Small molecules for the rescue of amyotrophic lateral sclerosis (ALS) and frontotemporal degeneration (FTD) by targeting c9orf72 hexanucleotide g-quadrupexes

Abstract

The present invention relates to a pharmaceutical composition comprising a series of compounds that selectively target a G-quadruplex (G4) formed by the GGGGCC (G4C2) hexanucleotide repeat sequence (HRE) (G4C2) n of C9orf72. The composition comprises a compound G4008, a compound G4009, a compound G4010, a compound G4012 and a compound G4013, or pharmaceutically acceptable salts, solvates or hydrates of the compound G4008, the compound G4009, the compound G4010, the compound G4012 and the compound G4013; and at least one pharmaceutically acceptable carrier and / or excipient. The invention also relates to a method for treating amyotrophic lateral sclerosis (ASL) and / or frontotemporal dementia (FTD) comprising administering to a subject an effective amount of the composition. The compounds can improve the pathological characteristics of an ALS model caused by the C9orf72 (G4C2) n sequence.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202610040607.5 (22) Application Date 2026.01.13 (30) Priority Data 63 / 744,866 2025.01.14 US (71) Applicant Hong Kong University of Science and Technology Address Clear Water Bay, Kowloon, Hong Kong, China (72) Inventors Zhu Guang, Liu Changdong, Sun Qing (74) Patent Agency Beijing Tianhao United Intellectual Property Agency Co., Ltd. 11112 Patent Attorney Sun Wei, Yin Likun (51) Int.Cl. A61K 31 / 4745 (2006.01) A61K 31 / 4985 (2006.01) A61P 25 / 00 (2006.01) A61P 25 / 28 (2006.01) (54) Invention Title: Treatment of Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD) by Targeting the C9ORF72 Hexanucleotide G-Quadruplex (57) Abstract: This invention relates to a pharmaceutical composition comprising a series of compounds selectively targeting the G-quadruplex (G4) formed by the hexanucleotide repeat sequence (HRE) (G4C2)n of C9orf72. The composition comprises: compounds G4008, G4009, G4010, G4012, and G4013, or pharmaceutically acceptable salts, solvates, or hydrates thereof; and at least one pharmaceutically acceptable carrier and / or excipient. The invention also relates to a method for treating amyotrophic lateral sclerosis (ASL) and / or frontotemporal dementia (FTD), comprising administering an effective amount of the composition to a subject. These compounds can improve the pathological features of an ALS model induced by the C9orf72 (G4C2)n sequence. Claims 1 page, Description 17 pages, Sequence List (electronic publication), Drawings 10 pages, CN 122376593 A 2026.07.14 CN 1 22 37 65 93 A 1. A pharmaceutical composition comprising: one or more compounds selected from the group consisting of G4008, G4009, G4010, G4012 and G4013, or a pharmaceutically acceptable salt, solvate or hydrate thereof; and at least one pharmaceutically acceptable carrier and / or excipient. 2. A method of treating a subject with amyotrophic lateral sclerosis and / or frontotemporal dementia, the method comprising administering to the subject an effective amount of the composition according to claim 1. 3. The method according to claim 2, wherein the composition comprises G4008, G4010, G4012 or G4013. 4. The method according to claim 2, wherein the composition comprises G4010 or G4013.5. The method of claim 2, wherein the composition comprises compound G4013. 6. The method of claim 2, wherein the composition is administered once, twice, three times, four times, or five times per week. 7. The method of claim 2, wherein the composition is administered at a dose of about 0.1 mg / kg to about 200 mg / kg. 8. The method of claim 2, wherein the composition is administered locally via intravitreal, intracranial, or intradiscal administration, or systemically via intramuscular, intravascular (e.g., intravenous), intraoral, intradermal, intranasal, intrathecal, or subcutaneous administration. 9. The method of claim 2, wherein the subject is a human. 10. The method of claim 2, wherein one or more compounds of the composition selectively target and bind to the G-quadruplex (G4) formed by the hexanucleotide repeat sequence (G4C2)n of C9orf72. 11. The method of claim 2, wherein administration of the composition results in an improvement of about 70% in one or more symptoms of ASL and / or FTD. 12. The method of claim 2, wherein the application of the composition reduces the progression of ASL and / or FTD by about 70%. 13. The method of claim 3, wherein the number of RNA aggregates is significantly reduced by applying a composition comprising one or more compounds selected from the group consisting of G4008, G4010, G4012, and G4013. 14. The method of claim 13, wherein the number of RNA aggregates is reduced by about 50%. 15. The method of claim 2, wherein the application of the composition reduces the reactive oxygen species (ROS) level of the subject by about 40%. 16. The method of claim 2, wherein G4013 is applied to target C9orf72 G4C2 G4 with high binding affinity. 17. The method of claim 16, wherein the binding affinity (Kd) of G4013 to C9orf72 DNA (G4C2)4 G4 is about 114 μM. 18. The method of claim 16, wherein the binding affinity (Kd) of G4013 to C9orf72 RNA (G4C2G4) G4 is about 68 μM. 19. The method of claim 2, wherein the composition comprises G4013 as the sole active agent. Claims 1 / 1 page 2 CN 122376593 A Small molecule for treating amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) by targeting the C9ORF72 hexanucleotide G-quadruplex

[0001] Cross-reference to related applications

[0002] This application claims the benefit of U.S. Provisional Application Sequence No. 63 / 744,866, filed January 14, 2025.The entire contents of this application are incorporated herein by reference, including any tables, figures, or drawings. Technical Field

[0003] This application relates to the biomedical field. More specifically, this application relates to a pharmaceutical composition comprising a compound selectively targeting a G-quadruplex formed by the hexanucleotide repeat sequence (G4C2)n of C9orf72 GGGGCC (G4C2), and a method of using the pharmaceutical composition to salvage amyotrophic lateral sclerosis (ALS) and / or frontotemporal dementia (FTD). Background Art

[0004] Amyotrophic lateral sclerosis (ALS), also known as Lugris disease, is a devastating neurodegenerative disease characterized by the rapid, progressive loss of motor neurons, ultimately leading to respiratory failure within 3 to 5 years after the onset of symptoms (see P. Masrori, P. Van Damme, Amyotrophic lateral sclerosis: a clinical review, Eur J Neurol, 27 (2020) 1918–1929). In particular, approximately 50% of ALS patients have cognitive impairment, and 13% of them develop frontotemporal dementia (FTD) (see O. Hardiman, A. Al-Chalabi, A. Chio, EM Corr, G. Logroscino, W. Robberecht, PJ Shaw, Z. Simmons, LH van den Berg, Amyotrophic lateral sclerosis, Nat Rev Dis Primers, 3 (2017) 17071). Approximately two out of every 100,000 people suffer from ALS, characterized by the degeneration of motor neurons, leading to muscle weakness and atrophy (see references: M. DeJesus-Hernandez, I.R. Mackenzie, BF. Boeve, AL. Boxer, M. Baker, N.J. Rutherford, AM. Nicholson, NA. Finch, H. Flynn, J. Adamson, N. Kouri, A. Wojtas, P. Sengdy, G.Y. Hsiung, A. Karydas, W.W. Seeley, K.A. Josephs, G. Coppola, D.H. Geschwind, Z.K. Wszolek, H. Feldman, D.S. Knopman, R..C. Petersen, B.L. Miller, D.W. Dickson, K.B. Boylan, N.R. Graff‑Radford, R. Rademakers, Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 chromosome 9p‑linked FTD and ALS, Neuron, 72 (2011) 245-256).

