[go: up one dir, main page]

WO2018170796A1 - Trim72 en tant que cible thérapeutique potentielle pour la sla par l'ubiquitination de la protéine fus mutante - Google Patents

Trim72 en tant que cible thérapeutique potentielle pour la sla par l'ubiquitination de la protéine fus mutante Download PDF

Info

Publication number
WO2018170796A1
WO2018170796A1 PCT/CN2017/077712 CN2017077712W WO2018170796A1 WO 2018170796 A1 WO2018170796 A1 WO 2018170796A1 CN 2017077712 W CN2017077712 W CN 2017077712W WO 2018170796 A1 WO2018170796 A1 WO 2018170796A1
Authority
WO
WIPO (PCT)
Prior art keywords
fus
scavenger
pharmaceutical composition
mutant
als
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2017/077712
Other languages
English (en)
Inventor
Yichang Jia
Xue ZHANG
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Tsinghua University
Original Assignee
Tsinghua University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Tsinghua University filed Critical Tsinghua University
Priority to PCT/CN2017/077712 priority Critical patent/WO2018170796A1/fr
Priority to CN201780091092.0A priority patent/CN110753557B/zh
Priority to CN202410175594.3A priority patent/CN117982625A/zh
Publication of WO2018170796A1 publication Critical patent/WO2018170796A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered

