，水杨酸盐，琥珀酸盐，硫酸盐(例如与硫酸形成 的盐)，磺酸盐(如本文提及的那些)，酒石酸盐，硫氰酸盐，甲苯磺酸盐例如甲磺酸盐，十一酸盐等。
所公开的含有酸性部分的化合物可以与各种有机和无机碱形成盐。示例性碱性盐包括铵盐、碱金属盐例如钠盐、锂盐和钾盐，碱土金属盐例如钙盐和镁盐，与有机碱(例如，有机胺)诸如benzathine、二环己胺，hydrabamine(与N,N-二(去氢枞基)乙二胺形成)、N-甲基-D-葡糖胺、N-甲基-D-葡糖酰胺、叔丁基胺的盐，以及与氨基酸诸如精氨酸、赖氨酸的盐等。
碱性含氮基团可以用诸如例如低级烷基卤化物(例如，甲基、乙基、丙基和丁基氯化物、溴化物和碘化物)、二烷基硫酸酯(例如，二甲基、二乙基、二丁基和二戊基硫酸酯)、长链卤化物(例如，癸基、月桂基、肉豆蔻基和硬脂基氯化物、溴化物和碘化物)、芳烷基卤化物(例如，苄基和苯乙基溴化物)等的试剂季铵化。
术语“取代”是指其中至少一个氢原子被取代基替换的分子。当被取代时，一个或多个基团是“取代基”。分子可以是多取代的。在氧代取代基(“＝O”)的情况下，两个氢原子被取代。在此上下文范围内的实例取代基可以包含卤素，羟基，烷基，烷氧基，硝基，氰基，氧代，碳环基，碳环烷基，杂碳环基，杂碳环烷基，芳基，芳基烷基，杂芳基，杂芳基烷基，-NRaRb，-NRaC(＝O)Rb，-NRaC(＝O)NRaNRb，-NRaC(＝O)ORb，-NRaSO2Rb，-C(＝O)Ra，-C(＝O)ORa，-C(＝O)NRaRb，-OC(＝O)NRaRb，-ORa，-SRa，-SORa，-S(＝O)2Ra，-OS(＝O)2Ra和-S(＝O)2ORa。在此上下文中的Ra和Rb可以是相同或不同的，并且独立地为氢，卤素羟基，烷基，烷氧基，烃基，氨基，烷基氨基，二烷基氨基，碳环基，碳环烷基，杂碳环基，杂碳环烷基，芳基，芳基烷基，杂芳基和杂芳基烷基。
本发明公开内容的化合物的溶剂合物也包括在本文中。化合物的溶剂合物优选是水合物。
在所公开的活性化合物及其盐可以以它们的互变异构形式存在的程度上，所有这样的互变异构形式在本文被考虑作为本公开内容的一部分。
本发明的化合物的所有立体异构体，例如可能由于各种取代基上的不对称碳原子上而存在的那些，包括对映异构形式(其甚至可以在不存在不对称碳的情况下存在)和非对映体形式，在本公开内容的范围内。本公开内容的化合物的各个立体异构体可以例如基本上不含其它异构体，或可以例如作为外消旋体混合或与所有其它的或另外 选择的立体异构体混合。本公开内容的化合物的手性中心可具有由IUPAC 1974Recommendation所定义的S或R构型。
术语“包含”、“诸如”、“例如”等旨在引用示例性实施方案，而不是限制本公开内容的范围。
讨论
本发明公开内容的实施方案包括筛选方法、用于筛选化合物文库的试剂盒、包含一种或多种活性化合物的活性组合物、包含一种或多种活性化合物的药物组合物、治疗和/或预防肌萎缩侧索硬化和额颞叶痴呆的方法、用于治疗和/或预防肌萎缩侧索硬化和额颞叶痴呆的试剂盒。
一般而言，本发明公开内容的实施方案包括筛选可以用于治疗肌萎缩侧索硬化和额颞叶痴呆的化合物(例如，药物候选物)的高通量方法。筛选是基于果蝇(例如，果蝇(fruit fly))的化合物筛选，其中化合物的功效通过对在一定的培养条件下蛹形成的数量或成年果蝇的出现进行打分来确定。满足筛选过程的化合物可以被选择作为活性化合物，其可以用于组合物和药物组合物以用有效量的活性组合物来治疗需要这样的治疗的宿主。
筛选方法的实施方案是有利的，因为它们提供了唯一已知的方法用于筛选可逆转rGGGGCC重复片段介导的毒性的临床后果的化合物。此外，使用果蝇提供了复杂生物体的筛选，其比较简单的筛选例如基于细胞的筛选更可能提供的有用的化合物。此外，含有C9orf72中的rGGGGCC重复片段的转基因果蝇显示与人表型一致的神经元细胞死亡。转基因果蝇模型已先前建立，它在报告基因EGFP的上游表达扩增的重复片段，并显示神经元细胞死亡在人ALS的发病机制中的直接参与。在此模型中，GAL4-UAS系统被用来驱动转基因在不同的组织(包括果蝇眼)中的表达。果蝇显示模拟人表型的眼和运动神经元的神经变性。
一般而言，本发明公开内容的实施方案提供包含可用于治疗和/或预防ALS和FTD的一种或多种活性化合物的活性组合物。
筛选
本发明公开内容的实施方案包括筛选可用于治疗ALS和FTD的化合物(例如，药物候选物)的高通量方法。
具体地，本公开内容的实施方案包括使用基于果蝇(fruit fly)的化合物筛选来 筛选化合物以鉴定用于治疗ALS和FTD的药物候选物的方法。携带在C9ord72中外显子1a和1b之间的GGGGCC30重复片段的果蝇表现出与在小鼠模型中和在人患者中所报道的那些相似的神经元和行为缺陷。已发现，rGGGGCC重复片段的表达导致在早期胚胎发育中的致死性。产生了用携带hs-hid转基因的Cyo平衡子平衡的UAS-GGGGCC30-EGFP转基因的果蝇株。在热休克后，hid的表达被激活，其导致GGGGCC30果蝇胚胎中的细胞死亡和致死性。如在图1所显示的，在早期胚胎发育期间的热休克后，没有产生活的果蝇或蛹。