[0005] Regarding the etiology of ALS, it has been reported that sporadic ALS (sALS) accounts for approximately 90% of all ALS cases, while the remaining 10% of ALS patients have familial ALS (fALS), which is caused by gene mutations (see reference X. Liu, X. Zhao, J. He, S. Wang, X. Shen, Q. Liu, S. Wang, Advances in the Structure of GGGGCC Repeat RNA Sequence and Its Interaction with Small Molecules and Protein Partners, Molecules, 28 (2023)). Abnormalities in the function of SOD1, FUS / TLS, and TDP-43 caused by mutations lead to neurotoxicity, resulting in fALS (see E.L. Feldman, SA. Goutman, S. Petri, L. Mazzini, MG. Savelieff, PJ. Shaw, G. Sobue, Amyotrophic lateral sclerosis, Lancet, 400 (2022) 1363-1380). Furthermore, it has been shown that amplification of the GGGGCC (G4C2) hexanucleotide repeat sequence in the non-coding region of the C9orf72 gene is a pathogenic factor for ALS and FTD (see CM. Rodriguez, PK. Todd, New pathologic mechanisms in nucleotide repeat expansion disorders, manual 1 / 17 page 3 CN 122376593 A Neurobiol Dis, 130 (2019) 104515; Q. Zhu, J. Jiang, TF).Gendron, M. McAlonis-Downes, L. Jiang, A. Taylor, S. Diaz Garcia, S. Ghosh Dastidar, M.J. Rodriguez, P. King, Y. Zhang, AR La Spada, H. Xu, L. Petrucelli, J. Ravits, S. Da Cruz, C. Lagier-Tourenne, D.W. Cleveland, Reduced C9ORF72 function exacerbates gain of toxicity from ALS / FTD‑causing repeat expansion in C9orf72, Nat Neurosci , 23 (2020) 615‑624). Aberrant amplification of the G4C2 repeat sequence was observed in 8% of sporadic ALS (sALS) patients and over 40% of familial ALS (fALS) cases (see references: AE Renton, E. Majounie, A. Waite, J. Simón-Sánchez, S. Rollinson, J.R. Gibbs, J.C. Schymick, H. Laaksovirta, J.C. van Swieten, L. Myllykangas, H. Kalimo, A. Paetau, Y. Abramzon, AM Remes, A. Kaganovich, SW Scholz, J. Duckworth, J.H. Ding, DW Harmer, DG. Hernandez, J.O. Johnson, K. Mok, M. Ryten, D. Trabzuni, RJ Guerreiro). RW Orrell, J. Neal, A. Murray, J. Pearson, IE Jansen, D. Sondervan, H. Seelaar, D. Blake, K. Young, N. Halliwell, J.B. Callister, G. Toulson, A. Richardson, A. Gerhard, J.Snowden, D. Mann, D. Neary, MA Nalls, T. Peuralinna, L. Jansson, VM Isoviita, AL Kaivorinne, M. Hölttä‑Vuori, E. Ikonen, R. Sulkava, M. Benatar, J. Wuu, A. Chiò, G. Restagno, G. Borghero, M. Sabatelli, D. Heckerman, E. Rogaeva, L. Zinman, J.D. Rothstein, M. Sendtner, C. Drepper, E.E. Eichler, C. Alkan, Z. Abdullaev, S.D. Pack, A. Dutra, E. Pak, J. Hardy, A. Singleton, N .M.Williams, P. Heutink, S. Pickering‑Brown, H.R. Morris, PJ. Tienari, BJ. Traynor, I. Consortium, A Hexanucleotide Repeat Expansion in Is the Cause of Chromosome 9p21‑Linked ALS‑FTD, Neuron, 72 (2011) 257‑268). The average number of repeat sequences in individuals with ALS typically ranges from 700 to 1600, while healthy individuals have fewer than 25 repeat sequences (see A. Nordin, C. Akimoto, A. Wuolikainen, H. Alstermark, P. Jonsson, A. Birve, S.L. Marklund, K.S. Graffmo, K. Forsberg, T. Brannstrom, P.M. Andersen, Extensive size variability of the GGGGCC expansion in C9orf72 in both neuronal and non-neuronal tissues in 18 patients).With ALS or FTD, Hum Mol Genet, 24 (2015) 3133-3142. In particular, aberrant amplification of short nucleotide repeat sequences has been identified in many neurodegenerative diseases, such as aberrant amplification of (CAG)n leading to spinocerebellar ataxia 3 (SCA3) (see I. Malik, C.P. Kelley, E.T. Wang, P.K. Todd, Molecular mechanisms underlying nucleotide repeat expansion disorders, Nat Rev Mol Cell Biol, 22 (2021) 589-607). Therefore, elucidating the pathological mechanisms by which these aberrant nucleotide repeat sequences lead to fatal diseases is a core research focus with the aim of discovering effective treatments.

[0006] Currently, three mechanisms have been proposed to elucidate the pathological basis of aberrant amplification of the hexanucleotide repeat sequence (G4C2)n (see R. Balendra, A.M. Isaacs, C9orf72-mediated ALS and FTD: multiple pathways to disease, Nat Rev Neurol, 14 (2018) 544-558). First, aberrant G4C2 amplification can lead to the gain or loss of C9orf72 gene function. Second, transcribed RNA G4C2 amplification can recruit RNA-binding proteins (RBPs) to form toxic RNA aggregates (RNA foci), resulting in impaired protein function. Third, non-AUG translation of RNA G4C2 amplification can produce neurotoxic dipeptide repeat sequences (DPRs) in the central nervous system, leading to neuronal death. Notably, the formation mechanism of toxic RNA aggregates has received the most attention, as noted in the specification 2 / 17 page 4 CN 122376593 A. In particular, C9orf72 G4C2 repeat sequences (including both DNA and RNA sequences) can fold into secondary structures such as G4 and hairpin structures (see AR. Haeusler, CJ Donnelly, G. Periz, EAJ Simko, PG Shaw, MS Kim, NJ Maragakis, J.C. Troncoso, A. Pandey, R. Sattler, J.D. Rothstein, J. Wang, nucleotide repeat structures).initiate molecular cascades of disease, Nature, 507 (2014) 195‑+;Y.Y. Geng , C.D. Liu, Q.X . Cai , Z.P. Luo , H.T. Miao , X . Shi , N.N. Xu, C .P . Fung , T .T . Choy , B . Yan , N . Li , P .Y . Qian , B . Zhou , G . Zhu , Crystal structure of parallel G‑quadruplex formed by the two‑repeat ALS‑ and FTD‑ related GGGGCC sequence , Nucleic Acids Res , 49 (2021) 5881‑5890;Y.Y. Geng , C .D. Liu , N .N . Xu , M .C . Suen , H .T . Miao , Y .Y . Xie , B.C . Zhang , X .Q . Chen , Y.J. Song, Z.X. Wang, Q.X. Cai, G. Zhu, Crystal structure of a tetrameric RNA G‑quadruplex formed by hexanucleotide repeat expansions of C9orf72 in ALS / FTD, Nucleic Acids Res , 52 (2024) 7961‑7970;Z.F. Wang, A. Ursu, J.L. Childs‑ Disney , R . Guertler , W .Y . Yang , V . Bernat , S .G . Rzuczek , R . Fuerst , Y .J . Zhang, T.F. Gendron, I. Yildirim, B.G. Dwyer, J.E. Rice, L. Petrucelli, M.D. Disney, The Hairpin Form of r(G4C2)exp in c9ALS / FTD Is Repeat‑Associated Non‑ ATG Translated and a Target for Bioactive SmallMolecules, Cell Chem Biol , 26 (2019) 179‑190e12). Most importantly, G4 formation is a key aspect of the toxicity of the C9orf72 G4C2 repeat sequence in ALS / FTD, and it has been reported that RNA aggregates formed by the C9orf72 G4C2 repeat sequence are mainly composed of RNA G4 (see X. Liu, X. Zhao, J. He, S. Wang, X. Shen, Q. Liu, S. Wang, Advances in the Structure of GGGGCC Repeat RNA Sequence and Its Interaction with Small Molecules and Protein Partners, Molecules, 28 (2023); EG Conlon, L. Lu, A. Sharma, T. Yamazaki, T. Tang, NA Shneider, JL Manley, The C9ORF72 GGGGCC expansion forms RNA G-quadruplex inclusions and sequesters hnRNP H to disrupt splicing in ALS brains, Elife, 5 (2016)). Therefore, structural studies of the C9orf72 G4C2 repeat sequence are currently a very important research area, as the corresponding structures may become potential drug targets for treatment.

[0007] Currently, due to insufficient understanding of the disease mechanism of ALS / FTD, it remains an incurable disease. The U.S. Food and Drug Administration (FDA) has currently approved seven drugs for the treatment of ALS: Qalsody®, Relyvrio®, Radicava®, Rilutek®, Tiglutik®, Exservan®, and Nuedexta® (see literature RJ Mead, N. Shan, H.J. Reiser, F. Marshall, P.J. Shaw, Amyotrophic lateral sclerosis: a neurodegenerative disorder poised for successful therapeutic translation, Nat Rev Drug Discov, 22 (2023) 185-212; U.Ansari, M. Alam, D. Nadora, Z. Muttalib, V. Chen, I. Taguinod, M. FitzPatrick, J. Wen, Z. Ansari, F. Lui, Assessing the efficacy of amyotrophic lateral sclerosis drugs in slowing disease progression: A literature review, AIMS Neurosci, 11 (2024) 166‑177; SA Johnson, T. Fang, F. De Marchi, D. Neel, D. Van Weehaeghe, JD Berry, S. Paganoni, Pharmacotherapy for Amyotrophic Lateral Sclerosis: A Review of Approved and Upcoming Agents, Drugs, 82 (2022) 1367‑1388). Qalsody® or tofersen was approved in 2023 as an antisense oligonucleotide targeting the mRNA of the mutant superoxide dismutase 1 (SOD1) gene (see reference HA Blair, Tofersen: First Approval, Drugs, 83 (2023) 1039-1043). Relyvrio® is a fixed combination of sodium phenylbutyrate and taurine glycol that prevents neuronal cell death by inhibiting stress signals in intracellular mitochondria and endoplasmic reticulum. Relyvrio® was approved in 2022 (see DB Tiz, L. Bagnoli, O. Rosati, F. Marini, C. Santi, L. Sancineto, FDA-Approved Small Molecules in 2022: Clinical Uses and Their Synthesis, Pharmaceutics, 14 (2022)), but was discontinued and withdrawn from the US and Canada in 2024 based on the results of a Phase 3 trial. Radicava® or similar products...Daravone was first manufactured and marketed in Japan in 2015 and received FDA approval for the treatment of ALS in 2017. Radicava® is a potent free radical scavenger that combats reactive oxygen species-driven motor neuron death and inflammation (see P. Neupane, P.K. Thada, P. Singh, A.R. Faisal, N. Rai, P. Poudel, M.S. Waleed, J. Quinonez, S. Ruxmohan, E. Jain, Investigating Edaravone Use for Management of Amyotrophic Lateral Sclerosis (ALS): A Narrative Review, Cureus, 15 (2023) e33746). Rilutek®, or riluzole, was the first ALS drug approved by the FDA in 1995. It is an inhibitor of the sodium channel α subunit and has been shown to extend the lifespan of ALS patients by about three months (see MC Bellingham, A Review of the Neural Mechanisms of Action and Clinical Efficiency of Riluzole in Treating Amyotrophic Lateral Sclerosis: What have we Learned in the Last Decade?, Cns Neurosci Ther, 17 (2011) 4-31). Tiglutik®, an oral suspension variant of riluzole designed for ALS patients with difficulty swallowing tablets, was reapproved by the FDA in 2018 (see B.R. Brooks, P. Bettica, S. Cazzaniga, Riluzole Oral Suspension: Bioavailability Following Percutaneous Gastrostomy Tube-modeled Administration Versus Direct Oral Administration, Clin Ther, 41 (2019) 2490-2499). Exservan®, an oral film formulation of riluzole designed for ALS patients with severe dysphagia, was approved in 2019 (see J. Wymer, S. Apple)., A. Harrison, B.A. Hill, Pharmacokinetics, Bioavailability, and Swallowing Safety With Riluzole Oral Film, Clin Pharmacol Drug Dev, 12 (2023) 57‑64). Nuedexta®, approved in 2010, is an oral combination of dextromethorphan hydrobromide and quinidine sulfate for the treatment of excessive emotional instability known as pseudobulbar mood (PBA) (see reference Y. Sun, M. Benatar, J. Mascias Cadavid, D. Ennist, P. Wicks, K. Staats, M. Beauchamp, S. Jhooty, G. Pattee, A. Brown, T. Bertorini, P. Barkhaus, M. Bromberg, G. Carter, R. Bedlack, X. Li, ALSUntangled #71: Nuedexta, Amyotroph Lateral Scler Frontotemporal Degener, 25 (2024) 218–222). However, the detailed mechanism of action of these drugs in treating ALS remains unclear. In particular, more than 5,000 new cases of ALS are diagnosed globally each year. Therefore, there is an urgent need to discover and identify new effective targets and drugs for the treatment of ALS. Summary of the Invention