Definitions

  • Embodiments of the present disclosure generally relate to biological medicine, more particularly, to a pharmaceutical composition, use of the scavenger used for clearing away mutant FUS in the manufacture of a medicament and a method for treating or preventing or alleviating ALS.
  • ALS Amyotrophic lateral sclerosis
  • MND motor neuron disease
  • ALS is a specific disease that causes the death of neurons which control voluntary muscles.
  • ALS is characterized by stiff muscles, muscle twitching, and gradually worsening weakness due to muscles decreasing in size. This results in difficulty in speaking, swallowing, and eventually breathing. The cause is not known in 90%to 95%of cases. About 5–10%of cases are inherited from a person's parents. About half of these genetic cases are due to four specific genes, SOD1, TDP-43, FUS, and C9orf72.
  • ALS a chronic respiratory disease
  • Physical therapy plays a large role in rehabilitation for individuals with ALS.
  • physical and occupational therapists can set goals and promote benefits for individuals with ALS by delaying loss of strength, maintaining endurance, limiting pain, improving speech and swallowing, preventing complications, and promoting functional independence.
  • Occupational therapy and special equipment such as assistive technology can also enhance people's independence and safety throughout the course of ALS.
  • Gentle, low-impact aerobic exercise such as performing activities of daily living, walking, swimming, and stationary bicycling can strengthen unaffected muscles, improve cardiovascular health, and help people fight fatigue and depression.
  • Range of motion and stretching exercises can help prevent painful spasticity and shortening (contracture) of muscles.
  • Physical and occupational therapists can recommend exercises that provide these benefits without overworking muscles.
  • Occupational therapists can provide or recommend equipment and adaptations to enable ALS people to retain as much safety and independence in activities of daily living as possible.
  • RNA metabolism abnormalities including gain-of-function of RBPs, loss-of-function of RNA helicases, and misprocessing of pre-mRNA splicing, lead to neurodegenerative diseases.
  • mutations in genes encoding two structurally similar RBPs, TDP-43 and FUS have been associated with ALS, the neurodegenerative disorders sharing genetic and pathological overlaps. More strikingly, the ubiquitin-positive and mislocalized TDP-43 and FUS were found in a large proportion of ALS, even though many of them do not carry these two RBP mutations, underscoring the critical roles of RBP dysfunction in the pathogenesis.
  • the disease mechanisms underlying neurodegeneration caused by dysfunction of these RBPs are still largely unknown.
  • TDP-43 and FUS among many other RBPs, which contain low complexity domain (LCD) also known as intrinsically disordered region (IDR)
  • LCD low complexity domain
  • IDR intrinsically disordered region
  • SG stress granule
  • mutations in those RBP genes have also been linked to neurodegenerative diseases, including ALS and FTD.
  • ALS and FTD patient specimen the mislocalized TDP-43 co-localized with SG markers, and in cultured cells, overexpressed mutant TDP-43 and FUS were found in the SG induced by stresses.
  • VCP a gene encodes protein involved in autophagic clearance of SGs
  • ALS a gene encodes protein involved in autophagic clearance of SGs
  • Embodiments of the present disclosure seek to solve at least one of the problems existing in the related art to at least some extent, or to provide a consumer with a useful commercial choice.
  • Embodiments of a first broad aspect of the present disclosure provide a pharmaceutical composition.
  • the pharmaceutical composition comprising scavenger, wherein the scavenger is used for clearing away mutant FUS.
  • the inventors of the patent surprisingly discover that scavenger used for clearing away mutant FUS can increase the motor ability, the number of motor neurons and the motor learning ability in ALS mice model.
  • the pharmaceutical composition disclosed here can treat or prevent or alleviate ALS effectively.
  • the pharmaceutical composition may further include at least one of the following additional technical features.
  • the scavenger is used for clearing away mutant FUS by ubiquitination and proteasome-mediated degradation.
  • the pharmaceutical composition disclosed here can treat or prevent or alleviate ALS more effectively and safely.
  • the scavenger is Trim72 or the nucleic acid encoding Trim72.
  • the inventors of the patent surprisingly discover that Trim72 can target the mutant FUS for ubiquitination and proteasome-mediated degradation.
  • the pharmaceutical composition comprising Trim72 or the nucleic acid encoding Trim72 can treat or prevent or alleviate ALS more effectively and safely.
  • mutant FUS is human ALS mutant FUS.
  • the pharmaceutical composition disclosed here is more suitable for human.
  • the mutant FUS has mutation in FUS nuclear localization signal.
  • the amino acid site of the nuclear localization signal of FUS is 513-526 (NP_004951.1) .
  • the mutant FUS is inclined to localizing in cytosol.
  • the mutant FUS disclosed herein has close relationship with ALS and is key pathogenic mutant.
  • Pharmaceutical composition comprising the scavenger used for clearing away the mutant FUS described above will be used for treating or preventing or alleviating ALS more effectively.
  • the pharmaceutical composition disclosed here is used for treating or preventing or alleviating ALS.
  • the inventors of the patent surprisingly discover that scavenger used for clearing away mutant FUS can increase the motor ability, the number of motor neurons and the motor learning ability in ALS mice model effectively.
  • the pharmaceutical composition disclosed here can treat or prevent or alleviate ALS effectively.
  • the scavenger is in at least one of the following forms: (a) protein or functional fragment; (b) Nucleic acid molecule encoding (a) ; (c) construct having (b) .
  • the scavenger can be used for clearing away mutant FUS by ubiquitination and proteasome-mediated degradation, such as Trim72. Trim72 or the functional fragment of Trim72 used for ubiquitination and proteasome-mediated degradation of mutant FUS or the Nucleic acid molecule encoding Trim72 or the functional fragment or the construct having the Nucleic acid molecule can be used as scavenger for clearing away mutant FUS effectively.
  • nucleic acid molecule comprising at least one of DNA and RNA.
  • DNA or RNA has double or single train encoding protein, such as Trim72 or the functional fragment thereof can be used as mutant FUS scavenger effectively.
  • the construct comprising at least one of plasmid and virus.
  • the construct can carry the nucleic acid molecule encoding protein, such as Trim72 or the functional fragment thereof and the protein, such as Trim72 or the functional fragment thereof can express in suitable environment.
  • the construct can be at least one of plasmid and virus.
  • the pharmaceutical composition further comprising pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier can change the way the drug enters the body and the distribution in the body, control the release rate of the drug and deliver the drug to the target organ. And then, the pharmaceutical composition disclosed here can treat ALS more effectively.
  • the acceptable carrier is drugs, toxins, cytokines, radioactive elements, carrier proteins, enzymes, lectins, fluorescent quantum dots, or high absorption coefficient of chromophore.
  • the acceptable carrier disclosed above can change the way the drug enters the body and the distribution in the body, control the release rate of the drug and deliver the drug to the target organ more effectively.
  • Embodiments of a second broad aspect of the present disclosure provide use of the scavenger used for clearing away mutant FUS in the manufacture of a medicament for treating or preventing or alleviating ALS.
  • the inventors of the patent surprisingly discover that scavenger used for clearing away mutant FUS can increase the motor ability, the number of motor neurons and the motor learning ability in ALS mice model.
  • the scavenger is used in the manufacture of a medicament and the medicament can treat or prevent or alleviate ALS effectively.
  • the use may further include at least one of the following additional technical features.
  • the scavenger is used for clearing away mutant FUS by ubiquitination and proteasome-mediated degradation.
  • the medicament disclosed herein can treat or prevent or alleviate ALS more effectively and safely.
  • the scavenger is Trim72 or the nucleic acid encoding Trim72.
  • the inventors of the patent surprisingly discover that Trim72 can target the mutant FUS for ubiquitination and proteasome-mediated degradation.
  • the medicament prepared with Trim72 or the nucleic acid encoding Trim72 can treat or prevent or alleviate ALS more effectively and safely.
  • mutant FUS is human ALS mutant FUS.
  • the medicament disclosed here is more suitable for human.
  • the mutant FUS has mutation in FUS nuclear localization signal.
  • the amino acid site of the nuclear localization signal of FUS is 513-526 (NP_004951.1) .
  • the mutant FUS is inclined to localizing in cytosol.
  • the mutant FUS disclosed herein has close relationship with ALS and is key pathogenic mutant. medicament prepared with the scavenger used for clearing away the mutant FUS described above will be used for treating or preventing or alleviating ALS more effectively.
  • the scavenger is in at least one of the following forms: (a) protein or functional fragment; (b) Nucleic acid molecule encoding (a) ; (c) construct having (b) .
  • the scavenger can be used for clearing away mutant FUS by ubiquitination and proteasome-mediated degradation, such as Trim72. Trim72 or the functional fragment of Trim72 used for ubiquitination and proteasome-mediated degradation of mutant FUS or the Nucleic acid molecule encoding Trim72 or the functional fragment or the construct having the Nucleic acid molecule can be used as scavenger for clearing away mutant FUS effectively.
  • nucleic acid molecule comprising at least one of DNA and RNA.
  • DNA or RNA has double or single train encoding protein, such as Trim72 or the functional fragment thereof can be used as mutant FUS scavenger effectively.
  • the construct comprising at least one of plasmid and virus.
  • the construct can carry the nucleic acid molecule encoding protein, such as Trim72 or the functional fragment thereof and the protein, such as Trim72 or the functional fragment thereof can express in suitable environment.
  • the construct can be at least one of plasmid and virus.
  • the medicament further comprising pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier can change the way the drug enters the body and the distribution in the body, control the release rate of the drug and deliver the drug to the target organ. And then, the medicament disclosed here can treat ALS more effectively.
  • the acceptable carrier is drugs, toxins, cytokines, radioactive elements, carrier proteins, enzymes, lectins, fluorescent quantum dots, or high absorption coefficient of chromophore.
  • the acceptable carrier disclosed above can change the way the drug enters the body and the distribution in the body, control the release rate of the drug and deliver the drug to the target organ more effectively.
  • Embodiments of a third broad aspect of the present disclosure provide a method for treating or preventing or alleviating ALS, wherein the method comprising administration of the scavenger used for clearing away FUS protein mutants to a patient in need.