突变的胚胎被放入含有具有或不具有来自药物候选物文库的化合物的食物的适当容器(例如，96孔容器)中。筛选了来自2,000个FDA批准的药物和天然产品的文库(The Spectrum CollectionTM)的5μM和40μM个体化合物。随后将小瓶保持在25℃下进行10至15天以针对活力进行打分。药物候选物的功效通过对在一定时间范围后蛹形成的数量或成年果蝇的出现进行打分来测定。本公开内容的实施方案提供了针对直接和间接调节rGGGGCC重复片段介导的毒性和挽救突变果蝇避免热休克诱导的致死性的化合物的筛选。关于活性组合物和病症的其它细节描述于下文的实施例中。
物质的组合物
一般而言，包含一种或多种活性化合物的活性组合物可用于治疗和/或预防ALS和FTD。
在某些实施方案中，化合物是OEM-34，OEM-62或OEM-77。
基于本发明公开内容的实施方案和实施例中的讨论，开发了用于活性组合物的剂量方案。一般而言，大多数I期临床试验的起始剂量基于临床前试验，且通常是相当保守的。在临床前测试中对药物毒性的标准量度是由于治疗死亡的动物(啮齿动物)的百分率。10％的动物死亡时的剂量被称为LD10，这在过去常常与人中的最大耐受剂量(MTD)关联(针对体表面积进行调整)。针对体表面积的调整包括宿主因素，例如，表面积、体重、代谢、组织分布、吸收率和排泄率。因此，标准的保守起始剂量为鼠LD10的十分之一，尽管如果其它物种(即，狗)对于药物更敏感，它可能会更低。预期在人中I期临床试验中的活性组合物的起始剂量会以这种方式来确定。此给药方案更详细地讨论于Freireich E J,et al.,Cancer Chemother Rep 50:219-244,1966中，其通过引用并入本文。
如上所述，治疗有效剂量水平将取决于许多因素。此外，在本领域的技术范围内 的是，以相对低的水平起始活性组合物的剂量，并增加剂量直至达到预期效果为止。
药物组合物
本发明公开内容的实施方案提供了组合物和药物组合物，其包含有效量的活性组合物(例如，一种或多种活性化合物)以治疗和/或预防诸如本文描述的那些病症。
本发明公开内容的药物活性组合物和剂型包括所公开的化合物的药学上可接受的盐或其药学上可接受的多晶型物、溶剂合物、水合物、去水合物、共晶体、无水或无定形形式。所公开的化合物的具体的盐包括，但不限于，其钠盐、锂盐、钾盐及其水合物。
本发明公开内容的药物组合物和单位剂型通常还包含一种或多种药学上可接受的赋形剂或稀释剂。由本公开内容的具体的化合物提供的优势例如但不限于增加的溶解度和/或增强的流动、纯度或稳定性(例如，吸湿性)特性可以使它们比现有技术更好地适合用于药物配制和/或施用至患者。
本发明公开内容的化合物的药物单位剂型适合用于口服、粘膜(例如，经鼻、舌下、阴道、含服或直肠)、肠胃外(例如，肌内、皮下、静脉内、动脉内或弹丸注射)、局部、或透皮施用至患者。剂型的实例包括，但不限于：片剂；囊片；胶囊，例如硬明胶胶囊和软弹性明胶胶囊；扁囊剂；糖锭剂；锭剂；分散剂；栓剂；软膏剂；泥敷剂(泥罨剂)；糊剂；粉剂；敷料；乳膏剂；硬膏剂；溶液；贴剂；气雾剂(例如，鼻喷雾剂或吸入剂)；凝胶；适合于口服或粘膜施用至患者的液体剂型，包括混悬剂(例如，水性或非水性液体悬浮液，水包油乳剂或油包水液体乳剂)、溶液和酏剂；适合于肠胃外施用至患者的液体剂型；和无菌固体(例如，晶体或无定形固体)，其可被重建以提供适合于肠胃外施用至患者的液体剂型。
本发明公开内容的组合物的剂型的组成、形状和类型通常将取决于它们的用途而变化。例如，在疾病或病症的急性治疗中使用的剂型可比在相同疾病或病症的慢性治疗中使用的剂型包含更大量的活性成分，例如所公开的化合物或其组合。类似地，肠胃外剂型可以比用于治疗相同疾病或病症的口服剂型包含更少量的活性成分。其中本公开内容涵盖的特定剂型将彼此变化的这些和其它方式对于本领域技术人员而言将是显而易见的。参见，例如，Remington's Pharmaceutical Sciences,18th ed.,Mack Publishing,Easton,Pa.(1990)。
典型的药物组合物和剂型包含一种或多种赋形剂。合适的赋形剂是配药学或制药 学领域中的本领域技术人员所公知的，并且在本文中提供了合适的赋形剂的非限制性实例。一种特定的赋形剂是否适合用于掺入药物组合物或剂型取决于多种本领域公知的因素，包含但不限于，其中剂型将被施用至患者的方式。例如，口服剂型如片剂或胶囊剂可含有不适合用于肠胃外剂型的赋形剂。特定赋形剂的适合性还可取决于剂型中的具体活性成分。例如，一些活性成分的分解可以通过一些赋形剂例如乳糖或当暴露于水时加速。含有伯胺或仲胺的活性成分对于这样的加速分解是特别易感的。
本发明公开内容还包括含有一种或多种降低活性成分的分解速率的化合物的药物组合物和剂型。这样的化合物(其在本文中称为“稳定剂”)包含但不限于抗氧化剂如抗坏血酸、pH缓冲剂或盐缓冲剂。此外，本公开内容的药物组合物或剂型可含有一种或多种的溶解度调节剂，例如氯化钠、硫酸钠、磷酸钠或磷酸钾或有机酸。特别的溶解度调节剂是酒石酸。
