[go: up one dir, main page]

WO2025106527A1 - Compositions et méthodes pour le traitement de l'ataxie de friedreich - Google Patents

Compositions et méthodes pour le traitement de l'ataxie de friedreich Download PDF

Info

Publication number
WO2025106527A1
WO2025106527A1 PCT/US2024/055696 US2024055696W WO2025106527A1 WO 2025106527 A1 WO2025106527 A1 WO 2025106527A1 US 2024055696 W US2024055696 W US 2024055696W WO 2025106527 A1 WO2025106527 A1 WO 2025106527A1
Authority
WO
WIPO (PCT)
Prior art keywords
frataxin
peptide
sequence
levels
tid1l
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.)
Pending
Application number
PCT/US2024/055696
Other languages
English (en)
Inventor
David R. Lynch
Yina DONG
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.)
Childrens Hospital of Philadelphia CHOP
Original Assignee
Childrens Hospital of Philadelphia CHOP
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 Childrens Hospital of Philadelphia CHOP filed Critical Childrens Hospital of Philadelphia CHOP
Publication of WO2025106527A1 publication Critical patent/WO2025106527A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22

Definitions

  • an isolated peptide comprising an amino acid sequence KRSTGN of SEQ ID NO: 1 operably linked to a synthetic functional sequence
  • the synthetic functional sequence is a cell penetrating peptide sequence, a tag sequence which facilitates purification of said peptide, or a linker sequence.
  • the synthetic functional sequence is a tat sequence and said peptide comprises KRSTGNYGRKKRRQRRR of SEQ ID NO: 2.
  • a pharmaceutical composition comprising a purified peptide and a pharmaceutically acceptable carrier, wherein the purified peptide comprises SEQ ID NO: 2.
  • the peptide in the pharmaceutical composition can comprise at least 1, 2, or 3 modified amino acids.
  • the pharmaceutical compositions described above are formulated for delivery across the blood brain bander.
  • nucleic acid sequence that encodes SEQ ID NOS: 1 or SEQ ID NO: 2, or an siRNA targeting TID1S, present in a pharmaceutically acceptable carrier is provided.
  • the nucleic acid can be contained within a vector which is optionally attached to, complexed with, or encapsulated by, a micro or nanoparticle.
  • the invention also provides a method of treating Friedreich’s ataxia (FDRA) and related mitochondrial dysfunction caused by frataxin deficiency in a subject in need thereof.
  • An exemplary method comprises administering an effective amount of the composition or carrier described above, wherein said administration reduces one or more symptoms of FDRA.
  • Symptoms to be alleviated include one or more of trouble walking, fatigue, loss of sensation that starts in the legs and spreads to the arms and trunk, loss of reflexes, slow or slurred speech, hearing loss, vision loss, chest pain, shortness of breath, heart palpitations and diabetes symptoms.
  • the method can further comprise administering a second therapeutic agent that treats or inhibits progress of FDRA.
  • the composition or carrier is formulated to cross the blood brain barrier and administration is via an intracranial, intraparenchymal, ccrcbro-ventricular, or transmeningeal route.
  • An exemplary method comprises determining levels of one or more of cellular parameters associated with FRDA in a biological sample from the subject, comparing said levels of said one or more parameters to levels observed i) prior to administration of the pharmaceutical composition or carrier, or ii) after administration of the pharmaceutical composition or carrier, wherein identification of agents which alter levels of one or more cellular parameters selected from of frataxin level, TID1 level, TID1S level, TID1L level, mitochondrial fragmentation level, mitochondrial dysfunction level, reactive oxygen species production level, and lipid peroxidation level, having efficacy for the treatment of FRDA and related mitochondrial disease.
  • FIGs 1A -IF Tumorous imaginal disc 1 (TID1) physically interacts with frataxin both in vivo in mouse cortex and in vitro in cortical neurons.
  • TID1L/S antibody In both cortical homogenates (Fig. 1A) and neuronal lysates (Fig. IB), frataxin was immunoprecipitated by a TID1L/S antibody but not control IgG.
  • *P ⁇ 0.05, **P ⁇ 0.0L Data were shown as mean ⁇ SE.
  • FIGS 3A - 3H TID1L protein levels are reduced in FRDA patient-derived cells.