  • the inventors of the patent surprisingly discover that scavenger used for clearing away mutant FUS can increase the motor ability, the number of motor neurons and the motor learning ability.
  • the method may further include at least one of the following additional technical features.
  • the scavenger is used for clearing away mutant FUS by ubiquitination and proteasome-mediated degradation.
  • the method disclosed herein is more effective and safety.
  • the scavenger is Trim72 or the nucleic acid encoding Trim72.
  • the inventors of the patent surprisingly discover that Trim72 can target the mutant FUS for ubiquitination and proteasome-mediated degradation. The method disclosed herein is more effectively and safely.
  • mutant FUS is human ALS mutant FUS.
  • the method disclosed herein is more suitable for human.
  • the mutant FUS has mutation in FUS nuclear localization signal.
  • the amino acid site of the nuclear localization signal of FUS is 513-526 (NP_004951.1) .
  • the mutant FUS is inclined to localizing in cytosol.
  • the mutant FUS disclosed herein has close relationship with ALS and is key pathogenic mutant. Administration of the scavenger used for clearing away FUS protein mutants described above to a patient in need will be more effective in treating or preventing or alleviating ALS.
  • the scavenger is in at least one of the following forms: (a) protein or functional fragment; (b) Nucleic acid molecule encoding (a) ; (c) construct having (b) .
  • the scavenger can be used for clearing away mutant FUS by ubiquitination and proteasome-mediated degradation, such as Trim72. Trim72 or the functional fragment of Trim72 used for ubiquitination and proteasome-mediated degradation of mutant FUS or the Nucleic acid molecule encoding Trim72 or the functional fragment or the construct having the Nucleic acid molecule can be used as scavenger for clearing away mutant FUS effectively.
  • nucleic acid molecule comprising at least one of DNA and RNA.
  • DNA or RNA has double or single train encoding protein, such as Trim72 or the functional fragment thereof can be used as mutant FUS scavenger effectively.
  • the construct comprising at least one of plasmid and virus.
  • the construct can carry the nucleic acid molecule encoding protein, such as Trim72 or the functional fragment thereof and the protein, such as Trim72 or the functional fragment thereof can express in suitable environment.
  • the construct can be at least one of plasmid and virus.
  • the scavenger is supplied in the form of a pharmaceutical composition
  • the pharmaceutical composition further comprising pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier can change the way the drug enters the body and the distribution in the body, control the release rate of the drug and deliver the drug to the target organ. And then, the pharmaceutical composition disclosed here can treat ALS more effectively.
  • the acceptable carrier is drugs, toxins, cytokines, radioactive elements, carrier proteins, enzymes, lectins, fluorescent quantum dots, or high absorption coefficient of chromophore.
  • the acceptable carrier disclosed above can change the way the drug enters the body and the distribution in the body, control the release rate of the drug and deliver the drug to the target organ more effectively.
  • Fig. 1 shows generation of mFUS-R513C KI mutant mouse for ALS by CRISPR/Cas9
  • A shows alignment of last 12 amino acids of FUS in different mammal species.
  • the amino acids are highly conserved from rodent to human.
  • the human FUS sequences are used as a reference for amino acid positioning.
  • the R521 is labeled with asterisk;
  • the mouse FUS R513 corresponds to human R521.
  • Two-nucleotide mutations labeled in red were introduced into mouse FUS locus 2bp upstream of PAM site (3 capital letters in dark black) .
  • the underlined genomic DNA sequences correspond to gRNA sequences.
  • a novel PstI cutting site was built into the locus for future genotyping;
  • D shows gel electrophoresis.
  • the PCR products including the insertion were treated with PstI. +/+, wild type C57BL/6J mouse, C/+ and C/C, hetero-and homozygous for mFUS-R513C KI mice ;
  • E shows the expression of FUS in mouse various tissues.
  • the tissues from wild type (+/+) and mFUS-R513C KI mutant (C/C) mice at 8 months of age were blotted with a homemade FUS antibody.
  • the GAPDH was used as loading control. Hippo. , hippocampus; S. C. spinal cord.
  • Fig. 2 shows the aged mFUS-R513C mutant KI mice showed motor decline and the reduced number of motor nerve fibers
  • Open field test the standing time (seconds in 10-minute interval) on the hind legs in aged (6.5 months) mice was calculated. The value are presented as mean ⁇ SEM. *p ⁇ 0.05, **p ⁇ 0.01 (one-way ANOVA or t-test, SPSS) . NS, no statistical significance;
  • C and D show rotarod performance (4-day interval, Med Associates Inc. , USA) was carried out in wild type (+/+) , the hetero- (C/+) and/or homozygous (C/C) KI mutant mice at 4 (C) and 6.5 (D) months of age.
  • the stay time on the rotarod was significantly decreased in the aged KI mutant (C/+ and C/C, 6.5 months) mice, but not in the younger (4 months) mutant animals.
  • 4 months, n 8 (+/+ and C/+, respectively) , male.
  • the value are presented as mean ⁇ SEM. *p ⁇ 0.05, **p ⁇ 0.01 (one-way ANOVA or t-test, SPSS) . NS, no statistical significance;
  • E shows toluidine blue-stained cross sections of femoral nerve motor branches from wild type (+/+) and KI mutant (C/C) animals at 8 months of age.
  • Axon degeneration (arrow) was shown in high magnification image.
  • Scale bar 50 ⁇ m in low and 100 ⁇ m in high magnification images.
  • F shows the big size (diameter ⁇ 5 ⁇ m) , but not the small size ( ⁇ 5 ⁇ m) , nerve fiber numbers of femoral motor branches were significantly reduced in the KI mutant (C/C, 8 months) animals, compared to their wild type (+/+) littermate controls.
  • A, B, C and D the value are presented as mean ⁇ SEM. *p ⁇ 0.05, **p ⁇ 0.01 (one-way ANOVA, SPSS) . NS, no statistical significance; and
  • Fig. 3 shows Trim72, encoding an E3 ligase, was upregulated in the mFUS-R513C KI spinal cords and the E3 ligase targeted mutant FUS for degradation,
  • A shows the differentially expressed genes in the spinal cords at 1.5 and 7 month of age between the wild type and KI mutant (C/C) genotypes were identified by RNA-seq.
  • Trim72 was the only differentially expressed gene at both time points;
  • D shows the HEK293 cells were infected with Myc-BirA*-Trim72 expression lentivirus.
  • the expression of Myc-BirA*-Trim72 was induced, and the biotinylation of hFUS-R521C was detected by streptavidin M-280 beads.
  • the interaction was largely attenuated when the MG132 was removed from the culture medium, suggesting that Trim72 ubiquitinate the ALS-associated hFUS-R521C and result in its degradation;
  • E and F show compared to hFUS-R521C, wild type hFUS and hFUS truncation ( ⁇ 513-526) had much less interactions with Trim72.
  • the interactions were quantitated by relative Flag intensity (the band density ratio between the biotinylated and total input) from at least three independent experiments. The value are presented as mean ⁇ SEM. **p ⁇ 0.01 (t-test, SPSS) .
  • the present invention provides a pharmaceutical composition comprising scavenger, wherein the scavenger is used for clearing away mutant FUS.
  • the pharmaceutical composition can further comprise pharmaceutically acceptable excipient, carrier, adjuvant, solvent and a combination thereof.
  • the present invention provides a method of treating, preventing or ameliorating a disease or disorder, comprising administrating a safe and effective amount of a combination of drugs containing the scavenger and one or more therapeutic active agents.
  • the combination of drugs comprises one or more additional drugs for treatment ALS.
  • Other drugs for treatment of ALS are not limited to: Riluzole or antisense oligoes against mutant FUS RNA.
  • the scavenger is in at least one of the following forms: (a) protein or functional fragment; (b) Nucleic acid molecule encoding (a) ; (c) construct having (b) .
  • AAV-mediated gene therapy is also included herein. Recently, AAV-mediated gene therapy has been used for delivering the interested genes for therapy.
  • AAV-mediated gene therapy becomes promising for delivering the scavenger gene into the patients: 1) the AAV apparent lack of pathogenicity and also present very low immunogenicity; 2) not like retroviruses, random integration of AAV DNA into the host genome occurs at very low frequency; 3) for non-dividing cell, like neuron, AAV-based gene therapy vectors form episomal concatemers in the host cell nucleus, and these concatemers remain intact for the life of the host cell; 4) one limitation for AAV delivery is the cloning capacity, which is limited to about 4.8kb for the virial vector. However, the human Trim72 recombinant protein is about 477 a. a.
  • the DNA size for expressing the scavenger is suitable for AAV-mediated gene delivery. Therefore, due to these advantages, AAV-mediated gene therapy will be feasible means to delivery target human Trim72 into the patients.
  • the single stranded (ss) and self-complementary (sc) AAV9 were used to deliver DNA encoding the SMN protein for SMA patients. Therefore, the similar means will be used for the scavenger.
  • the amount of the scavenger in the pharmaceutical composition disclosed herein refers to an amount which can be effectively clearing away mutant FUS.
  • the dosage of active ingredient in the compositions of this invention may be varied, however, it is necessary that the amount of the active ingredient be such that a suitable dosage form is obtained.
  • the active ingredient may be administered to patients (animals or human) in need of such treatment in dosage that will provide optimal pharmaceutical efficacy.
  • the selected dosage upon the desired therapeutic effect, on the route of administration, and on the duration of the treatment.
  • the dosage will vary from patient to patient depending upon the nature and severity of disease, the patient's weight, special diet then being followed by a patient, concurrent medication, and other factors which those skilled in the art will recognize.
  • the dosage range will generally be about 0.5 mg to 1.0 g per patient per day which may be administered in single or multiple doses. In one embodiment, the dosage range will be about 0.5 mg to 500 mg per patient per day; in anther embodiment about 0.5 mg to 200 mg per patient per day; and in yet another embodiment about 5 mg to 50 mg per patient per day.
  • compositions of the invention may be prepared and packaged in bulk form wherein a safe and effective amount of a compound of Formula (I) disclosed herein can be extracted and then given to the patient, such as with powders or syrups. Generally, dosage levels of between 0.0001 to 10 mg/kg of body weight daily are administered to the patient to obtain effective clearing away mutant FUS.
  • the pharmaceutical compositions of the invention may be prepared and packaged in unit dosage form wherein each physically discrete unit contains a safe and effective amount of the scavenger disclosed herein. When prepared in unit dosage form, the pharmaceutical compositions of the invention commonly contain from about 0.5 mg to 1 g, or 1 mg to 700 mg, or 5 mg to 100 mg, of the compound.