类似于赋形剂的量和类型，剂型中活性成分的量和具体类型可取决于因素例如但不限于将其施用至患者的途径而不同。然而，本公开内容的化合物的典型剂型包括药学上可接受的盐，或其药学上可接受的多晶型物、溶剂合物、水合物、去水合物、共晶体、无水或无定形形式，其量为约10mg至约1000mg，优选约25mg至约750mg，更优选50mg至500mg。
此外，化合物和/或组合物可以使用基于脂质或聚合物的纳米颗粒递送。例如，纳米颗粒可以被设计来改善肠胃外施用的药物的药理学和治疗性质(Allen,T.M.,Cullis,P.R.Drug delivery systems:entering the mainstream.Science.303(5665):1818-22(2004))。
口服剂型
本发明公开内容的适合于口服给药的药物活性组合物可以被呈递作为不连续的剂型，例如但不限于，片剂(包括但不限于刻痕片剂或包衣片剂)、丸剂、囊片、胶囊、咀嚼片、粉末包、扁囊剂、糖锭剂、糯米纸剂、气雾喷雾剂，或液体，例如但不限于，糖浆剂、酏剂、水性液体、非水性液体中的溶液或悬浮液、水包油乳剂或油包水乳剂。这样的组合物包含所公开的化合物的药学上可接受的盐的预定量，并且可以通过本领域技术人员公知的配药学方法制备。一般地参见，Remington's Pharmaceutical Sciences,18th ed.,Mack Publishing,Easton,Pa.(1990)。
本发明公开内容的组合物的典型口服剂型通过按照常规药物复合技术将公开的化 合物的药学上可接受的盐与至少一种赋形剂组合在紧密掺合物中来制备。赋形剂可以采取各种形式，取决于给药所需的组合物的形式。例如，适用于口服液体或气雾剂剂型的赋形剂包括，但不限于水、二醇类、油类、醇类、调味剂、防腐剂和着色剂。适用于固体口服剂型(例如粉剂、片剂、胶囊和囊片)的赋形剂的实例包括，但不限于，淀粉、糖、微晶纤维素、高岭土、稀释剂、制粒剂、润滑剂、粘合剂和崩解剂。
由于其易于给药，片剂和胶囊代表最有利的固体口服单位剂型，在这种情况下，使用固体药物赋形剂。如果需要，片剂可以通过标准水性或非水性技术进行包衣。这些剂型可通过任何配药学方法制备。一般而言，药物组合物和剂型可以如下制备：将活性成分与液体载体、细碎的固体载体或两者均匀和紧密掺合，然后如果必要的话，将产物成型为所需的形式。
例如，片剂可以通过压制或模制来制备。压缩片可通过在合适的机器中压缩活性成分在自由流动形式例如粉末或颗粒中来制备，任选与一种或多种赋形剂混合。模制片剂可通过在合适的机器中将用惰性液体稀释剂润湿的粉状化合物的混合物模制来制备。
可以在本发明公开内容的口服剂型中使用的赋形剂的实例包括，但不限于粘合剂、填充剂、崩解剂和润滑剂。适合用于药物组合物和剂型的粘合剂包括，但不限于，玉米淀粉、马铃薯淀粉或其它淀粉，明胶，天然和合成的树胶如阿拉伯胶，海藻酸钠，海藻酸，其它海藻酸盐，黄蓍胶粉，瓜尔胶，纤维素及其衍生物(例如，乙基纤维素、醋酸纤维素、羧甲基纤维素钙、羧甲基纤维素钠)，聚乙烯吡咯烷酮，甲基纤维素，预胶化淀粉，羟丙基甲基纤维素，(例如，No.2208,2906,2910)，微晶纤维素，及其混合物。
微晶纤维素的适当形式包括但不限于以AVICEL-PH-101、AVICEL-PH-103 AVICEL RC-581和AVICEL-PH-105出售的材料(可购自FMC Corporation,American Viscose Division,Avicel Sales,Marcus Hook,Pa.,U.S.A.)，及其混合物。示例性的合适粘合剂是作为AVICEL RC-581出售的微晶纤维素和羧甲基纤维素钠的混合物。合适的无水或低水分赋形剂或添加剂包括
Figure PCTCN2015100061-appb-000001
和Starch 1500LM。
适合用于本文公开的药物组合物和剂型的填充剂的实例包括但不限于：滑石，碳酸钙(例如，颗粒或粉末)，微晶纤维素，粉状纤维素，葡萄糖结合剂，高岭土，甘露醇，硅酸，山梨糖醇，淀粉，预胶化淀粉，以及它们的混合物。在本公开内容的药 物组合物中的粘合剂或填充剂通常以药物组合物或剂型的约50至约99重量百分比存在。
崩解剂用于本发明公开内容的组合物中，以提供当暴露于水性环境时崩解的片剂。含有过多崩解剂的片剂可能会膨胀，开裂，或在存储时崩解，而含有太少崩解剂的片剂可能不足以发生崩解并因而可改变活性成分从剂型释放的速率和程度。因此，应使用既不过少也不过多的足够量的崩解剂(不会不利地改变活性成分的释放)来形成本公开内容的固体口服剂型。所使用的崩解剂的量基于配制类型和给药方式而变化，并且是本领域普通技术人员容易辨别。典型的药物组合物包含约0.5至约15重量百分比的崩解剂，优选约1至约5重量百分比的崩解剂。
可以用于形成本发明公开内容的药物组合物和剂型的崩解剂包括但不限于：琼脂，海藻酸，碳酸钙，微晶纤维素，交联羧甲纤维素钠，交聚维酮，波拉克林钾，淀粉羟乙酸钠，马铃薯或木薯淀粉，其它淀粉，预胶化淀粉，粘土，其它海藻胶，其它纤维素，树胶，以及它们的混合物。