[0008] In one aspect, the present invention relates to a novel composition for the treatment of ALS comprising a derivative of 7H-pyridocarbazole and 7H-pyrazinocarbazole, selectively targeting a G-quadruplex (G4) formed by the hexanucleotide repeat sequence (HRE) (G4C2) n of C9orf72. In some embodiments, the composition comprises: one or more compounds including G4008, G4009, G4010, G4012, and G4013, or pharmaceutically acceptable salts, solvates, or hydrates thereof; and at least one pharmaceutically acceptable carrier and / or excipient.

[0009] In another aspect, the present invention relates to a novel method for the treatment of ALS, comprising administering to a subject an effective amount of the composition of the present invention. (Page 4 / 17, CN 122376593 A) In some embodiments, the novel compounds of the present invention target C9orf72G4C2G4. In preferred embodiments...In some embodiments, the composition comprises: one or more compounds selected from the group consisting of G4008, G4010, G4012, and G4013, or a pharmaceutically acceptable salt, solvate, or hydrate thereof; and at least one pharmaceutically acceptable carrier and / or excipient. In a more preferred embodiment, the composition comprises: one or more compounds selected from the group consisting of G4010 and G4013, or a pharmaceutically acceptable salt, solvate, or hydrate thereof; and at least one pharmaceutically acceptable carrier and / or excipient. In the most preferred embodiment, the composition comprises: compound G4013 or a pharmaceutically acceptable salt, solvate, or hydrate thereof; and at least one pharmaceutically acceptable carrier and / or excipient.

[0010] In some embodiments, administration of a composition comprising one or more compounds selected from the group consisting of G4008, G4010, G4012, and G4013 significantly reduced the number of RNA aggregates and reactive oxygen species (ROS) levels in subjects.

[0011] In some embodiments, administration of the composition can significantly improve the pathological features of an ALS model induced by the C9orf72 (G4C2)n sequence. In other embodiments, administration of the composition can significantly improve one or more symptoms and / or the progression of ALS and / or FTD in a subject.

[0012] Figure 1 shows the structures of six compounds designed based on chrexanthomycin A. Figure 1 shows small molecules G4008 to G4013 designed based on the chemical structural backbone of chrexanthomycin A, 7H-pyridocarbazole, and derivatives of 7H-pyrazinocarbazole.

[0013] Figure 2 shows the NMR titration of the compounds with C9orf72DNA (G4C2)4G4. For small molecules G4008, G4009, G4010, G4011, G4012, and G4013, the imino regions of the 1D 1H spectra of C9orf72 DNA (G4C2)4 titrated with compounds (without G4: black, 1:1 red, 1:5 green, 1:10 purple) are shown.

[0014] Figure 3 shows the NMR titration of the compounds with C9orf72 RNA (G4C2G4) G4. For small molecules G4008, G4009, G4010, G4011, G4012, and G4013, the imino regions of the 1D 1H spectra of C9orf72 RNA (G4C2G4) G4 titrated with compounds (without G4: black, 1:1 red, 1:5 green, 1:10 purple) are shown.

[0015] Figures 4A and 4B show the cellular assays performed on compounds in HT22 cells using MTT and ROS detection. Figure 4A shows the MTT assays for six compounds (the six data points correspond to logarithmic concentrations of 0.064 µM, 0.32 µM, and 0.42 µM respectively).(µM, 1.6 µM, 8 µM, 40 µM, and 200 µM). Figure 4B shows the ROS detection of the six compounds (x-axis: six compounds and related control group, y-axis: normalized ROS fluorescence intensity).

[0016] Figures 5A and 5B show the treatment of C9orf72(G4C2)29 amplification-related symptoms in cell models using the compounds. Figure 5A shows representative RNA FISH images of Neuro2a cells transfected with C9orf72 DNA (G4C2)29 in either the DMSO control or the new compound (8 µM treatment). RNA aggregates of C9orf72 G4C2 repeat sequences were detected using Cy3-labeled probes, with DAPI used as a counterstain (blue). Figure 5B shows the quantification of the number of C9orf72 RNA G4C2 aggregates per cell in cells expressing (G4C2)29 treated with the new compound. n = 15–20 cells, p-value determined by one-way ANOVA.

[0017] Figures 6A and 6B show the treatment of C9orf72(G4C2)49 amplification-related symptoms in a Drosophila model using the compounds. Figure 6A shows representative external eye images and scanning electron microscopy (SEM) images of 7-day-old wild-type (WT) Drosophila and larval GMR-GAL4-(G4C2)49 Drosophila fed with DMSO (100 μM), compounds G4010 (100 μM), and G4013 (100 μM). Figure 6B shows the quantification of the percentage of eye degeneration in WT and GMR-GAL4-(G4C2)49 Drosophila fed with DMSO (100 μM), compounds G4010 (100 μM), and G4013 (100 μM). n = 7–18 animals; p-values ​​were determined by one-way ANOVA (Tukey multiple comparison test).

[0018] Figure 7 shows the ITC experiments of G4010 and G4013 with C9orf72 DNA (G4C2)4 and RNA (G4C2G4) G4. The ITC experiments were performed in a buffer containing 20 mM potassium phosphate (pH 7.0) and 70 mM KCl for G4010 + DNA (G4C2)4, G4010 + RNA (G4C2G4), G4013 + DNA (G4C2)4, and G4013 + RNA (G4C2G4). The compounds were dissolved in DMSO to a stock solution concentration of 10 mM.

[0019] Brief description of the sequence

[0020] SEQ ID NO: 1 DNA sequence corresponding to C9orf72 DNA (G4C2)4[GGGGCCGGGGCCGGGGCCGGGCC], consisting of 24 nucleotides, was purchased from Accurate Biotechnology (Hunan) Co., Ltd.

[0021] SEQ ID NO: 2 corresponds to the RNA sequence r[GGGGCCGGGG] of C9orf72 RNA (G4C2G4), consisting of 10 nucleotides, and was synthesized by Accurate Biotechnology (Hunan) Co., Ltd.

[0022] SEQ ID NO: 3 corresponds to the DNA sequence d[GGGGCCGGGGCCGGGGCCG GGGCCGG ... Detailed Description

[0023] Selected Definitions

[0024] As used herein, unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” are intended to also include the plural forms. Furthermore, in relation to the use of the terms “comprising,” “including,” “containing,” “having,” “with,” or variations thereof in the detailed description and / or claims, such terms are intended to be inclusive in a manner similar to the term “comprising.” The transitional terms / phrases (and any grammatical variations thereof) “comprising,” “including,” “substantially consisting of,” and “consisting of” are used interchangeably.

[0025] The phrase “substantially consisting of” indicates that the claim covers embodiments that include the specified materials or steps as well as materials and steps that do not substantially affect the essential and novel features of the claim.

[0026] The term “about” refers to an acceptable error range for a particular value as determined by a person skilled in the art, where the error is produced in part depending on how that value is measured, i.e., limitations of the measurement system. In compositions containing a certain amount of an ingredient, with the term "about" used, these compositions contain said amount of the ingredient, the variation (error range) of which is 0% to 10% (X ± 10%) around a given value. In other cases, the term "about" provides for a variation (error range) of 0% to 10% around a given value.(X±10%). Clearly, this variation represents a range that is 10% higher or lower than a given value, such as X±1%, X±2%, X±3%, X±4%, X±5%, X±6%, X±7%, X±8%, X±9%, or X±10%.