  • the weight ratio of the compound of the present invention to the second active ingredient may be varied and depend upon the effective dose of each ingredient. Generally, an effective dose of each will be used. Thus, for example, when a compound of the present invention is combined with another agent, the weight ratio of the compound of the present invention to the other agent will generally range from about 1000: 1 to about 1: 1000, such as about 200: 1 to 1: 200. Combinations of a compound of the present invention and other active ingredients will generally also be within the aforementioned range, but in each case, an effective dose of each active ingredient should be used.
  • “Pharmaceutically acceptable excipient” as used herein means a pharmaceutically acceptable material, composition or vehicle involved in giving form or consistency to the pharmaceutical composition. Each excipient must be compatible with the other ingredients of the pharmaceutical composition when commingled, such that interactions which would substantially reduce the efficacy of the compound of the invention when administered to a patient and would result in pharmaceutically unacceptable compositions are avoided. In addition, each excipient must of course be of sufficiently high purity to render it pharmaceutically acceptable.
  • Suitable pharmaceutically acceptable excipients will vary depending upon the particular dosage form chosen.
  • suitable pharmaceutically acceptable excipients may be chosen for a particular function that they may serve in the composition.
  • certain pharmaceutically acceptable excipients may be chosen for their ability to facilitate the production of uniform dosage forms.
  • Certain pharmaceutically acceptable excipients may be chosen for their ability to facilitate the production of stable dosage forms.
  • Certain pharmaceutically acceptable excipients may be chosen for their ability to facilitate the carrying or transporting the compound of the present invention once administered to the patient from one organ, or portion of the body, to another organ, or portion of the body.
  • Certain pharmaceutically acceptable excipients may be chosen for their ability to enhance patient compliance.
  • Suitable pharmaceutically acceptable excipients include the following types of excipients: diluents, fillers, binders, disintegrants, lubricants, glidants, granulating agents, coating agents, wetting agents, solvents, co-solvents, suspending agents, emulsifiers, sweetners, flavoring agents, flavor masking agents, coloring agents, anticaking agents, humectants, chelating agents, plasticizers, viscosity increasing agents, antioxidants, preservatives, stabilizers, surfactants, and buffering agents.
  • excipients may serve more than one function and may serve alternative functions depending on how much of the excipient is present in the formulation and what other ingredients are present in the formulation.
  • Skilled artisans possess the knowledge and skill in the art to enable them to select suitable pharmaceutically acceptable excipients in appropriate amounts for use in the invention.
  • resources that are available to the skilled artisan which describe pharmaceutically acceptable excipients and may be useful in selecting suitable pharmaceutically acceptable excipients. Examples include Remington's Pharmaceutical Sciences (Mack Publishing Company) , The Handbook of Pharmaceutical Additives (Gower Publishing Limited) , and The Handbook of Pharmaceutical Excipients (the American Pharmaceutical Association and the Pharmaceutical Press) .
  • compositions of the invention are prepared using techniques and methods known to those skilled in the art. Some of the methods commonly used in the art are described in Remington's Pharmaceutical Sciences (Mack Publishing Company) .
  • Another aspect of the present invention is related to a method for preparing a pharmaceutical composition.
  • the pharmaceutical composition contains the compound disclosed herein and pharmaceutically acceptable excipient, carrier, adjuvant, vehicle or a combination thereof, the method comprises mixing various ingredients.
  • the pharmaceutical composition containing the compound disclosed herein can be prepared at for example environment temperature and under barometric pressure.
  • dosage forms include those adapted for (1) oral administration such as tablets, capsules, caplets, pills, troches, powders, syrups, elixers, suspensions, solutions, emulsions, sachets, and cachets; (2) parenteral administration such as sterile solutions, suspensions, and powders for reconstitution; (3) transdermal administration such as transdermal patches; (4) rectal administration such as suppositories; (5) inhalation such as aerosols, solutions, and dry powders; and (6) topical administration such as creams, ointments, lotions, solutions, pastes, sprays, foams, and gels.
  • oral administration such as tablets, capsules, caplets, pills, troches, powders, syrups, elixers, suspensions, solutions, emulsions, sachets, and cachets
  • parenteral administration such as sterile solutions, suspensions, and powders for reconstitution
  • transdermal administration such as transdermal patches
  • rectal administration such as sup
  • the compounds disclosed herein can be prepared to oral. In the other embodiment, the compounds disclosed herein can be prepared to inhalation. In the still other embodiment, the compounds disclosed herein can be prepared to nasal administration. In the yet other embodiment, the compounds disclosed herein can be prepared to transdermal administration. In the still yet other embodiments, the compounds disclosed herein can be prepared to topical administration.
  • compositions provided herein may be provided as compressed tablets, tablet triturates, chewable lozenges, rapidly dissolving tablets, multiple compressed tablets, or enteric-coating tablets, sugar-coated, or film-coated tablets.
  • Enteric-coated tablets are compressed tablets coated with substances that resist the action of stomach acid but dissolve or disintegrate in the intestine, thus protecting the active ingredients from the acidic environment of the stomach.
  • Enteric-coatings include, but are not limited to, fatty acids, fats, phenylsalicylate, waxes, shellac, ammoniated shellac, and cellulose acetate phthalates.
  • Sugar-coated tablets are compressed tablets surrounded by a sugar coating, which may be beneficial in covering up objectionable tastes or odors and in protecting the tablets from oxidation.
  • Film-coated tablets are compressed tablets that are covered with a thin layer or film of a water-soluble material.
  • Film coatings include, but are not limited to, hydroxyethylcellulose, sodium carboxymethylcellulose, polyethylene glycol 4000, and cellulose acetate phthalate. Film coating imparts the same general characteristics as sugar coating.
  • Multiple compressed tablets are compressed tablets made by more than one compression cycle, including layered tablets, and press-coated or dry-coated tablets.
  • the tablet dosage forms may be prepared from the active ingredient in powdered, crystalline, or granular forms, alone or in combination with one or more carriers or excipients described herein, including binders, disintegrants, controlled-release polymers, lubricants, diluents, and/or colorants. Flavoring and sweetening agents are especially useful in the formation of chewable tablets and lozenges.
  • the pharmaceutical compositions provided herein may be provided as soft or hard capsules, which can be made from gelatin, methylcellulose, starch, or calcium alginate.
  • the hard gelatin capsule also known as the dry-filled capsule (DFC) , consists of two sections, one slipping over the other, thus completely enclosing the active ingredient.
  • the soft elastic capsule (SEC) is a soft, globular shell, such as a gelatin shell, which is plasticized by the addition of glycerin, sorbitol, or a similar polyol.
  • the soft gelatin shells may contain a preservative to prevent the growth of microorganisms.
  • Suitable preservatives are those as described herein, including methyl-and propyl-parabens, and sorbic acid.
  • the liquid, semisolid, and solid dosage forms provided herein may be encapsulated in a capsule.
  • Suitable liquid and semisolid dosage forms include solutions and suspensions in propylene carbonate, vegetable oils, or triglycerides. Capsules containing such solutions can be prepared as described in U.S. Pat. Nos. 4,328,245; 4,409,239; and 4,410,545.
  • the capsules may also be coated as known by those of skill in the art in order to modify or sustain dissolution of the active ingredient.
  • compositions provided herein may be provided in liquid and semisolid dosage forms, including emulsions, solutions, suspensions, elixirs, and syrups.
  • An emulsion is a two-phase system, in which one liquid is dispersed in the form of small globules throughout another liquid, which can be oil-in-water or water-in-oil.
  • Emulsions may include a pharmaceutically acceptable non-aqueous liquids or solvent, emulsifying agent, and preservative.
  • Suspensions may include a pharmaceutically acceptable suspending agent and preservative.
  • Aqueous alcoholic solutions may include a pharmaceutically acceptable acetal, such as a di (lower alkyl) acetal of a lower alkyl aldehyde, e.g., acetaldehyde diethyl acetal; and a water-miscible solvent having one or more hydroxy groups, such as propylene glycol and ethanol.
  • Elixirs are clear, sweetened, and hydroalcoholic solutions.
  • Syrups are concentrated aqueous solutions of a sugar, for example, sucrose, and may also contain a preservative.
  • a solution in a polyethylene glycol may be diluted with a sufficient quantity of a pharmaceutically acceptable liquid carrier, e.g., water, to be measured conveniently for administration.
  • liquid and semisolid dosage forms include, but are not limited to, those containing the active ingredient (s) provided herein, and a dialkylated mono-or poly-alkylene glycol, including, 1, 2-dimethoxymethane, diglyme, triglyme, tetraglyme, polyethylene glycol-350-dimethyl ether, polyethylene glycol-550-dimethyl ether, polyethylene glycol-750-dimethyl ether, wherein 350, 550, and 750 refer to the approximate average molecular weight of the polyethylene glycol.
  • a dialkylated mono-or poly-alkylene glycol including, 1, 2-dimethoxymethane, diglyme, triglyme, tetraglyme, polyethylene glycol-350-dimethyl ether, polyethylene glycol-550-dimethyl ether, polyethylene glycol-750-dimethyl ether, wherein 350, 550, and 750 refer to the approximate average molecular weight of the polyethylene glycol.
  • formulations may further comprise one or more antioxidants, such as butylated hydroxytoluene (BHT) , butylated hydroxyanisole (BHA) , propyl gallate, vitamin E, hydroquinone, hydroxycoumarins, ethanolamine, lecithin, cephalin, ascorbic acid, malic acid, sorbitol, phosphoric acid, bisulfite, sodium metabisulfite, thiodipropionic acid and its esters, and dithiocarbamates.
  • antioxidants such as butylated hydroxytoluene (BHT) , butylated hydroxyanisole (BHA) , propyl gallate, vitamin E, hydroquinone, hydroxycoumarins, ethanolamine, lecithin, cephalin, ascorbic acid, malic acid, sorbitol, phosphoric acid, bisulfite, sodium metabisulfite, thiodipropionic acid and its esters, and dithiocarbamates
  • dosage unit formulations for oral administration can be microencapsulated.
  • the formulation can also be prepared to prolong or sustain the release as for example by coating or embedding particulate material in polymers, wax, or the like.
  • compositions provided herein for oral administration may be also provided in the forms of liposomes, micelles, microspheres, or nanosystems.
  • Miccellar dosage forms can be prepared as described in U.S. Pat. No. 6,350,458.
  • compositions provided herein may be provided as non-effervescent or effervescent, granules and powders, to be reconstituted into a liquid dosage form.