可以用于形成本发明公开内容的药物组合物和剂型的润滑剂包括但不限于，硬脂酸钙，硬脂酸镁，矿物油，轻质矿物油，甘油，山梨糖醇，甘露醇，聚乙二醇，其它二醇类，硬脂酸，月桂基硫酸钠，滑石，氢化植物油(例如，花生油、棉籽油、葵花籽油、芝麻油、橄榄油、玉米油和大豆油)，硬脂酸锌，油酸乙酯，月桂酸乙酯，琼脂，及其混合物。其它的润滑剂包括，例如，syloid硅胶(
Figure PCTCN2015100061-appb-000002
200，由W.R.Grace Co.of Baltimore,Md.制造)，合成氧化硅的凝结气雾剂(由Degussa Co.of Plano,Tex.销售)，
Figure PCTCN2015100061-appb-000003
(由Cabot Co.of Boston,Mass.销售的烧结二氧化硅产品)，及其混合物。如果使用，润滑剂通常以其所掺入的药物组合物或剂型的少于约1重量百分比的量使用。
本发明公开内容还包括不含乳糖的药物组合物和剂型，其中这样的组合物优选含有很少(如果有的话)乳糖或其它单糖或二糖。如本文所用，术语“不含乳糖”表示存在的乳糖的量(如果有的话)不足以实质上增加活性成分的降解速率。
本发明公开内容的不含乳糖的组合物可包含本领域公知的且列于USP(XXI)/NF(XVI)(其通过引用并入本文)中的赋形剂。一般而言，不含乳糖的组合物包含活性组合物中的化合物的药学上可接受的盐、粘合剂/填充剂以及以药物相容和药学上可接受量的润滑剂。优选的不含乳糖的剂型包含所公开的化合物的药学上可接受的盐、微晶 纤维素、预胶化淀粉和硬脂酸镁。
本发明公开内容还包括包含所公开的化合物作为活性成分的无水药物组合物和剂型，因为水可以促进一些化合物的降解。例如，加入水(例如，5％)在制药学领域中被广泛接受为模拟长期贮存的方式以测定制剂随时间的特性如货架期或稳定性。参加，例如，Jens T.Carstensen,Drug Stability:Principles&Practice,379-80(2nd ed.,Marcel Dekker,NY,N.Y.:1995)。水和热加速一些化合物的分解。因此，水对制剂的作用可具有重要意义，因为水分和/或湿度在制剂的制造、处理、包装、储存、运输和使用过程中经常遇到。
本发明公开内容的无水药物组合物和剂型可以使用无水或含有低水分的成分和低水分或低湿度条件来制备。包含乳糖和至少一种包含伯胺或仲胺的活性成分的药物组合物和剂型优选是无水的，如果预期在制造、包装和/或储存过程中与水分和/或湿气发生实质性接触。
无水药物组合物应该被制备和储存以使得其无水性质得到保持。因此，无水组合物优选使用已知防止暴露于水的材料包装，以使得它们可以包括在合适的制药试剂盒中。合适的包装的实例包括但不限于密封的箔、塑料、具有或不具有干燥剂的单位剂量容器(例如，小瓶)、泡罩包装和条带包装。
控释和缓释剂型
所公开的活性化合物的药学上可接受的盐可通过控释或缓释装置施用。控释药物产品具有相对于其非控释对应物改善药物治疗的共同目标。理想地，在医学治疗中使用最佳设计的控释制剂的特征在于最小化所使用的药物物质以在最小的时间量中治愈或控制病状。控释制剂的优点包含：1)延长的药物活性；2)降低给药频率；3)提高患者依从性；4)使用较少的药物总量；5)减少局部或全身的副作用；6)最小化药物的积累；7)减少血液水平波动；8)改善治疗功效；9)减少药物活性的增强或损失；和10)改善的控制疾病或病状的速度。Kim,Chemg-ju,Controlled Release Dosage Form Design,2(Technomic Publishing,Lancaster,Pa.:2000)。
常规剂型通常提供从制剂快速或立即释放药物。取决于药物的药理学和药物动力学，常规剂型的使用可导致该药物在患者的血液和其它组织中的浓度的大范围波动。这些波动可影响许多参数，例如给药频率、作用起始、药效持续时间、治疗血液水平的维持、毒性、副作用等。有利地，控释制剂可用于控制药物的作用起始、作用持续 时间、治疗窗口内的血浆水平以及峰值血液水平。特别地，控释或缓释剂型或制剂可以用于确保实现药物的最大效果，同时最小化潜在的不利影响和安全问题，其可能会由于药物剂量不足(即，低于最低治疗水平)以及超过药物的毒性水平而发生。
大多数控释制剂被设计成最初释放迅速产生所需的治疗效果的量的药物(活性成分)，并且逐渐和连续释放其它量的药物以在延长的时间周期上维持该水平的治疗或预防效果。为了在体内维持药物此恒定水平，药物必须以将取代被代谢和从体内排出的药物的量的速率从剂型释放。活性成分的控释可通过各种条件来刺激，包括但不限于，pH、离子强度、渗透压、温度、酶、水、和其它生理条件或化合物。
各种已知的控释或缓释剂型、制剂和装置可调整用于与本公开内容的盐和组合物使用。实例包括，但不限于，在美国专利No.3,845,770；3,916,899；3,536,809；3,598,123；4,008,719；5674,533；5,059,595；5,591,767；5,120,548；5,073,543；5,639,476；5,354,556；5,733,566；和6,365,185B1中描述的那些；其每一个通过引用并入本文。这些剂型可用于提供一种或多种活性成分的缓慢或受控释放，其使用例如羟丙基甲基纤维素、其它聚合物基质、凝胶、可渗透的膜、渗透系统(例如