[0027] In this disclosure, ranges are stated in a simplified form to avoid having to state and describe every and all values ​​within the range in detail. Where appropriate, any suitable value within the range may be chosen as the upper limit, lower limit, or endpoint of the range. For example, a range of 0.1 to 1.0 represents the endpoints 0.1 and 1.0, and the intermediate values ​​0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and all intermediate ranges included within 0.1 to 1.0, such as 0.2 to 0.5, 0.2 to 0.8, 0.7 to 1.0, and so on. It is conceivable that a value having at least two significant figures within a range, for example, a range of 5 to 10 represents all values ​​between 5.0 and 10.0 and between 5.00 and 10.00, including end values. When ranges are used herein, combinations and sub-combinations of ranges (e.g., sub-ranges within the disclosed ranges) and specific embodiments thereof are explicitly included.

[0028] As used herein, the term “subject” refers to an animal, particularly a human, that needs or expects to receive the benefits provided by a therapeutic compound. The term “subject” as used herein encompasses both humans and non-human animals. The term “non-human animal” includes all vertebrates, such as mammals, such as non-human primates (particularly higher primates), sheep, dogs, rodents (such as mice or rats), guinea pigs, goats, pigs, cats, rabbits, cows; and non-mammals, such as chickens, amphibians, reptiles, etc. These benefits may include, but are not limited to, treating a health condition, disease, or symptom; preventing a health condition, disease, or symptom; immune health; and enhancing the function of organs, tissues, or systems in the body. In one embodiment, the subject is a human. In another embodiment, the subject is an experimental animal or animal substitute used as a disease model. In some embodiments, the term "subject" refers to a mammal, including but not limited to rats, apes, humans, felines, canines, horses, cattle, farm mammals, racing mammals, and pet mammals. In the context of this invention, the subject is preferably a human. The term does not indicate a specific sex. Therefore, it is intended to cover both male and female subjects. Subjects may be at any age or developmental stage, including infancy, childhood, adolescence, youth, adulthood, or old age.

[0029] As used herein, the terms "therapeutic effective amount," "therapeutic effective dose," "effective amount," and "effective dose" are used to refer to an amount or dose of a compound or composition that, when administered to a subject, is capable of treating, preventing, inhibiting, or improving the condition of the subject.Condition, disease, or symptom. In other words, the amount is “therapeutic” when administered to a subject. The actual amount will vary depending on many factors, including but not limited to the specific condition, disease, or symptom being treated or improved; the severity of the condition; the specific organ, tissue, or body system for which health or function is to be enhanced; the patient’s weight, height, age, and health status; and the route of administration.

[0030] As used herein, the term “treatment” means eradication; reduction; inhibition; improvement; elimination; relief; symptom reduction or delay of symptom onset; slowing of the rate of degeneration or deterioration; lessening of the degree of weakness at the endpoint of degeneration; and / or improvement of the physical or mental health of the subject, or reversal of the signs or symptoms of a health condition, disease, or symptom to any extent, and includes, but does not require, a complete cure of the condition, disease, or symptom. Treatment may cure, improve, or partially improve a symptom. “Treatment” may also include improving or enhancing a condition or characteristic, for example, bringing the function of a specific system in the body to a higher state of health or homeostasis.

[0031] As used herein, the terms “prevent,” “reduce,” “inhibit,” “block,” “prevent,” “relieve,” “delay,” “preemptively block,” “minimize,” or “mitigate” refer to the onset of a specific sign or symptom of a condition, disease, or symptom. Inhibition may be, but is not required to be, absolute or complete; this means that the sign or symptom may still progress later. Inhibition may include reducing the severity of an onset of such a condition, disease, or symptom, and / or inhibiting the progression of a condition, disease, or symptom to a more severe condition, disease, or symptom.

[0032] When referring to a compound, “inhibition” means that the compound is able to reduce the incidence, severity, lesion size, volume, or associated symptoms of ALS / FTD by at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, or 100% compared to the normal presentation of pathological features in an ALS / FTD model without the application of the compound or a composition containing the compound.

[0033] As used herein, the terms “nucleic acid” or “polynucleotide” refer to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form. Unless specifically limited, the term covers those nucleic acids comprising known analogs of natural nucleotides, which have similar binding properties to the mentioned nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise stated, a particular nucleic acid sequence also implies coverage of variants of its conserved modifications (e.g., degenerate codon substitutions), alleles, homologous sequences, single nucleotide polymorphisms (SNPs), and complementary sequences, as well as explicitly stated sequences. Specifically, degenerate codon substitutions can be achieved by generating sequences in which one or more selected (or all) codons are substituted with a mixture of bases and / or deoxyinosine residues at the third position. The term nucleic acid can be related to the basic...Because, cDNA and mRNA encoded by genes are used interchangeably.

[0034] As used herein, the terms “low nucleotide” and “oligonucleotide” are used interchangeably to describe short single strands of synthetic DNA or RNA, such as (e.g.) sequences of about 5 nucleic acid bases to about 500 nucleic acid bases. “Low nucleotide delivery” and “oligonucleotide delivery” are also used interchangeably to describe methods of delivering oligonucleotides into cells or targeting specific cell types, tissues or organs.

[0035] The term “G-tetrachain” refers to its common and general meaning. A “G-tetrachain” is a square structure of four guanines stabilized by Husstan hydrogen bonds. A “G-tetrachain” is helical and is formed by stacks of interconnected guanine tetrads (or “G-tetrads”), which are flattened ring structures formed by four guanines and can also be stabilized by the presence of a monovalent cation (e.g., potassium) at the center of the tetrad. “G-quadruplexes” can be formed from DNA, RNA, or any combination thereof.

[0036] As used herein, “isolated” or “purified” compounds are substantially free of other compounds. In some embodiments, the purified compound is at least 60% by weight (dry weight) of the target compound. Preferably, the formulation is at least 75% by weight, more preferably at least 90% by weight, and most preferably at least 99% by weight of the target compound. For example, the purified compound is a compound in which the desired compound is at least 90% by weight, 91% by weight, 92% by weight, 93% by weight, 94% by weight, 95% by weight, 98% by weight, 99% by weight, or 100% by weight. Purity is determined by any suitable standard method, such as by column chromatography, thin-layer chromatography, or high-performance liquid chromatography (HPLC).

[0037] In some embodiments, treatment is administered to a subject to significantly reduce or eliminate the formation and number of RNA aggregates in neurons. Compared to an untreated subject, this term refers to a reduction of at least (or at least about) 30% in the number of aggregates. Therefore, treated subjects exhibited a significant reduction or elimination of the number of RNA aggregates, ranging from about 30% to about 99.99%, about 35% to about 99.99%, about 40% to about 99.99%, about 45% to about 99.99%, about 50% to about 99.99%, about 60% to about 99.99%, and about 70% to about 99.99%.

[0038] The term “gene” refers to a DNA segment involved in the production of a polypeptide chain; a gene includes regions before and after the coding region (leader and tail) involved in the transcription / translation and transcription / translation regulation of the gene product, as well as insertion sequences (introns) between individual coding segments (exons).

[0039] “Pharmaceutically acceptable salt” means a salt of the compounds of the present invention that is pharmaceutically acceptable and hasThe desired parent compound has pharmacological activity. In particular, this non-toxic salt can be an inorganic or organic acid addition salt and a base addition salt.

[0040] "Pharmaceutical-acceptable carrier" means a diluent, adjuvant, excipient, or carrier used for administering the compound of the present invention. "Pharmaceutical-acceptable carrier" means a substance that is non-toxic, biologically tolerable, and also biologically suitable for administration to a subject, such as an inert substance, added to or otherwise used to facilitate the administration of the drug, and is compatible with it. Examples of carriers include, but are not limited to, calcium carbonate, calcium phosphate, various sugars and starches, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycol.

[0041] "Decrease" means a negative change of at least 1%, 5%, 10%, 25%, 50%, 75%, or 100%.

[0042] "Increase" means a positive change of at least 1%, 5%, 10%, 25%, 50%, 75%, or 100%.

[0043] As used herein, the terms “determine,” “measure,” “evaluate,” and “test” are used interchangeably and include both quantitative and qualitative determination.

[0044] In some embodiments of the invention, the method includes administering multiple doses of the composition of the invention. The method may include administering a therapeutically effective dose of a composition comprising a compound or composition thereof described herein at frequencies of twice daily, once daily, every other day, three times weekly, once weekly, once monthly, or less. In some embodiments, a dose is administered over a period of 1 week, 2 weeks, 3 weeks, 4 weeks, 2 months, 6 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, or more than 10 years. Furthermore, treating a subject with a therapeutically effective amount of the composition of the invention may include a single treatment or may include a series of treatments. It should also be understood that the effective dose of the compound or composition thereof used for treatment may be increased or decreased during a particular treatment. As is known in the art, variations in dose can be determined by the results of diagnostic tests and are readily apparent. In some embodiments of the invention, the method includes applying the composition once daily to several times daily, including but not limited to twice daily, three times daily, and four times daily.

[0045] In this document, the description of any chemical group listed in the definition of a variant includes the definition of a variant as any single group or combination of the listed groups. In this document, the description of an embodiment of a variant or situation includes an embodiment as any single embodiment or in combination with any other embodiment or part thereof.

[0046] Any composition or method provided herein may be combined with one or more other compositions and methods provided herein.

[0047] Method of the Invention

[0048] In one aspect, the present invention relates to a novel composition for treating amyotrophic lateral sclerosis (ALS) and / or frontotemporal dementia (FTD), comprising: one or more compounds including, but not limited to, G4008, G4009, G4010, G4012, and G4013, or pharmaceutically acceptable salts, solvates, or hydrates thereof; and at least one pharmaceutically acceptable carrier and / or excipient.