  • Pharmaceutically acceptable carriers and excipients used in the non-effervescent granules or powders may include diluents, sweeteners, and wetting agents.
  • Pharmaceutically acceptable carriers and excipients used in the effervescent granules or powders may include organic acids and a source of carbon dioxide.
  • Coloring and flavoring agents can be used in all of the above dosage forms.
  • the scavenger disclosed herein can also be coupled to soluble polymers as targeted medicament carriers.
  • Such polymers may encompass polyvinylpyrrolidone, pyran copolymer, polyhydroxypropylmethacrylamidophenol, polyhydroxyethylaspartamidophenol or polyethylene oxide polylysine, substituted by palmitoyl radicals.
  • the compounds may furthermore be coupled to a class of biodegradable polymers which are suitable for achieving controlled release of a medicament, for example polylactic acid, poly-epsilon-caprolactone, polyhydroxybutyric acid, polyorthoesters, polyacetals, polydihydroxypyrans, polycyanoacrylates and crosslinked or amphipathic block copolymers of hydrogels.
  • compositions provided herein may be formulated as immediate or modified release dosage forms, including delayed-, sustained, pulsed-, controlled, targeted-, and programmed-release forms.
  • compositions provided herein may be co-formulated with other active ingredients which do not impair the desired therapeutic action, or with substances that supplement the desired action.
  • compositions provided herein may be administered parenterally by injection, infusion, or implantation, for local or systemic administration.
  • Parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, intrasternal, intracranial, intramuscular, intrasynovial, and subcutaneous administration.
  • compositions provided herein may be formulated in any dosage forms that are suitable for parenteral administration, including solutions, suspensions, emulsions, micelles, liposomes, microspheres, nanosystems, and solid forms suitable for solutions or suspensions in liquid prior to injection.
  • dosage forms can be prepared according to conventional methods known to those skilled in the art of pharmaceutical science (see, Remington: The Science and Practice of Pharmacy, supra) .
  • compositions intended for parenteral administration may include one or more pharmaceutically acceptable carriers and excipients, including, but not limited to, aqueous vehicles, water-miscible vehicles, non-aqueous vehicles, antimicrobial agents or preservatives against the growth of microorganisms, stabilizers, solubility enhancers, isotonic agents, buffering agents, antioxidants, local anesthetics, suspending and dispersing agents, wetting or emulsifying agents, complexing agents, sequestering or chelating agents, cryoprotectants, lyoprotectants, thickening agents, pH adjusting agents, and inert gases.
  • aqueous vehicles water-miscible vehicles
  • non-aqueous vehicles non-aqueous vehicles
  • antimicrobial agents or preservatives against the growth of microorganisms stabilizers, solubility enhancers, isotonic agents, buffering agents, antioxidants, local anesthetics, suspending and dispersing agents, wetting or emuls
  • Suitable aqueous vehicles include, but are not limited to, water, saline, physiological saline or phosphate buffered saline (PBS) , sodium chloride injection, Ringers injection, isotonic dextrose injection, sterile water injection, dextrose and lactated Ringers injection.
  • Non-aqueous vehicles include, but are not limited to, fixed oils of vegetable origin, castor oil, corn oil, cottonseed oil, olive oil, peanut oil, peppermint oil, safflower oil, sesame oil, soybean oil, hydrogenated vegetable oils, hydrogenated soybean oil, and medium-chain triglycerides of coconut oil, and palm seed oil.
  • Water-miscible vehicles include, but are not limited to, ethanol, 1, 3-butanediol, liquid polyethylene glycol (e.g., polyethylene glycol 300 and polyethylene glycol 400) , propylene glycol, glycerin, N-methyl-2-pyrrolidone, N, N-dimethylacetamide, and dimethyl sulfoxide.
  • Suitable antimicrobial agents or preservatives include, but are not limited to, phenols, cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoates, thimerosal, benzalkonium chloride (e.g., benzethonium chloride) , methyl-and propyl-parabens, and sorbic acid.
  • Suitable isotonic agents include, but are not limited to, sodium chloride, glycerin, and dextrose.
  • Suitable buffering agents include, but are not limited to, phosphate and citrate.
  • Suitable antioxidants are those as described herein, including bisulfite and sodium metabisulfite.
  • Suitable local anesthetics include, but are not limited to, procaine hydrochloride.
  • Suitable suspending and dispersing agents are those as described herein, including sodium carboxymethylcelluose, hydroxypropyl methylcellulose, and polyvinylpyrrolidone.
  • Suitable emulsifying agents include those described herein, including polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate 80 and triethanolamine oleate.
  • Suitable sequestering or chelating agents include, but are not limited to EDTA.
  • Suitable pH adjusting agents include, but are not limited to, sodium hydroxide, hydrochloric acid, citric acid, and lactic acid.
  • Suitable complexing agents include, but are not limited to, cyclodextrins, including ⁇ -cyclodextrin, ⁇ -cyclodextrin, hydroxypropyl- ⁇ -cyclodextrin, sulfobutylether- ⁇ -cyclodextrin, and sulfobutylether 7- ⁇ -cyclodextrin ( CyDex, Lenexa, Kans. ) .
  • compositions provided herein may be formulated for single or multiple dosage administration.
  • the single dosage formulations are packaged in an ampoule, a vial, or a syringe.
  • the multiple dosage parenteral formulations must contain an antimicrobial agent at bacteriostatic or fungistatic concentrations. All parenteral formulations must be sterile, as known and practiced in the art.
  • the pharmaceutical compositions are provided as ready-to-use sterile solutions.
  • the pharmaceutical compositions are provided as sterile dry soluble products, including lyophilized powders and hypodermic tablets, to be reconstituted with a vehicle prior to use.
  • the pharmaceutical compositions are provided as ready-to-use sterile suspensions.
  • the pharmaceutical compositions are provided as sterile dry insoluble products to be reconstituted with a vehicle prior to use.
  • the pharmaceutical compositions are provided as ready-to-use sterile emulsions.
  • the pharmaceutical compositions may be formulated as a suspension, solid, semi-solid, or thixotropic liquid, for administration as an implanted depot.
  • the pharmaceutical compositions provided herein are dispersed in a solid inner matrix, which is surrounded by an outer polymeric membrane that is insoluble in body fluids but allows the active ingredient in the pharmaceutical compositions diffuse through.
  • Suitable inner matrixes include polymethylmethacrylate, polybutyl-methacrylate, plasticized or unplasticized polyvinylchloride, plasticized nylon, plasticized polyethylene terephthalate, natural rubber, polyisoprene, polyisobutylene, polybutadiene, polyethylene, ethylene-vinyl acetate copolymers, silicone rubbers, polydimethylsiloxanes, silicone carbonate copolymers, hydrophilic polymers, such as hydrogels of esters of acrylic and methacrylic acid, collagen, cross-linked polyvinyl alcohol, and cross-linked partially hydrolyzed polyvinyl acetate.
  • Suitable outer polymeric membranes include polyethylene, polypropylene, ethylene/propylene copolymers, ethylene/ethyl acrylate copolymers, ethylene/vinyl acetate copolymers, silicone rubbers, polydimethyl siloxanes, neoprene rubber, chlorinated polyethylene, polyvinylchloride, vinyl chloride copolymers with vinyl acetate, vinylidene chloride, ethylene and propylene, ionomer polyethylene terephthalate, butyl rubber epichlorohydrin rubbers, ethylene/vinyl alcohol copolymer, ethylene/vinyl acetate/vinyl alcohol terpolymer, and ethylene/vinyloxyethanol copolymer.
  • the pharmaceutical composition of the invention is prepared to a dosage form adapted for administration to a patient by inhalation, for example as a dry powder, an aerosol, a suspension, or a solution composition.
  • the invention is directed to a dosage form adapted for administration to a patient by inhalation as a dry powder.
  • the invention is directed to a dosage form adapted for administration to a patient by inhalation as a dry powder.
  • Dry powder compositions for delivery to the lung by inhalation typically comprise a compound disclosed herein or a pharmaceutically acceptable salt thereof as a finely divided powder together with one or more pharmaceutically-acceptable excipients as finely divided powders.
  • compositions particularly suited for use in dry powders are known to those skilled in the art and include lactose, starch, mannitol, and mono-, di-, and polysaccharides.
  • the finely divided powder may be prepared by, for example, micronisation and milling.
  • the size-reduced (e. g. micronised) compound can be defined by a D50 value of about 1 to about 10 microns (for example as measured using laser diffraction) .
  • Aerosols may be formed by suspending or dissolving a compound disclosed herein or a pharmaceutically acceptable salt thereof in a liquified propellant.
  • Suitable propellants include halocarbons, hydrocarbons, and other liquified gases.
  • Representative propellants include: trichlorofluoromethane (propellant 11) , dichlorofluoromethane (propellant 12) , dichlorotetrafluoroethane (propellant 114) , tetrafluoroethane (HFA-134a) , 1, 1-difluoroethane (HFA-152a) , difluoromethane (HFA-32) , pentafluoroethane (HFA-12) , heptafluoropropane (HFA-227a) , perfluoropropane, perfluorobutane, perfluoropentane, butane, isobutane, and pentane.
  • the aerosol may contain additional pharmaceutically-acceptable excipients typically used with MDIs such as surfactants, lubricants, cosolvents and other excipients to improve the physical stability of the formulation, to improve valve performance, to improve solubility, or to improve taste.
  • additional pharmaceutically-acceptable excipients typically used with MDIs such as surfactants, lubricants, cosolvents and other excipients to improve the physical stability of the formulation, to improve valve performance, to improve solubility, or to improve taste.
  • compositions adapted for transdermal administration may be presented as discrete patches intended to remain in intimate contact with the epidermis of the patient for a prolonged period of time.
  • the active ingredient may be delivered from the patch by iontophoresis as generally described in Pharmaceutical Research, 3 (6) , 318 (1986) .
  • compositions adapted for topical administration may be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils.
  • Ointments, creams and gels may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agent and/or solvents.
  • bases may thus, for example, include water and/or an oil such as liquid paraffin or a vegetable oil such as arachis oil or castor oil, or a solvent such as polyethylene glycol.
  • Thickening agents and gelling agents which may be used according to the nature of the base include soft paraffin, aluminium stearate, cetostearyl alcohol, polyethylene glycols, woolfat, beeswax, carboxypolymethylene and cellulose derivatives, and/or glyceryl monostearate and/or non-ionic emulsifying agents.
  • Lotions may be formulated with an aqueous or oily base and will in general also contain one or more emulsifying agents, stabilising agents, dispersing agents, suspending agents or thickening agents.
  • Powders for external application may be formed with the aid of any suitable powder base, for example, talc, lactose or starch.