Figure PCTCN2015100061-appb-000004
(Alza Corporation,Mountain View,Calif.USA))、多层包衣、微粒、脂质体、或微球或其组合，以通过不同的比例提供所需的释放特征谱。此外，可以使用离子交换材料来制备所公开的化合物固定的、吸附的盐形式，从而实现药物的受控递送。具体的阴离子交换剂的实例包括，但不限于，
Figure PCTCN2015100061-appb-000005
A568和
Figure PCTCN2015100061-appb-000006
AP143(Rohm&Haas,Spring House,Pa.USA)。
本发明公开内容的一个实施方案包括一种单位剂型，其包含所公开的化合物的药学上可接受的盐(例如，钠盐、钾盐或锂盐)，或其多晶型物、溶剂合物、水合物、去水合物、共晶体，无水或无定形形式，以及一种或多种药学上可接受的赋形剂或稀释剂，其中所述药物组合物或剂型被配制用于受控释放。具体的剂型利用渗透药物递送系统。
一种具体的和公知的渗透药物递送系统被称为
Figure PCTCN2015100061-appb-000007
(Alza Corporation,Mountain View,Calif.USA)。这种技术可以容易地调整用于本公开内容的化合物和组合物的递送。该技术的各个方面公开于美国专利No.6,375,978 B1；6,368,626 B1；6,342,249 B1；6,333,050 B2；6,287,295 B1；6,283,953 B1；6,270,787 B1；6,245,357 B1；和6,132,420；其每一个通过引用并入本文。可以用于施用本公开内容的化合物和 组合物的
Figure PCTCN2015100061-appb-000008
的特定适应体包括但不限于，
Figure PCTCN2015100061-appb-000009
Delayed
Figure PCTCN2015100061-appb-000010
Multi-Layer
Figure PCTCN2015100061-appb-000011
和
Figure PCTCN2015100061-appb-000012
System，所有这些都是公知的。参见，例如，世界范围的网站alza.com。可用于本发明公开内容的化合物和组合物的受控口服递送的另外
Figure PCTCN2015100061-appb-000013
系统包括
Figure PCTCN2015100061-appb-000014
和
Figure PCTCN2015100061-appb-000015
参见，Delivery Times,vol.11,issue II(Alza Corporation)。
常规
Figure PCTCN2015100061-appb-000016
口服剂型通过压缩药物粉末(例如，活性组合物的化合物的盐)至硬片剂、用纤维素衍生物涂覆片剂以形成一个半渗透膜、然后在包衣上钻孔(例如用激光)来制备。Kim,Chemg-ju,Controlled Release Dosage Form Design,231-238(Technomic Publishing,Lancaster,Pa.:2000)。这样的剂型的优点是，药物的递送速率不受生理或实验条件的影响。即使是具有pH依赖性溶解度的药物也可以以恒定速率递送而不论递送介质的pH。但由于这些优点通过给药后在剂型内建立渗透压提供，故常规
Figure PCTCN2015100061-appb-000017
药物递送系统不能用于有效地递送具有低水溶性的药物。因为本公开内容的活性组合物和复合物的化合物的盐(例如，活性组合物的化合物钠盐)可以是比活性化合物本身远更溶于水的，因而它们可非常适合于基于渗透递送给患者。然而，本公开内容确实包括将活性化合物及其非盐异构体和异构体混合物掺入
Figure PCTCN2015100061-appb-000018
剂型。
本发明公开内容的活性组合物的具体的剂型包括：限定腔的壁，所述壁具有在其中形成或可形成的出口孔并且至少壁的一部分是半渗透的；位于远离出口孔的腔内并与壁的半渗透部分流体连通的可扩张层；位于邻近出口孔的腔内并与可扩张层处于直接或间接的接触关系的干燥或基本上干燥状态的药物层；和介于壁的内表面与位于腔内的药物层的至少外表面之间的流动促进层，其中所述药物层包含活性组合物的化合物的盐，或其多晶型物、溶剂合物、水合物、去水合物、共晶体、无水或无定形形式。参见美国专利No.6,368,626，其全部内容通过引用并入本文。