[0049] In another aspect, the present invention relates to a method for treating ALS and / or FTD, comprising administering to a subject an effective amount of a composition comprising, but not limited to, one or more compounds including, G4008, G4009, G4010, G4012, and G4013. In some embodiments, one or more compounds of the composition may selectively recognize C9orf72 G4C2 G4 in cells. In a preferred embodiment, administration of the composition to a subject results in a significant reduction, inhibition, or improvement of one or more symptoms of ALS and / or FTD by about 70%. In a preferred embodiment, the progression of ALS and / or FTD is significantly reduced, inhibited, or improved by about 70%.

[0050] In a preferred embodiment, the composition includes, but is not limited to: compounds G4008, G4010, G4012, and G4013, or pharmaceutically acceptable salts, solvates, or hydrates thereof; and at least one pharmaceutically acceptable carrier and / or excipient. In a more preferred embodiment, the composition includes, but is not limited to: compounds G4010 and G4013, or pharmaceutically acceptable salts, solvates, or hydrates thereof; and at least one pharmaceutically acceptable carrier and / or excipient. In the most preferred embodiment, the composition comprises: compound G4013, or pharmaceutically acceptable salts, solvates, or hydrates thereof; and at least one pharmaceutically acceptable carrier and / or excipient. In some embodiments, the composition comprises: compound G4013 as the sole active agent, or a pharmaceutically acceptable salt, solvate, or hydrate thereof; and at least one pharmaceutically acceptable carrier and / or excipient. In some embodiments, the composition comprises, but is not limited to: compound G4013 or a pharmaceutically acceptable salt, solvate, or hydrate thereof; and at least one pharmaceutically acceptable carrier and / or excipient.

[0051] In some embodiments, by administering a composition comprising one or more of compounds G4008, G4010, G4012, and G4013, the number of RNA aggregates in the cells of subjects suffering from ALS and / or FTD is significantly reduced. In some embodiments, the number of aggregates is reduced by at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, or more. In a preferred embodiment, the number of aggregates is reduced by an average of about 50% or more.

[0052] In some embodiments, application of the composition significantly reduces the level of reactive oxygen species (ROS) in the subject's cells.

[0053] In a preferred embodiment, application of G4013 targets C9orf72 G4C2 G4 with high binding affinity, wherein G4013 has a binding affinity (Kd) of about 114 μM for C9orf72 DNA (G4C2)4 G4 and a binding affinity of about 68 μM for C9orf72 RNA (G4C2G4) G4.

[0054] In some embodiments, the subject is a mammal. In a preferred embodiment, the mammal is a primate. In a more preferred embodiment, the primate is a human. In some embodiments, the subject suffers from ALS and / or FTD. Instructions for Use, Page 9 / 17, CN 122376593 A

[0055] In some embodiments, the composition of the present invention can be formulated in a therapeutically effective amount for administration by injection, such as bolus, parenteral administration, intravenous administration, intraperitoneal administration, or continuous infusion, orally, rectally, bronchially, nasally, topically, orally, sublingually, percutaneously, transvaginally, intramuscularly, intraarterially, intracerebrally, or intraocularly, or in a form suitable for inhalation or blowing, including powders and liquid aerosols, or by a semi-permeable membrane matrix of a sustained-release system such as a solid hydrophobic polymer. Administration may also be by other carriers or loading agents, such as patches, micelles, liposomes, vesicles, implants (e.g., microimplants), synthetic polymers, microspheres, nanoparticles, etc.

[0056] Depending on the method of administration, the composition can be administered in various unit dosage forms. Suitable unit dosage forms include, but are not limited to, powders, tablets, pills, capsules, lozenges, suppositories, patches, nasal sprays, injections, implantable sustained-release formulations, lipid complexes, etc.

[0057] In various embodiments, the active agents described herein (e.g., G4008, G4009, G4010, G4012, and G4013) may be combined with pharmaceutically acceptable carriers (excipients) to form a pharmacological composition. In some embodiments, pharmaceutically acceptable carriers include carriers approved by federal or state regulatory agencies or listed in the United States Pharmacopeia or other recognized pharmacopoeias for use in animals, and more particularly for humans. “Carrier” means, for example, a diluent, adjuvant, excipient, auxiliary agent, or transporter used for administering the active agents of the present invention.

[0058] Furthermore, with or without the addition of preservatives, the composition may be present in unit dose form in ampoules, pre-filled syringes, and small-volume infusions, or may be present in multi-dose containers. The composition may be in the form of a suspension, solution, or emulsion in an oily or aqueous carrier. The composition may also contain formulation reagents such as suspending agents, stabilizers, and / or dispersants. In other embodiments, the active ingredient of the composition according to the invention may be in powder form prior to use.The active ingredient is obtained by aseptic separation of a sterile solid or by lyophilization of a solution with a suitable carrier, such as sterile pyrogen-free water.

[0059] The compositions of the present invention may also contain one or more pharmaceutically acceptable carriers and / or excipients and may be formulated into liquid forms, such as solutions or suspensions for injection. The solutions or suspensions may contain suitable non-toxic, parenteral-acceptable diluents or solvents, such as mannitol, 1,3-butanediol, water, Ringer's solution or isotonic sodium chloride solution; or suitable dispersants or wetting agents and suspending agents, such as sterile, non-irritating fixed oils, including synthetic monoglycerides or diglycerides, and fatty acids including oleic acid.

[0060] The carriers and / or excipients according to the invention may include any and all solvents, diluents, buffers (e.g., neutral buffered saline, phosphate buffered saline or optionally Tris-HCl, acetate or phosphate buffer), oil-in-water or water-in-oil emulsions, aqueous compositions suitable for, for example, intravenous use, with or without organic cosolvents, solubilizers (e.g., polysorbate 65, polysorbate 80), colloids, dispersion media, carriers, fillers, chelating agents (e.g., EDTA or glutathione), amino acids (e.g., glycine), proteins, disintegrants, binders, lubricants, wetting agents, emulsifiers, sweeteners, colorants, flavoring agents, aroma agents, thickeners (e.g., carbomer, gelatin or sodium alginate), coatings, preservatives (e.g., thimerosal, benzyl alcohol, polyquaternium), antioxidants (e.g., ascorbic acid, sodium metabisulfite), tension control agents, absorption delay agents, adjuvants, compatibilizers (e.g., lactose, mannitol), etc. The use of carriers and / or excipients in the pharmaceutical and supplement fields is well known. The use of carriers or excipients in the compositions of the present invention may be considered, except for any conventional media or reagents incompatible with the target health-promoting substance or composition.

[0061] Pharmaceutically acceptable carriers may contain one or more physiologically acceptable compounds that act, for example, to stabilize the composition or increase or decrease the absorption of the active agent. Physiologically acceptable compounds may include, for example, carbohydrates such as glucose, sucrose, or dextran; antioxidants such as ascorbic acid or glutathione; chelating agents; low molecular weight proteins; protective and uptake enhancers such as lipids; compositions that reduce the scavenging or hydrolysis of the active agent; or excipients or other stabilizers and / or buffers.

[0062] Other physiologically acceptable compounds include wetting agents, emulsifiers, dispersants, or preservatives specifically designed to inhibit the growth or action of microorganisms. Various preservatives are known and include, for example, phenol and ascorbic acid. Those skilled in the art will understand that the choice of pharmaceutically acceptable carriers, including physiologically acceptable compounds, depends, for example, on the route of administration of the active agent and the specific physicochemical properties of the active agent.

[0063] In one embodiment, the excipient is sterile and generally free of unwanted substances. These compositions can be sterilized using conventionally known sterilization techniques.

[0064] In some therapeutic applications, the compound or composition is therapeutically administered to a subject scheduled to receive chemotherapy in an amount sufficient to reduce and / or inhibit the symptoms, morbidity, severity, and / or progression of the disease. The amount sufficient to achieve this objective is defined as the “therapeutic effective dose.” The effective dose for therapeutic use will depend on the subject’s expected response to the active agent. Single or multiple administrations of the active agent may be used, depending on the dose and frequency required and tolerated by the subject. In any case, treatment should provide an adequate amount of the compound or composition to effectively reduce, inhibit, or improve one or more symptoms and the progression of ASL and / or FTD.

[0065] The concentration / amount of the compound or composition can vary widely and is selected primarily based on the activity of the active ingredient, body weight, etc., according to the specific route of administration chosen and the patient’s needs. However, concentrations typically chosen provide doses ranging from about 0.0005 mg / kg / day to about 1000 mg / kg / day, and sometimes higher doses. Typical dosage ranges are from about 1.0 mg / kg / day to about 250 mg / kg / day, preferably from about 1.0 mg / kg / day to about 200 mg / kg / day, more preferably from about 1.0 mg / kg / day to about 150 mg / kg / day, and most preferably from about 1.0 mg / kg / day to about 100 mg / kg / day. Other dosage ranges are preferably from about 600 mg / kg / day to about 1000 mg / kg / day, more preferably from 600 mg / kg / day to about 750 mg / kg / day; and most preferably from 600 mg / kg / day to about 650 mg / kg / day. It should be understood that such dosages can be varied to optimize the treatment regimen for a particular subject.

[0066] In some instances, the composition is administered at a dose of about 10 mg / kg to about 250 mg / kg of the subject's body weight. In preferred instances, the composition is administered at a dose of about 15 mg / kg to about 200 mg / kg of the subject's body weight. In a more preferred embodiment, the dosage of the composition is from about 25 mg / kg to about 200 mg / kg of the subject's body weight.