  • Drops may be formulated with an aqueous or non-aqueous base also comprising one or more dispersing agents, solubilising agents, suspending agents or preservatives.
  • Topical preparations may be administered by one or more applications per day to the affected area; over skin areas occlusive dressings may advantageously be used. Continuous or prolonged delivery may be achieved by an adhesive reservoir system.
  • the therapies disclosed herein comprise administrating a safe and effective amount of the compound or the pharmaceutical composition containing the scavenger used for clearing away mutant FUS to patients in need.
  • Each example disclosed herein comprises the method of treating the diseases above comprising administrating a safe and effective amount of the compound of the inhibitor or the pharmaceutical composition containing the compound of the inhibitor to patients in need.
  • the pharmaceutical composition thereof may be administered by any suitable route of administration, including both systemic administration and topical administration.
  • Systemic administration includes oral administration, parenteral administration, transdermal administration and rectal administration.
  • Parenteral administration refers to routes of administration other than enteral or transdermal, and is typically by injection or infusion.
  • Parenteral administration includes intravenous, intramuscular, and subcutaneous injection or infusion.
  • Topical administration includes application to the skin as well as intraocular, otic, intravaginal, inhaled and intranasal administration.
  • the compound of the invention or the pharmaceutical composition thereof may be administered orally.
  • the compound of the invention or the pharmaceutical composition thereof may be administered by inhalation.
  • the compound of the invention or the pharmaceutical composition thereof may be administered intranasally.
  • the pharmaceutical composition thereof may be administered once or according to a dosing regimen wherein a number of doses are administered at varying intervals of time for a given period of time. For example, doses may be administered one, two, three, or four times per day. In one embodiment, a dose is administered once per day. In a further embodiment, a dose is administered twice per day. Doses may be administered until the desired therapeutic effect is achieved or indefinitely to maintain the desired therapeutic effect. Suitable dosing regimens for the compound of the invention or the pharmaceutical composition thereof depend on the pharmacokinetic properties of that compound, such as absorption, distribution, and half-life, which can be determined by the skilled artisan.
  • suitable dosing regimens including the duration such regimens are administered, for the compound of the invention or the pharmaceutical composition thereof depend on the disorder being treated, the severity of the disorder being treated, the age and physical condition of the patient being treated, the medical history of the patient to be treated, the nature of concurrent therapy, the desired therapeutic effect, and like factors within the knowledge and expertise of the skilled artisan. It will be further understood by such skilled artisans that suitable dosing regimens may require adjustment given an individual patient's response to the dosing regimen or over time as individual patient needs change.
  • the pharmaceutical composition of the present invention may be administered either simultaneously with, or before or after, one or more other therapeutic agents.
  • the compounds of the present invention may be administered separately, by the same or different route of administration, or together in the same pharmaceutical composition as the other agents.
  • the pharmaceutical composition or combination of the present invention can be in unit dosage of about 1-1000 mg of active ingredients for a subject of about 50-70 kg, preferably about 1-500 mg or about 1-250 mg or about 1-150 mg or about 0.5-100 mg or about 1-50 mg of active ingredients.
  • the therapeutically effective dosage of a compound, the pharmaceutical composition, or the combinations thereof is dependent on the species of the subject, the body weight, age and individual condition, the disorder or disease or the severity thereof being treated. A physician, clinician or veterinarian of ordinary skill can readily determine the effective amount of each of the active ingredients necessary to prevent, treat or inhibit the progress of the disorder or disease.
  • the above-cited dosage properties are demonstrable in vitro and in vivo tests using advantageously mammals, e.g., mice, rats, dogs, monkeys or isolated organs, tissues and preparations thereof.
  • the compounds of the present invention can be applied in vitro in the form of solutions, e.g., preferably aqueous solutions, and in vivo either enterally or parenterally, advantageously intravenously, e.g., as a suspension or in aqueous solution.
  • a therapeutically effective dosage of the pharmaceutical composition disclosed herein from about 0.1 mg to about 2,000 mg per day.
  • the pharmaceutical compositions should provide a dosage of from about 0.1 mg to about 2000 mg of the compound.
  • pharmaceutical dosage unit forms are prepared to provide from about 1 mg to about 2,000 mg, about 10 mg to about 1,000 mg, about 20 mg to about 500 mg, or about 25 mg to about 250 mg of the active ingredient or a combination of essential ingredients per dosage unit form.
  • pharmaceutical dosage unit forms are prepared to provide about 10 mg, 20 mg, 25 mg, 50 mg, 100 mg, 250 mg, 500 mg, 1000 mg or 2000 mg of the active ingredient.
  • Embodiments of a second broad aspect of the present disclosure provide use of the scavenger used for clearing away mutant FUS in the manufacture of a medicament for treating or preventing or alleviating ALS.
  • the inventors of the patent surprisingly discover that scavenger used for clearing away mutant FUS can increase the motor ability, the number of motor neurons and the motor learning ability in ALS mice model.
  • the scavenger is used in the manufacture of a medicament and the medicament can treat or prevent or alleviate ALS effectively.
  • Embodiments of a third broad aspect of the present disclosure provide a method for treating or preventing or alleviating ALS, wherein the method comprising administration of the scavenger used for clearing away FUS protein mutants to a patient in need.
  • the inventors of the patent surprisingly discover that scavenger used for clearing away mutant FUS can increase the motor ability, the number of motor neurons and the motor learning ability.
  • the terms "administration of” and “administering a” the scavenger should be understood to mean providing the scavenger to an individual in need thereof. It is recognized that one skilled in the art can treat a patient presently afflicted with ALS disease with an effective amount of scavenger used for clearing away mutant FUS.
  • the donor DNA fragment contains the R513C mutation (tcg to ctg) , and the flanking left and right homology arms ( ⁇ 1kb) , respectively.
  • the donor DNA, gRNA (gcgagcacagacaggatcgcAGG, PAM site capitalized) , and Cas9 mRNA were injected into C57BL/6J embryos.
  • the injected embryos were transferred into the oviduct ampulla of the pseudo-pregnant ICR (JAX, Stock No. 009122) female recipients.
  • the right genotype offsprings were backcrossed to C57BL/6J for at least five generations to establish the line.
  • Open Field behavior test the single animal was placed in the center of an open field area (60 ⁇ 60 cm) and tracked with multiple parameters, including total distance, average speed, and distance traveled in the center region, by TopScan behavioral analysis system (CleverSys., USA) in a 10-minute interval.
  • the rotarod performance was measured by an automated system (Med Associates, Inc) .
  • the animal was placed on an accelerating spindle (4-40rpm) in 5 minutes per trail and 3 consecutive trials per day. A 20-minute break was set in between each trial.
  • the test was repeated for four days.
  • the fall time from the spindle was auto-calculated by the system when the mouse fell off the spindle within the 5-minute interval.
  • the stay time was calculated by subtraction of the fall time from the 5 minutes, and the mean value of the stay time from 3 consecutive trials per day was used for statistical analysis.
  • Dissection of femoral nerves was performed as described previously.
  • the femoral nerve was exposed in the sacrificed mice and briefly fixed (2%glutaraldehyde/2%paraformaldehyde in 0.1 M cacodylate buffer) before dissection.
  • the isolated nerves were postfixed overnight in the same fixative.
  • Dissected nerves were processed for plastic embedding and transmission electron microscopy by standard procedures. Nerve cross sections were stained with toluidine blue and examined by light microscopy.
  • RNA-seq and deferentially expressed gene identification.
  • RNAs were extracted by TRIzol (Invitrogen) , and the cDNA library preparation for high-throughput sequencing (Illumina HiSeq 2500) was prepared as described in the manual (NEBNext Ultra) .
  • Reads were aligned to mouse reference genome mm10 by HISAT2 with known splice sites from Ensembl database as a parameter. Differentially expressed genes were called by DESeq2 .
  • a previous lentivirus-based inducible system (Addgene, #50661) was modified for doxycycline-induced expression of Myc-BirA*-mTrim72 in HEK293 cells.
  • blasticidin Sigma
  • the infected cells were transiently transfected with plasmids (pCMV-3Tag, Stratagene) expressing human wild type FUS, FUS-R521C, and FUS 513-526 truncation.
  • Doxycycline (1 ⁇ g/ml, Sigma) were added in the cells to induce the expression of Myc-BirA*-mTrim72 8 hours after transfection.
  • the R521C mutation locates in FUS C-terminal, a non-classical PY nuclear localization signal (NLS) , in which clusters more than half of ALS-associated FUS mutations. Among these mutations, R521C is the most frequent one and accounts for 30%of the total. Second, the R521C mutation has been found in both familiar and sporadic ALS patients with high disease penetration. Third, the FUS R521C transgenic mouse and rat models present strong ALS-like pathological features.
  • NLS non-classical PY nuclear localization signal
  • Fig. 1A The C-terminal NLS encoded by the last exon of mouse FUS is identical to that of human (515GEHRQDRRERPY526) .
  • the mouse corresponding position for human R521 is R513.
  • a PAM site for Cas9 recognition appears (Fig. 1B) .
  • the correct genome insertion and germ line transmission were confirmed by breeding the KI founders with C57BL/6J wild type mice (Fig. 1C and 1D) . Meanwhile, inventors backcrossed the KI mutant mouse to C57BL/6J at least 5 generations, in order to reduce the potential off-target effect introduced by CRISPR/Cas9.
  • mutant FUS was slightly upregulated in spinal cord (Fig. 1E) , although the mRNA level of mutant FUS was comparable to that of wild type (data not shown) , suggesting that the R513C mutation may increase the protein stability and/or decrease the turnover rate in the target tissue.
  • the Trim72 preferentially interacted with FUS-R521C, compared to wild type FUS, but had little interaction with the FUS truncation ( ⁇ 513-526) , which lacks the nuclear localization signal (NLS) , suggesting that the NLS of FUS is required for the interaction (Fig. 3C and 3D) .
  • the localization of the expressed Trim72 in the cytosol Fig. 3E
  • Trim72 may target the mislocalized FUS for ubiquitination and proteasome-mediated degradation.
  • the interaction between Trim72 and FUS-R521C was sensitive to MG132, a proteasome inhibitor (Fig. 3F) .