本发明公开内容的另一特定剂型包括：限定腔的壁，所述壁具有在其中形成或可形成的出口孔并且至少壁的一部分是半渗透的；位于远离出口孔的腔内并与壁的半渗透部分流体连通的可扩张层；位于邻近出口孔的腔内并与可扩张层处于直接或间接的接触关系的药物层；所述药物层包含吸附在多孔颗粒中的液体、活性剂制剂，所述多孔颗粒被调整用于耐受足以形成没有液体、活性剂制剂的显著渗出的压实药物层的压紧力，剂型任选在出口孔和药物层之间具有安慰剂层，其中活性剂制剂包含活性化合物的化合物的盐，或其多晶型物、溶剂合物、水合物、去水合物、共晶体、无水或无 定形形式。参见美国专利No.6,342,249，其全部内容通过引用并入本文。
肠胃外剂型
肠胃外剂型可以通过各种途径施用至患者，包括但不限于皮下、静脉内(包含弹丸注射)、肌内和动脉内途径。因为肠胃外剂型的施用通常绕开患者对污染物的天然防御，故肠胃外剂型优选是无菌的，或者能够在对患者施用之前进行灭菌。肠胃外剂型的实例包括但不限于，准备用于注射的溶液、准备溶解或悬浮在药学上可接受的注射用媒介物中的干燥产品、准备用于注射的悬浮液、以及乳剂。此外，可以制备控释肠胃外剂型用于患者的给药，包括但不限于，施用
Figure PCTCN2015100061-appb-000019
型剂型，以及剂量倾泻。
可用于提供本发明公开内容的肠胃外剂型的合适的媒介物本领域技术人员所熟知的。实例包括，但不限于：无菌水；注射用USP水；盐溶液；葡萄糖溶液；水性媒介物，例如但不限于，氯化钠注射液、林格氏注射液、右旋糖注射液、右旋糖和氯化钠注射液以及乳酸林格氏注射液；水混溶媒介物，例如但不限于，乙醇、聚乙二醇和丙二醇；和非水性媒介物，例如但不限于，玉米油、棉籽油、花生油、芝麻油、油酸乙酯、肉豆蔻酸异丙酯和苯甲酸苄酯。
可改变或修改本文公开的活性组合物的药学上可接受的盐的溶解度的化合物也可以掺入到本公开内容的肠胃外剂型中，包括常规和控释肠胃外剂型。
局部、透皮和粘膜剂型
本发明公开内容的局部剂型包括但不限于：乳膏剂，洗剂，软膏剂，凝胶，洗发剂，喷雾剂，气雾剂，溶液，乳剂，和本领域技术人员已知的其它形式。参见，例如，Remington's Pharmaceutical Sciences,18th ed.,Mack Publishing,Easton,Pa.(1990)；和Introduction to Pharmaceutical Dosage Forms,4th ed.,Lea&Febiger,Philadelphia,Pa.(1985)。对于非喷雾的局部剂型，通常使用包含与局部应用相容的载体或一种或多种赋形剂并且具有优选大于水的动力学粘度的粘性至半固体或固体形式。合适的制剂包括但不限于，溶液、悬浮液、乳剂、乳膏剂、软膏剂、粉剂、搽剂、油膏剂等，如果需要的话，对它们进行灭菌或与助剂(例如，防腐剂、稳定剂、润湿剂、缓冲剂或盐)混合用于影响各种性质，例如，渗透压。其它合适的局部剂型包括可喷雾的气雾剂制剂，其中活性成分(优选与固体或液体惰性载体组合)包装在与加压挥发物(例如，气体推进剂，例如氟利昂)的混合物中或挤压瓶中。如果需要的话，增湿剂或湿润剂也可以添加至药物组合物和剂型。这样的附加成分的实例是本 领域公知的。参见，例如，Remington's Pharmaceutical Sciences,18th Ed.,Mack Publishing,Easton,Pa.(1990)。
本发明公开内容的活性组合物的透皮和粘膜剂型包括但不限于眼用溶液、贴剂、喷雾剂、气雾剂、乳膏剂、洗剂、栓剂、软膏剂、凝胶、溶液、乳剂、悬浮液、或本领域技术人员已知的其它形式。参见，例如，Remington's Pharmaceutical Sciences,18th Ed.,Mack Publishing,Easton,Pa.(1990)；和Introduction to Pharmaceutical Dosage Forms,4th Ed.,Lea&Febiger,Philadelphia,Pa.(1985)。适合用于治疗口腔内的粘膜组织的剂型可以被配制成漱口剂、口腔凝胶、或口腔贴剂。另外的透皮剂型包括“贮库型”或“基质型”贴剂，其可被施加到皮肤上并佩戴特定的时间段，以允许渗透所需量的活性成分。
可用于施用本发明公开内容的活性成分的透皮剂型和给药方法的实例包括但不限于，在如下美国专利中公开的那些：No.4,624,665；4,655,767；4,687,481；4,797,284；4,810,499；4,834,978；4,877,618；4,880,633；4,917,895；4,927,687；4,956,171；5,035,894；5,091,186；5,163,899；5,232,702；5,234,690；5,273,755；5,273,756；5,308,625；5,356,632；5,358,715；5,372,579；5,421,816；5,466；465；5,494,680；5,505,958；5,554,381；5,560,922；5,585,111；5,656,285；5,667,798；5,698,217；5,741,511；5,747,783；5,770,219；5,814,599；5,817,332；5,833,647；5,879,322；和5,906,830，其每一个通过引用整体并入本文。