[0067] In some embodiments of the invention, the method comprises administering multiple doses of the compound of the invention. The method may comprise administering a therapeutically effective dose of the composition comprising the compound of the invention described herein once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times, fifteen times, twenty times, twenty-five times, thirty times, thirty-five times, forty times or more. In some embodiments, the dosage is for a process of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 14 days, 21 days, 30 days or more than 30 days.The compound may be administered orally, or at a frequency of once a week, once a month, once a quarter, twice a year, once a year, or less. Furthermore, treating a subject with a therapeutically effective amount of the compound of the present invention may comprise a single treatment or may comprise a series of treatments. It will also be understood that the effective dose of the compound used for treatment may be increased or decreased during a particular treatment course. Variations in dose may be determined by and are readily apparent from the results of diagnostic tests or imaging techniques known in the art for detecting the progression of ALS or FTD. In some embodiments of the invention, the method includes administering the compound multiple times daily, including but not limited to twice daily, three times daily, and four times daily. In some embodiments, the compound or composition is administered to the patient for approximately three to five days.

[0068] In some embodiments, at least once daily, three times a week, twice a week, once a week, twice a month, once a month, once a year, or at least every approximately 2 weeks, approximately 3 weeks, approximately 4 weeks, approximately 5 weeks, approximately 6 weeks, approximately 7 weeks, approximately 8 weeks, approximately 9 weeks, approximately 10 weeks, approximately 11 weeks, approximately 12 weeks, approximately 13 weeks, approximately 14 weeks, approximately 15 weeks, approximately 16 weeks, approximately 17 weeks, approximately 18 weeks, approximately 19 weeks, approximately 20 weeks, approximately 21 weeks, approximately 22 weeks, approximately 23 weeks, approximately 24 weeks, approximately 25 weeks, approximately 26 weeks, approximately 52 weeks, approximately 1 year, approximately 2 years, approximately 3 years, approximately 4 years, approximately 5 years, approximately 11 / 17 pages of the specification, 13 CN 122376593 A The composition is repeatedly administered for at least one dose for 6 years, about 7 years, about 8 years, about 9 years, about 10 years, about 11 years, about 12 years, about 13 years, about 14 years, about 15 years, about 16 years, or longer. In some embodiments, the repeated administration of at least one dose of the composition occurs for at least about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 13 weeks, about 14 weeks, about 15 weeks, about 16 weeks, about 17 weeks, about 18 weeks, about 19 weeks, about 20 weeks, about 21 weeks, about 22 weeks, about 23 weeks, about 24 weeks, about 25 weeks, about 26 weeks, about 52 weeks, or longer. In a preferred embodiment, at least one dose of the composition is repeated every about 2 to about 5 days for about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, or about 10 years. In a preferred embodiment, the composition is applied once daily for about 8 weeks to about 52 weeks. In a more preferred embodiment, at least one dose of the composition is repeated every about 2 to about 5 days for about 1 year to about 5 years. Other embodiments of the invention include, but are not limited to, the following experimental methods and results: Materials and Methods

[0069] G4 DNA / RNA Sample Preparation

[0070] DNA / RNA oligonucleotides were purchased from Hunan Aike Rui Biotechnology Co., Ltd. A 100 µM concentration of DNA / RNA sample was placed in a solution containing 20 mM potassium phosphate (pH 10).Annealed in annealing buffer of 7.0) and 70 mM KCl at 97 °C, then slowly cooled to room temperature overnight. For 1D NMR screening, samples were prepared with 5% D2O at a concentration of 50 µM.

[0071] NMR Spectroscopy

[0072] Experiments were performed on Varian spectrometers at 500 MHz and 800 MHz. All samples were diluted from the initial stock solution to 50 µM and the final volume was 400 µL for NMR detection. The imino protons of guanine were identified using 1H NMR spectroscopy and the data were analyzed by MestReNova. NMR titration was performed by adding a compound pre-dissolved in DMSO-d6 at a stock concentration of 100 mM.

[0073] One-Dimensional 1H-NMR Titration Experiment

[0074] To verify whether the compound interacts with G4 DNA / RNA, we performed NMR titration experiments at 25 °C by running one-dimensional (1D) 1H NMR spectra. All compounds were dissolved in isotopically labeled d6-DMSO at a concentration of approximately 100 mM as a stock solution. To avoid chemical shift changes in G4 DNA / RNA due to the addition of d6-DMSO, 10 μL or 15 μL of d6-DMSO was added to 400 μL of 0.1 mM G4 DNA / RNA NMR buffer (20 mM potassium phosphate buffer and 70 mM KCl, pH 7.0, 10% D2O), and then 1D 1H-NMR spectra were performed. In NMR titration experiments where each compound was titrated into the G4 DNA / RNA solution, the maximum volume of d6-DMSO solution for each compound, 10 μL or 15 μL, was considered the final data point.

[0075] Isothermal Titration Calorimetry (ITC)

[0076] ITC is commonly used to quantitatively analyze the interaction between two molecules. It provides valuable information, such as the binding stoichiometry, kinetics, and affinity of complex formation, through association and dissociation constants. Malvern Panalytical MicroCal PEAQ-ITC is used to determine the affinity between ligands and DNA / RNA. Since the cell volume is 280 µL, sample preparation requires at least 400 µL, while the ligand used as a titrant requires 40 µL. The molar ratio depends on the specific case and typically starts at 10–20:1 (titer:cells). All samples must be kept under the same buffer conditions (including additives). A default titration procedure is typically 18 injections, with each injection titrating 2 µL (the first injection being 0.4 µL).

[0077] Cell Culture

[0078] Neuro2a cells were initially seeded onto glass coverslips using 12-well plates. Lipofectamine 3000 (Thermo Fisher Scientific) was then used.(Addgene) Co-transfected the DNA plasmid pHR-Tre3G-29xGGGGCC-12xMS2 (Addgene # 99149) and pCAG-TetON-3G plasmid (Addgene # 96963) into cells. Following the manufacturer's protocol, 1 µg of plasmid was transfected per well (page 12 / 17, CN 122376593 A). The plasmid was incubated for over 10 hours for expression and induced overnight at 37°C with doxycycline (1000 ng / mL).

[0079] Transfection

[0080] The DNA construct was transfected at 7 DIV using Lipofectamine 2000 (Thermo Fisher Scientific, 11668019) or Lipofectamine LTX with Plus reagent (Thermo Fisher Scientific, 15338100). 4 to 6 h after transfection, the transfection medium was replaced with fresh medium, and the cells were incubated for another 48 h (HT22) or longer (primary neurons; 5 d to 8 d) to allow for recovery and construct expression.

[0081] MTT assay

[0082] The cytotoxicity levels of different concentrations of the compound were determined using the MTT assay (3-[4,5-dimethylthiazolyl-2-yl]-2,5-diphenyltetrazole bromide) to determine cell growth inhibition (IC50) (see van Meerloo J, Kaspers GJ, Cloos J. Cell sensitivity assays: the MTT assay. Methods Mol Biol, 2011; 731:237-45. doi: 10.1007 / 978-1-61779-080-5_20. PMID: 21516412). Mitochondrial activity of cells can be reflected by converting tetrazolium salt into formazan crystals, which are then further dissolved in DMSO for assay. First, 5 × 10³ HT22 cells were seeded in 96-well plates and cultured overnight. Then, the cells were treated with different concentrations of the added compound and diluted overnight in culture medium. 10 µL of MTT (5 mg / ml) diluted in PBS buffer was added to each well, and the plates were incubated at 37°C for 4 hours. Then, 100 µL of DMSO was added to each well to dissolve the thoroughly pre-washed crystals. The absorbance was then recorded at 570 nm using a CLARIOstar PLUS microplate reader (BMG LABTECH). IC50 was then analyzed using a GraphPad Prism.

[0083] Reactive Oxygen Spectroscopy (ROS) Detection

[0084] HT22 cells were prepared in 96-well plates in DMEM medium supplemented with 5 mM L-glutamate and incubated overnight at 37°C. Then, following the manufacturer's (Beyotime) instructions, the ROS detection kit was used to test reactive oxygen species levels. 10 µM of the DCFH-DA fluorescent probe was added to the cells, followed by pre-washing with DMEM-only medium at 37°C for 30 minutes, gently agitated every 5 minutes. The cells were then washed three times with DMEM-only medium to remove excess probe. Subsequently, the appropriate compound (2 µM) and control (Rosup: 50 µg / mL) were added, and incubation was performed for one hour. The fluorescence intensity of the oxidation product, dichlorofluorescein (DCF), was measured using a CLARIOstar PLUS microplate reader (BMG LABTECH) with a 488 / 525 nm filter.

[0085] Drosophila strains

[0086] (G4C2) n-repetitive sequence related Drosophila strains and eye development expression promoters were obtained from the Bloomington Drosophila Stock Centre (BDSC), while wild-type strains such as w (CS10) were kindly provided by Professor Yukinori's research group. All Drosophila were reared at 25°C and humidity controlled between 56% and 60%. Drosophila food was prepared one day before the addition of DMSO control and 100 µM of various promising compounds. Then, on the second day, parental Drosophila were crossbred on solidified food containing the test compounds, and larvae were also fed food containing the compounds until emergence.

[0087] Eye imaging using bright-field microscopy

[0088] All emerging adult Drosophila that were photographed were anesthetized with CO2 and on day 5 were photographed using a camera (Canon EOS Kiss X7 dual zoom kit) on a microscope (Nikon SMZ 745T) to capture the condition of the entire eye, which was then examined by scanning electron microscopy. Then, the level of eye degeneration was examined accordingly and collected separately.