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Engineering & Computer Science (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Medicinal Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Epidemiology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Neurology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Physical Education & Sports Medicine (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Marine Sciences & Fisheries (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne une composition pharmaceutique comprenant un piégeur, le piégeur étant utilisé pour éliminer la FUS mutante. L'invention concerne également un procédé de traitement ou de prévention ou de soulagement de la sclérose latérale amyotrophique (SLA).
PCT/CN2017/077712 2017-03-22 2017-03-22 Trim72 en tant que cible thérapeutique potentielle pour la sla par l'ubiquitination de la protéine fus mutante Ceased WO2018170796A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
PCT/CN2017/077712 WO2018170796A1 (fr) 2017-03-22 2017-03-22 Trim72 en tant que cible thérapeutique potentielle pour la sla par l'ubiquitination de la protéine fus mutante
CN201780091092.0A CN110753557B (zh) 2017-03-22 2017-03-22 Trim72通过泛素化fus蛋白突变体作为als的潜在治疗性靶标
CN202410175594.3A CN117982625A (zh) 2017-03-22 2017-03-22 Trim72在预防或治疗als中的用途

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2017/077712 WO2018170796A1 (fr) 2017-03-22 2017-03-22 Trim72 en tant que cible thérapeutique potentielle pour la sla par l'ubiquitination de la protéine fus mutante