可用于提供本发明公开内容包含的透皮和粘膜剂型的合适的赋形剂(例如，载体和稀释剂)和其它材料是制药领域的普通技术人员所公知的，并取决于给定药物组合物或剂型将被应用于特定组织或器官。考虑到该事实，典型的赋形剂包括但不限于：水，丙酮，乙醇，乙二醇，丙二醇，丁烷-1,3-二醇，肉豆蔻酸异丙酯，棕榈酸异丙酯，矿物油，及其混合物，以形成无毒的和药学上可接受的剂型。
取决于待治疗的具体组织，与本发明公开内容的活性组合物的化合物的药学上可接受的盐结合治疗之前或在用本发明公开内容的活性组合物的化合物的药学上可接受的盐治疗后使用另外的组分。例如，渗透增强剂可用于帮助递送活性成分至组织或穿过组织。合适的渗透增强剂包括但不限于：丙酮；各种醇，如乙醇，油醇，四氢呋喃；烷基亚砜如二甲基亚砜；二甲基乙酰胺；二甲基甲酰胺；聚乙二醇；吡咯烷酮，例如聚乙烯吡咯烷酮；聚乙烯吡咯烷酮级(聚乙烯吡咯酮，聚维酮)；尿素；和各种水溶 性或不溶性糖酯类，例如TWEEN 80(聚山梨酯80)和SPAN 60(脱水山梨醇单硬脂酸酯)。
药物组合物或剂型的pH或该药物组合物或剂型所应用于的组织的pH也可以进行调节以改善活性成分的递送。类似地，溶剂载体的极性、其离子强度或张力可进行调节以改善递送。化合物例如硬脂酸酯也可以被加入到药物组合物或剂型中，以有利地改变活性成分的亲水性或亲脂性，从而改善递送。在这点上，硬脂酸酯可以用作为制剂的脂质媒介物、作为乳化剂或表面活性剂、和作为递送增强剂或渗透增强剂。活性组合物的化合物的药学上可接受的盐的不同水合物、去水合物、共晶体、溶剂合物、多晶型物、无水或无定形形式可用于进一步调节所得的组合物的性质。
试剂盒
在一些实施方案中，本发明公开内容的药物组合物的活性成分可能不在同一时间或通过同一施用途径给患者施用。因此，本公开内容包括试剂盒，其在由医师使用时，可以简化适当量的活性成分至患者的施用。
典型的试剂盒包含活性组合物的活性化合物的药学上可接受的盐的单位剂型。具体地，活性组合物的活性化合物的药学上可接受的盐是钠盐、锂盐或钾盐，或其多晶型物、溶剂合物、水合物、去水合物、共晶体，无水或无定形形式。试剂盒可进一步包含可用于施用活性成分的装置。这样的装置的实例包括但不限于注射器、滴液袋、贴剂和吸入剂。试剂盒可以包含使用试剂盒的说明书。
本发明公开内容的试剂盒可进一步包含可用于施用一种或多种活性成分(例如，活性化合物)的药学上可接受的媒介物。例如，如果活性成分以必需经重建以用于肠胃外给药的固体形式提供，则试剂盒可包含合适的媒介物的密封容器，在其中可以溶解活性成分以形成适合用于肠胃外给药的不含颗粒的无菌溶液。
药学上可接受的媒介物的实例包括但不限于：注射用USP水；水性媒介物，例如，但不限于，氯化钠注射液、林格氏注射液、右旋糖注射液、右旋糖和氯化钠注射液、和乳酸林格氏注射液；水混溶媒介物例如但不限于，乙醇、聚乙二醇和丙二醇；和非水性媒介物，例如但不限于，玉米油、棉籽油、花生油、芝麻油、油酸乙酯、肉豆蔻酸异丙酯和苯甲酸苄酯。
在另一个实施方案中，用于筛选候选药物的化合物的试剂盒包含多个果蝇胚胎。每个果蝇胚胎包含与hs-hid转基因连锁的C9orf72中的GGGGCC30重复片段(其中热休克 对于GGGGCC30转基因的果蝇胚胎是致死的)和一套用于筛选化合物文库的说明书。
实施例
通过化学筛选对抑制由肌萎缩侧索硬化(ALS)排列rGGGGCC重复片段引起的毒性的化合物的鉴定
ALS和FTD的果蝇疾病模型提供了实验证据表明，C9orf72中的rGGGGCC重复片段单独地足以以重复剂量方式引起神经变性。参见Xu等人,PNAS,2013,110:7778-7783。具有C9orf72中的rGGGGCC30重复片段的表达的果蝇没有达到成年期。致死主要在幼虫形成之前的胚胎发育过程中发生。利用这种致死性，设计了高通量策略来与ALS和FTD相关的小分子筛选化学文库。UAS-GGGGCC30-EGFP转基因用携带与热休克诱导性凋亡hid(hs-hid)转基因重组的卷翅(Cyo)标记物的染色体平衡(w1118,UAS-GGGGCC30-EGFP/TM2Cyo:hs-hid)。然后，使用果蝇GAL4/UAS系统测试成年子代的活力。UAS-GGGGCC30-EGFP/TM2Cyo:hs-hid的杂交用纯合的泛神经元elav-Gal4驱动子来进行。存在杂合非Cyo成年子代(elav-GAL4/+；UAS-GGGGCC30-EGFP/+)的缺失，这证实了神经元中rGGGGCC30重复片段的表达导致致死性。唯一活着的子代将携带elav-GAL4/+；+/TM2Cyo:hs-hid。此外，在热休克后，携带elav-GAL4/+；+/TM2Cyo:hs-hid的子代也死亡，因为hid的表达导致可存活的胚胎阶段的细胞死亡和致死。