[0089] Eye imaging using scanning electron microscopy (SEM)

[0090] The fruit flies isolated in the previous step were further treated with 2.5% glutaraldehyde and fixed overnight at 4°C. Then, the fruit flies were dehydrated sequentially in a gradient of acetone for 12 hours each day (30%, 50%, 70%, 90%, 95%, 100% twice). The dehydrated fruit flies were then air-dried and the compound eyes were dissected using an optical microscope. Finally, the compound eyes were gold-plated before SEM (JSM-6390). Specification 13 / 17 pages 15 CN 122376593 A

[0091] All patents, patent applications, provisional applications and publications referenced or cited herein are incorporated herein by reference in their entirety, including all figures and tables, unless they contradict the express teachings of this specification.

[0092] The following are examples illustrating the process of carrying out the invention. These examples should not be construed as limiting. Unless otherwise stated, all percentages are by weight and all solvent mixture proportions are by volume.

[0093] Example 1—Design of a new compound based on chrexanthomycin A

[0094] ALS is a fatal degenerative neurological disease that is currently incurable. Abnormal amplification of hexanucleotides (G4C2)n (n ranging from 30 to several thousand) in the first non-coding region of the C9orf72 gene is the most common genetic cause of ALS / FTD. The C9orf72 DNA / RNA G4C2 repeat sequence can fold into the secondary structure G4. Several studies have shown that C9orf72 G4C2 G4 could be a novel drug target for small molecule therapy of ALS (see references Z. Su, Y. Zhang, TF Gendron, PO Bauer, J. Chew, W.Y. Yang, E. Fostvedt, K. Jansen-West, V.V. Belzil, P. Desaro, A. Johnston, K. Overstreet, S.Y. Oh, P.K. Todd, J.D. Berry, M.E. Cudkowicz, BF Boeve, D. Dickson, MK Floeter, BJ Traynor, C. Morelli, A. Ratti, V. Silani, R. Rademakers, RH Brown, JD Rothstein, KB Boylan, L. Petrucelli, MD Disney, Discovery of a Biomarker and Lead Small Molecules to Target r(GGGGCC)‑Associated Defects in c9FTD / ALS, Neuron, 84 (2014) 239; R. Simone, P. Fratta, S. Neidle, G.N. Parkinson, AM Isaacs, G‑quadruplexes: Emerging roles in neurodegenerative diseases and the non‑coding transcriptome, FEBS Lett, 589 (2015)1653–1668; A. Cheng, C. Liu, W. Ye, D. Huang, W. She, X. Liu, CP Fung, N. Xu, MC Suen, W. Ye, HHY Sung, ID Williams, G. Zhu, PY. Qian, Selective C9orf72 G-Quadruplex-Binding Small Molecules Ameliorate Pathological Signatures of ALS / FTD Models, J Med Chem, 65 (2022) 12825–12837). In our previous study, we identified the marine-derived natural product chrexa nthomycin A, which specifically recognizes C9orf72 DNA / RNA G4C2G4. However, chrexanthomycin A exhibits weak binding affinity, with Kd values ​​of ~ 2.2 ± 0.1 mM, 3.0 ± 0.1 mM, and 2.8 ± 0.1 mM. To develop lead compounds with high selectivity, specificity, and affinity for targeting C9orf72 DNA / RNA G4C2G4, we designed and synthesized six small molecules (G4008 to G4013) based on the structure of chrexanthomycin A. These are derivatives of 7H-pyridocarbazole and 7H-pyrazinocarbazole, and contain four fused rings (Figure 1).

[0095] Example 2—NMR titration of these designed compounds using C9orf72 DNA / RNA G4C2 G4

[0096] It is well known that the C9orf72 DNA G4C2 repeat sequence can fold into G4, including both antiparallel and forward parallel forms (see YY Geng, CD Liu, QX Cai, ZP Luo, HT Miao, X. Shi, N.N. Xu, C.P. Fung, T.T. Choy, B. Yan, N. Li, PY. Qian, B. Zhou, G. Zhu, Crystal structure of parallel G-quadruplex formed by the two-repeat ALS- and FTD-related GGGGCC sequence, Nucleic Acids Res, 49 (2021).5881-5890; B. Zhou, C. Liu, Y. Geng, G. Zhu, Topology of a G-quadruplex DNA formed by C9orf72 hexanucleotide repeats associated with ALS and FTD, Sci Rep, 5 (2015) 16673). We performed NMR titration on these compounds using C9orf72 DNA / RNA G4C2 G4. For the C9orf72 DNA G4C2 sequence, NMR titration was performed using d(G4C2)4. As shown in Figure 2, compounds G4008, G4009, G4010, G4012, and G4013 showed significant chemical shift changes in the imino proton region, indicating that these compounds bind to C9orf72 d(G4C2)4 (SEQ ID NO: 1). Instructions 14 / 17 pages 16 CN 122376593 A

[0097] For the C9orf72 RNA G4C2 sequence, by using r(G4C2G4) (SEQ ID NO: 2) for NMR titration, r(G4C2G4) can fold into a forward parallel G4 form (see reference YY Geng, CD Liu, NN Xu, MC Suen, HT Miao, YY Xie, BC Zhang, XQ Chen, YJ Song, ZX Wang, QX Cai, G. Zhu, Crystal structure of a tetrameric RNA G-quadruplex formed by hexanucleotide repeat expansions of C9orf72 in ALS / FTD, Nucleic Acids Res, 52 (2024) 7961-7970). As shown in Figure 3, five of the six compounds, namely G4008, G4009, G4010, G4012, and G4013, showed significant chemical shift changes in the imino proton region, indicating that these compounds bind to C9orf72r (G4C2G4). Further MTT assay for cytotoxicity showed that G4011 did not exhibit associated cytotoxicity.

[0098] Example 3—Compounds are able to rescue symptoms associated with the C9orf72G4C2 repeat sequence at the cellular level

[0099] Cytotoxicity is important for determining whether cells can withstand and tolerate high concentrations of drug treatment without causing significantCell death is crucial. To assess the cytotoxicity of these six novel compounds, the MTT assay was applied to HT22 cells, an immortalized mouse hippocampal neuronal cell line, to determine cell viability (Figure 4A). Initially, a high concentration of 200 µM was applied, followed by five consecutive dilutions to obtain a total of six data points (0.064 µM, 0.32 µM, 1.6 µM, 8 µM, 40 µM, 200 µM). The calculated IC50 values, ranked from highest to lowest toxicity, were as follows: G4008 (2.5 µM) > G4013 (10 µM) > G4009 (63 µM) > G4010 (79.4 µM) > G4012 (199.5 µM) > G4011 (no value). Compound G4011 showed no detectable cytotoxicity even at concentrations up to 200 µM, therefore no inhibitory concentration was expected.

[0100] It has been reported that in ALS / FTD, C9-mutant astrocytes produce insufficient antioxidants, leading to loss of motor neuron defense capabilities and increased oxidative stress, ultimately resulting in neurodegeneration (see A. Birger, I. Ben-Dor, M. Ottolenghi, T. Turetsky, Y. Gil, S. Sweetat, L. Perez, V. Belzer, N. Casden, D. Steiner, M. Izrael, E. Galun, E. Feldman, O. Behar, B. Reubinoff, Human iPSC-derived astrocytes from ALS patients with mutated C9ORF72 show increased oxidative stress and neurotoxicity, EBioMedicine, 50 (2019) 274-289). Therefore, it is crucial to test whether these compounds have the ability to protect neurons from oxidative stress. Comparing the positive control (Rosup) with increased oxidative stress levels to the negative control without drug treatment, all six compounds showed a significant reduction in glutamate-induced cellular ROS levels (Figure 4B). Overall, we selected G4008, G4009, G4010, G4012, and G4013 for further in vivo testing in a cell model expressing the C9orf72 (G4C2)29 sequence.

[0101] Because NMR titration studies revealed the interaction between C9orf72 DNA / RNA G4C2 G4 and these compounds.Therefore, it was assumed that these compounds had similar biological activities. Thus, a plasmid encoding the C9orf72 (G4C2)29 DNA sequence (SEQ ID NO:3) was transfected into Neuro2a cells used as an ALS / FTD cell model. The amplified G4C2 repeat sequence can lead to ALS / FTD by isolating RNA-binding proteins (RBPs) and forming nuclear RNA G4C2 aggregates, which recruit specific RBPs (such as hnRNP H). Based on NMR titration results combined with cell viability assays (MTT assays), compounds G4008, G4009, G4010, G4012, and G4013 were selected for RNA FISH experiments to determine their biological activity against RNA G4C2 aggregates via immunostaining (Figure 5A).

[0102] As shown in Figure 5B, the number of RNA aggregates was significantly reduced in cells treated overnight with G4008, G4010, G4012, and G4013. Considering the IC50 value in the MTT assay, we ultimately used G4010 and G4013 to test the bioactivity of a Drosophila model expressing the C9orf72 (G4C2)49 sequence.