Publications (1)

Publication Number Publication Date
WO2018170796A1 true WO2018170796A1 (fr) 2018-09-27

Family

ID=63584800

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2017/077712 Ceased WO2018170796A1 (fr) 2017-03-22 2017-03-22 Trim72 en tant que cible thérapeutique potentielle pour la sla par l'ubiquitination de la protéine fus mutante

Country Status (2)

Country Link
CN (2) CN110753557B (fr)
WO (1) WO2018170796A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109528684A (zh) * 2018-12-26 2019-03-29 牡丹江友搏药业有限责任公司 一种mg53蛋白/mg53突变体蛋白肠溶胶囊及其制备方法

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008054561A2 (fr) * 2006-07-11 2008-05-08 University Of Medicine And Dentistry Of New Jersey Protéines, acides nucléiques encodant celles-ci et leurs procédés d'utilisation associés
WO2010011283A2 (fr) * 2008-07-22 2010-01-28 The General Hospital Corporation Composés à base de fus/tls et méthodes de diagnostic, traitement et prévention d'une sclérose latérale amyotrophique et de maladies des motoneurones apparentées
CN101797375A (zh) * 2009-12-02 2010-08-11 北京大学 Mg53蛋白预防和/或治疗心脏缺血/再灌损伤的用途
WO2011142744A1 (fr) * 2010-05-11 2011-11-17 University Of Medicine And Dentistry Of New Jersey Compositions et méthodes pour le traitement et la prévention d'une lésion ischémique cardiaque
WO2012135868A2 (fr) * 2011-03-31 2012-10-04 University Of Medicine And Dentistry Of New Jersey Compositions et procédés pour le traitement et la prévention de lésion d'ischémie cardiaque
WO2013036610A2 (fr) * 2011-09-07 2013-03-14 University Of Medicine And Dentistry Of New Jersey Compositions comprenant le mg53 et procédés de traitement et de prévention de lésions au niveau des voies respiratoires
CN105079810A (zh) * 2015-07-16 2015-11-25 朱笠 一种开发fus蛋白病药物新靶点
WO2016109638A1 (fr) * 2014-12-30 2016-07-07 Rutgers, The State University Of New Jersey Compositions et méthodes pour la prévention et la réparation d'une lésion rénale aiguë

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101413005B1 (ko) * 2007-12-04 2014-07-02 유니버시티 오브 메디신 앤드 덴티스트리 오브 뉴 저지 세포막 재봉합을 조절하기 위한 조성물 및 방법
BR112017025574A2 (pt) * 2015-05-29 2018-08-14 Univ Pennsylvania composições e métodos para degradação de proteínas de má dobra

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008054561A2 (fr) * 2006-07-11 2008-05-08 University Of Medicine And Dentistry Of New Jersey Protéines, acides nucléiques encodant celles-ci et leurs procédés d'utilisation associés
WO2010011283A2 (fr) * 2008-07-22 2010-01-28 The General Hospital Corporation Composés à base de fus/tls et méthodes de diagnostic, traitement et prévention d'une sclérose latérale amyotrophique et de maladies des motoneurones apparentées
CN101797375A (zh) * 2009-12-02 2010-08-11 北京大学 Mg53蛋白预防和/或治疗心脏缺血/再灌损伤的用途
WO2011142744A1 (fr) * 2010-05-11 2011-11-17 University Of Medicine And Dentistry Of New Jersey Compositions et méthodes pour le traitement et la prévention d'une lésion ischémique cardiaque
WO2012135868A2 (fr) * 2011-03-31 2012-10-04 University Of Medicine And Dentistry Of New Jersey Compositions et procédés pour le traitement et la prévention de lésion d'ischémie cardiaque
WO2013036610A2 (fr) * 2011-09-07 2013-03-14 University Of Medicine And Dentistry Of New Jersey Compositions comprenant le mg53 et procédés de traitement et de prévention de lésions au niveau des voies respiratoires
WO2016109638A1 (fr) * 2014-12-30 2016-07-07 Rutgers, The State University Of New Jersey Compositions et méthodes pour la prévention et la réparation d'une lésion rénale aiguë
CN105079810A (zh) * 2015-07-16 2015-11-25 朱笠 一种开发fus蛋白病药物新靶点

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
ITO, D. ET AL.: "Nuclear Transport Impairment of Amyotrophic Lateral Sclerosis-Linked Mutations in FUS/TLS", ANNALS OF NEUROLOGY, vol. 69, no. 1, 1 January 2011 (2011-01-01), pages 152 - 162, XP055538558 *
VANCE, C. ET AL.: "ALS mutant FUS disrupts nuclear localization and sequesters wild-type FUS within cytoplasmic stress granules", HUMAN MOLECULAR GENETICS, vol. 22, no. 13, 7 March 2013 (2013-03-07), pages 2676 - 2688, XP055538530 *
WEISLEDER, N. ET AL.: "Recombinant MG 53 Protein can Increase Membrane Repair Capacity and Improve Pathology in Dystrophic Mouse Muscle", BIOPHYSICAL JOURNAL, vol. 102, no. 3, 31 January 2012 (2012-01-31), XP028893927 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109528684A (zh) * 2018-12-26 2019-03-29 牡丹江友搏药业有限责任公司 一种mg53蛋白/mg53突变体蛋白肠溶胶囊及其制备方法

Also Published As

Publication number Publication date
CN110753557A (zh) 2020-02-04
CN117982625A (zh) 2024-05-07
CN110753557B (zh) 2024-03-22

Similar Documents

Publication Publication Date Title
US20250032459A1 (en) Method for treating neurodegenerative diseases
JP6837429B2 (ja) Ｃｒｉｓｐｒ／ｃａｓ９媒介遺伝子編集による筋ジストロフィーの予防
US20250186429A1 (en) Methods and compositions for reducing tactile dysfunction and anxiety associated with autism spectrum disorder, rett syndrome, and fragile x syndrome
JP7366388B2 (ja) 抗Ｒｙｋ抗体およびその使用方法
JP2020500541A (ja) ヒト化デュシェンヌ型筋ジストロフィー変異を有するｄｍｄレポーターモデル
EP2200649A2 (fr) Compositions et procédés permettant d'améliorer l'inflammation du sn, la psychose, le délire, le ptsd ou le sspt
Johnson et al. Early postnatal administration of an AAV9 gene therapy is safe and efficacious in CLN3 disease
US20210017268A1 (en) Methods for treating alcoholic liver disease, alcohol-induced brain injury and reducing alcohol addiction
Fujita et al. UCP1 expression in the mouse adrenal gland is not upregulated by thermogenic conditions
WO2018170796A1 (fr) Trim72 en tant que cible thérapeutique potentielle pour la sla par l'ubiquitination de la protéine fus mutante
US20160015783A1 (en) Methods for modulating development and function of photoreceptor cells
US20200215032A1 (en) Methods and compositions for improving exercise endurance, performance, or tolerance
Hassan et al. The dose-response relationship of subretinal gene therapy with rAAV2tYF-CB-h RS1 in a mouse model of X-linked retinoschisis
US7393864B2 (en) Use of ClC3 chloride channel blockers to modulate vascular tone
Aghaeipour et al. 436P Optimizing the efficacy of antisense oligonucleotides for the treatment of Duchenne muscular dystrophy
US20250064770A1 (en) Method for preventing or treating cocaine addiction and applications thereof
Ye et al. XLRS Rat with Rs1-/Y Exon-1-Del Shows Failure of Early Postnatal Outer Retina Development. Genes 2022, 13, 1995
Secondulfo et al. Genome-Based Advances in Modelling Renal Ciliopathies and Enhancing Patient Care
Sawdai The Role of Vitamin D in the CNS and Immune Pathology of Multiple Sclerosis Research Thesis
Wolf et al. Ghrelin Enhances Tubular Magnesium Absorption in the Kidneys: FR-PO506
Myers Investigation of Pathological Processes in a Novel Murine Model of Triosephosphate Isomerase Deficiency and Assessment of Potential Therapeutics
Rong et al. Dual-Targeting Nanoparticles Enhance Microglial P2Y12R Expression to Promote Neuronal Mitophagy for Repairing Spinal Cord Injury
Santos Identifying the Critical Therapeutic Window for Molecular and Behavioural Correction of Angelman Syndrome
Dupont et al. Gene Therapy for X-Linked Myotubular Myopathy
Marrett Modification of the myostatin and nitric oxide signalling pathways as treatments for Duchenne Muscular Dystrophy

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17901889

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 17901889

Country of ref document: EP

Kind code of ref document: A1