由于致死的挽救是容易打分的表型，故能够使用该测定进行化学筛选以鉴定可以改善/恢复表达rGGGGCC重复片段的果蝇的活力的小分子。如示意图所描述的(图1)，在补充有来自2000种美国食品和药物管理局(FDA)批准的药物和天然产物(The Spectrum Collection,MicroSource Discovery Systems,Inc.)的40μm个体独特的化合物的食物中喂养elav-GAL4和UAS-GGGG30-EGFP/TM2Cyo:hs-hid之间的杂交体对照果蝇。随后在第2天，对杂交体进行热休克以清除(elav-GAL4/+；+/TM2Cyo:hs-hid)子代，随后将小瓶保持在25℃进行10-15天，以针对任何活的果蝇(elav-GAL4/+；UAS-GGGGCC30-EGFP/+)(其否则是致死的)进行打分。
在初步筛选的2000种化合物中，有24种化合物被发现导致蛹壳形成或成年非Cyo子代(elav-GAL4/+；UAS-GGGGCC30-EGFP/+)的出现。由于蛹壳形成还代表仅由于热休克导致的死亡子代(elav-GAL4/+；+/TM2Cyo:hs-hid)，基于从杂交体中恢复的果蝇或蛹的百分比选择前50％(24种化合物中的12种)用于使用运动测定的进一步验证。
验证可以改善由C9orf72中的rGGGGCC重复片段引起的运动缺陷的化合物
为了进一步验证这些化合物，开发了二次行为测定法。在该测定中，将UAS-GGGGCC30-EGFP转基因果蝇与运动神经元特异性驱动子Ok371-GAL4进行杂交，所述驱动子可以驱动rGGGGC重复片段在运动神经元中的表达，从而使我们能够就运动缺陷分析成年果蝇。rGGGGCC重复片段的表达导致运动障碍(图2)。如图2A所示的，在补充有各种化合物的食物中建立UAS-GGGGCC30-EGFP/TM2Cyo与Ok371-GAL4之间的杂交。收集子代果蝇(UAS-GGGGCC30-EGFP/Ok371-GAL4)以监测运动。为了持续监测果蝇的运动，建立了果蝇活动监测(DAM)系统以用于此目的。参见Pfeiffenberger等人，Locomotor activity level monitoring using the Drosophila Activity Monitoring(DAM)System.Cold Spring Harb.Protoc.2010。
对于每种化合物，每次同时监测每组中用包含该化合物的果蝇食物喂养的30只果蝇的运动。收集数据，并分析运动活性(图2)。每种药物处理重复至少三次。结果显示，OEM-34、OEM-62和OEM-77可显著改善由rGGGGCC重复片段诱导的运动障碍(图3和4)。

Claims (5)

  1. 抑制由C9orf72的第一内含子中的rGGGGCC重复片段引起的毒性的化合物在制备用于治疗肌萎缩侧索硬化(ALS)和额颞叶痴呆(FTD)的药物中的用途。
  2. 如权利要求1所述的用途，其特征在于，所述药物的受试者被诊断患有肌萎缩侧索硬化。
  3. 2.如权利要求1所述的用途，其特征在于，所述药物的受试者被诊断患有额颞叶痴呆。
  4. 如权利要求1所述的用途，其特征在于，所述化合物选自：OEM-34、OEM-62、OEM-77、OK-EM 026、OK-EM 027、OK-EM 028、OK-EM 029、OK-EM 033和/或OK-EM 036。
  5. 如权利要求1所述的用途，其特征在于，所述化合物选自：OEM-34、OEM-62、OEM-77、OK-EM 026、OK-EM 027、OK-EM 028、OK-EM 029、OK-EM 033和/或OK-EM 036的盐、酯或前药。
PCT/CN2015/100061 2015-12-29 2015-12-31 用于治疗肌萎缩侧索硬化和额颞叶痴呆的化合物与应用 Ceased WO2017113268A1 (zh)

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CN104684558A (zh) * 2012-07-31 2015-06-03 耶达研究及发展有限公司 诊断和治疗运动神经元疾病的方法
CN105637090A (zh) * 2013-10-11 2016-06-01 Ionis制药公司 用于调节c9orf72表达的组合物

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CN105637090A (zh) * 2013-10-11 2016-06-01 Ionis制药公司 用于调节c9orf72表达的组合物

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US20240215578A1 (en) * 2022-12-13 2024-07-04 Hubei Biopesticide Engineering Research Centre Polysubstituted nitrogen-containing heterocyclic derivatives, preparation method and use thereof

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