[0103] Example 4—Compounds can rescue symptoms associated with the C9orf72G4C2 repeat sequence in Drosophila

[0104] In addition, we tested the bioactivity of G4010, G4012, and G4013 on C9orf72 ALS Drosophila. Therefore, the GMR-GAL4-(G4C2)49 Drosophila was engineered to express (G4C2)49 RNA only in the eye. A Drosophila model of C9-ALS expressing the pathogenic 49-repetitive (G4C2)49 sequence confirmed that the amplified G4C2 repeat sequence induced eye degeneration (Figure 6B). During the larval stage, mutant and wild-type (WT) Drosophila were fed solid food containing compounds G4010 and G4013 at an effective concentration of 100 μM. In external eye experiments, we observed dark spots on the eyes of 7-day-old adult GMR-GAL4-(G4C2)49 Drosophila, which were much rougher than those of age-matched WT Drosophila, indicating eye degeneration. Interestingly, G4013 treatment made the eyes smoother and more reflective (Fig. 6A), significantly inhibiting eye degeneration in (G4C2)49 Drosophila and thus significantly treating the structurally disordered eye phenotype. In summary, these data indicate that G4013 rescues the eye defects in Drosophila caused by the G4C2 repeat sequence (Figs. 6A and 6B). However, G4010 failed to treat eye degeneration. Notably, ITC experiments showed that G4010 and G4013 are associated with C9orf72 DNA (G4C2) 4G4 and C9orf72 RNA (G4C2G4).The binding affinity of G4 is approximately 100 times stronger than that of chrexanthomycin A, with Kd values ​​of approximately 46 μM and 114 μM for G4010 and G4013 with C9orf72 DNA (G4C2)4 G4, respectively; and Kd values ​​of approximately 35 μM and 68 μM for G4010 and G4013 with C9orf72 RNA (G4C2G4) G4, respectively (Figure 7).

[0105] Therefore, this invention confirms the ability of these compounds to bind to specific targets of C9orf72 G4C2 amplification and to rescue pathological features in ALS / FTD cells and Drosophila models, where C9orf72 G4C2 amplification is the most common genetic cause of ALS. All these data indicate that G4013 can recognize C9orf72 G4C2 G4 with high binding affinity and rescue Drosophila eye defects caused by G4C2 repetitive sequences.

[0106] It should be understood that the embodiments and implementations described herein are for illustrative purposes only, and various modifications or changes will be apparent to those skilled in the art, and such modifications or changes will be included within the spirit and scope of this application and the scope of the appended claims. Furthermore, any element or limitation of any invention or implementation thereof disclosed herein may be combined with any and / or all other elements or limitations disclosed herein (alone or in any combination) or any other invention or implementation thereof, and all such combinations are within the scope of the invention but not limited thereto.

[0107] Exemplary Embodiments

[0108] Embodiment 1. A pharmaceutical composition comprising: one or more compounds selected from the group consisting of G4008, G4009, G4010, G4012 and G4013, or a pharmaceutically acceptable salt, solvate or hydrate thereof; and at least one pharmaceutically acceptable carrier and / or excipient.

[0109] Embodiment 2. A method of treating a subject with amyotrophic lateral sclerosis (ALS) and / or frontotemporal dementia (FTD), the method comprising administering to the subject an effective amount of the composition according to Embodiment 1.

[0110] Embodiment 3. The method according to Embodiment 2, wherein the composition comprises G4008, G4010, G4012, or G4013.

[0111] Embodiment 4. The method according to Embodiment 2, wherein the composition comprises G4010 or G4013.

[0112] Embodiment 5. The method according to Embodiment 2, wherein the composition comprises compound G4013.

[0113] Embodiment 6. The method according to Embodiments 2 to 5, wherein the composition is applied once, twice, three times, four times, or five times per week.

[0114] Embodiment 7. The method according to Embodiments 2 to 6, wherein the composition is administered at a dose of about 0.1 mg / kg to about 200 mg / kg.Administered at a dose of mg / kg.

[0115] Embodiment 8. The method according to Embodiments 2 to 7, wherein the composition is administered locally via intravitreal, intracranial, or intradiscal administration, or systemically via intramuscular, intravascular (e.g., intravenous), intraoral, intradermal, intranasal, intrathecal, or subcutaneous administration.

[0116] Embodiment 9. The method according to Embodiments 2 to 8, wherein the subject is a human. Specification 16 / 17 pages 18 CN 122376593 A

[0117] Embodiment 10. The method according to Embodiments 2 to 9, wherein one or more compounds of the composition selectively target and bind to the G-quadruplex (G4) formed by the hexanucleotide repeat sequence (HRE) (G4C2)n of C9orf72.

[0118] Embodiment 11. The method according to Embodiments 2 to 10, wherein administration of the composition improves one or more symptoms of ASL and / or FTD by about 70%.

[0119] Embodiment 12. The method according to Embodiments 2 to 10, wherein application of the composition reduces the progression of ASL and / or FTD by about 70%.

[0120] Embodiment 13. The method according to Embodiment 3, wherein the number of RNA aggregates is significantly reduced by application of a composition comprising one or more compounds selected from the group consisting of G4008, G4010, G4012, and G4013.

[0121] Embodiment 14. The method according to Embodiment 13, wherein the number of RNA aggregates is reduced by about 50%.

[0122] Embodiment 15. The method according to Embodiments 2 to 14, wherein application of the composition reduces the reactive oxygen species (ROS) level of the subject by about 40%.

[0123] Embodiment 16. The method according to Embodiment 5, wherein G4013 is applied to target C9orf72 G4C2 G4 with high binding affinity.

[0124] Embodiment 17. The method according to Embodiment 16, wherein the binding affinity (Kd) of G4013 to C9orf72 DNA (G4C2)4 G4 is about 114 μM.

[0125] Embodiment 18. The method according to Embodiment 16, wherein the binding affinity (Kd) of G4013 to C9orf72 RNA (G4C2G4) G4 is about 68 μM.

[0126] Embodiment 19. The method according to Embodiment 2, wherein the composition comprises G4013 as the sole active agent. Specification 17 / 17 pages 19 CN 122376593 A Figure 1 Specification Drawings 1 / 10 pages 20 CN 122376593 A Figure 2 Specification Drawings 2 / 10 pages 21CN 122376593 A Figure 3, Appendix 3 / 10, Page 22 CN 122376593 A Figure 4A, Appendix 4 / 10, Page 23 CN 122376593 A Figure 4B, Appendix 5 / 10, Page 24 CN 122376593 A Figure 5A, Appendix 6 / 10, Page 25 CN 122376593 A Figure 5B, Appendix 7 / 10, Page 26 CN 122376593 A Figure 6A, Appendix 8 / 10, Page 27 CN 122376593 A Figure 6B, Appendix 9 / 10, Page 28 CN 122376593 A Figure 7, Appendix 10 / 10, Page 29 CN 122376593 A Abstract The subject invention pertains to a composition comprising a series of compounds selectively targeting G-quadruplexes (G4s) formed by the GGGGCC (G4C2) hexanucleotide repeats (HREs) of C9orf72, (G4C2)n. The composition comprises compounds G4008, G4009, G4010, G4012, and G4013, or a pharmaceutically acceptable salt, solvate, or hydrate thereof and at least one pharmaceutically acceptable carrier and / or excipient. The subject invention further pertains to a method for treating amyotrophic Lateral sclerosis (ASL) and / or frontotemporal dementia (FTD), comprising administering to the subject an effective amount of the composition. The compounds canameliorate the pathological signatures of ALS model caused by C9orf72 (G4C2)n sequence.

Claims

1. A pharmaceutical composition comprising: one or more compounds selected from the group consisting of G4008, G4009, G4010, G4012 and G4013, or a pharmaceutically acceptable salt, solvate or hydrate thereof; and at least one pharmaceutically acceptable carrier and / or excipient.

2. A method for treating a subject with amyotrophic lateral sclerosis and / or frontotemporal dementia, the method comprising administering to the subject an effective amount of the composition according to claim 1.

3. The method of claim 2, wherein the composition comprises G4008, G4010, G4012 or G4013.

4. The method of claim 2, wherein the composition comprises G4010 or G4013.

5. The method of claim 2, wherein the composition comprises compound G4013.

6. The method of claim 2, wherein the composition is applied once, twice, three times, four times, or five times per week.

7. The method of claim 2, wherein the composition is administered at a dose of about 0.1 mg / kg to about 200 mg / kg.

8. The method of claim 2, wherein the composition is administered locally via intravitreal, intracranial, or intradiscal application, or systemically via intramuscular, intravascular (e.g., intravenous), intraoral, intradermal, intranasal, intrathecal, or subcutaneous application.

9. The method of claim 2, wherein the subject is a human.

10. The method of claim 2, wherein one or more compounds of the composition selectively target and bind to... C9orf72 The G-quadruplex (G4) is formed by the hexanucleotide repeat sequence (G4C2)n of GGGGGCC (G4C2).

11. The method of claim 2, wherein the application of said composition improves one or more symptoms of ASL and / or FTD by about 70%.

12. The method of claim 2, wherein the application of the composition reduces the progress of ASL and / or FTD by about 70%.

13. The method of claim 3, wherein the number of RNA aggregates is significantly reduced by applying a composition comprising one or more compounds selected from the group consisting of G4008, G4010, G4012 and G4013.

14. The method of claim 13, wherein the number of RNA aggregates is reduced by about 50%.

15. The method of claim 2, wherein the administration of the composition reduces the reactive oxygen species (ROS) level of the subject by about 40%.

16. The method of claim 2, wherein G4013 is applied with high binding affinity to target C9orf72 G4C2G4.

17. The method of claim 16, wherein G4013 and C9orf72 DNA (G4C2)4 G4 binding affinity ( K d The value is approximately 114 μM.

18. The method of claim 16, wherein G4013 and C9orf72 RNA (G4C2G4) G4 binding affinity ( K d The value is approximately 68 μM.

19. The method of claim 2, wherein the composition comprises G4013 as the sole active agent.

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