  • FRDA patient buccal cells, skin fibroblasts, platelets or PBMCs were lysed and subjected to Western blotting with the indicated antibodies. The amount of immunoreactivity in the lysates was quantified as a percentage of the controls.
  • FIGS 4A - 4C Effect of TID1 overexpression on frataxin protein levels.
  • HEK293 cells transfected with frataxin and TID1 plasmid DNAs were lysed and subjected to Western blotting with the indicated antibodies. The amount of immunoreactivity in the lysates was quantified as a percentage of vector control.
  • FIG. 5A - 5C Mechanistic study of TID1 overexpression-caused changes in frataxin levels.
  • HEK293 cells transfected with frataxin and TID1 plasmid DNAs were subject to mitochondria fractionation followed by Western blotting analysis.
  • Frataxin precursor is predominantly localized in the mitochondrial fraction upon TID1L or TID1S overexpression (Fig. 5A).
  • Tom20 was used as a mitochondrial marker (Fig. 5A).
  • Treatment with MG132 (10 (1M) also had no effect on TID1L or TID1S overexpression-caused decrease in intermediate and mature frataxin (Fig. 5B).
  • Frataxin G130V mutant transfected HEK293 cells were used as a positive control (B). Neither TID1L nor TID1S interacted with MPP, in contrast to GRP75 (Fig. 5C).
  • FIGS 6A -6D TID1S overexpression decreases mature frataxin in human skin fibroblasts.
  • Human skin fibroblasts from healthy individuals were transduced with lentivirus carrying pHAGE- TID IS gene or vector control for 5 days before Western blotting or immunofluorescence.
  • TID1S transduction also caused mitochondria fractionation in fibroblasts (Fig. 6C and Fig. 6D).
  • Vector control was stained with an anti-GFP antibody and TID1S was stained with an anti-TIDlL/S antibody. *P ⁇ 0.05, **P ⁇ 0.01. Data were shown as mean+SE.
  • FIGS 7A- 7C TIDlS448-453Tat rescues frataxin deficiency and mitochondrial defect in FRD A patient-derived skin fibroblasts.
  • FRD A patient skin fibroblasts were treated with TIDlS448-453Tat and sTID1448-453Tat for 24 hours followed by Western blotting analysis or immunofluorescence.
  • Mitotracker was used to identify mitochondria. *P ⁇ 0.05, **P ⁇ 0.01. Data were shown as mean ⁇ SE.
  • FRDA Friedreich’s ataxia
  • GAA guanine-adenine-adenine
  • TID1 Tumorous imaginal disc 1
  • DNAJA3 DnaJ homolog subfamily A member 3, mitochondrial (DNAJA3)
  • Hsp heat shock protein
  • TID1 interacts with the Hsp70 family of chaperone proteins through its distinctive J domain, a highly conserved tctrahclical region, which increases their ATPasc activity for substrate binding and functions as a cochaperone and regulatory component for Hsp70 (Silver and Way, 1993; Hendrick et al, 1993, Liu T., 2012, Syken et al., 1999, Trentin et al., 2001). TID1 also affects cell survival, proliferation, and responses to stress (Lo et al., 2004; Syken et al., 2003; Chen 2009; Cheng 2016). TID1 null mutations are lethal (Lo et al., 2004).
  • TID1 encodes two mitochondrial matrix localized splice variants, TIDl-long (TID1L, 43 kDa) and -short (TID1S, 40 kDa), which differ only at their carboxyl termini (Lu et al., 2006).
  • TID1L and TID1S are distinct.
  • TID1S interacts with the agrin receptor and is involved in neuromuscular transmission
  • TID1L connects with the von Hippel-Lindau protein and is implicated in tumor suppression (Linnoila et al., 2008).
  • TID1L enhances external stimulus-induced apoptosis
  • TID1S suppresses it (Syken et al., 1999).
  • Our findings show that TID1 negatively modulates frataxin levels, thereby providing a novel therapeutic target for treating FRDA and modulating symptoms thereof.
  • FDRA FRDA
  • FDRA FRDA
  • FDRA a recessive genetic disorder caused by defects in the frataxin gene, FXN.
  • Physical symptoms include, without limitation, trouble walking, fatigue, loss of sensation that starts in the legs and spreads to the aims and trunk, loss of reflexes, slow or slurred speech, healing loss, vision loss, chest pain, shortness of breath, heart palpitations and in some case, diabetes symptoms.
  • Many FDRA patients develop scoliosis or foot deformities over time, which often require surgery.
  • FXN Fluxin (FXN) is a highly conserved protein required for efficient regulation of cellular iron homeostasis. Frataxin deficiency is associated with the cardio and neurodegenerative symptoms of FDRA, commonly resulting from a GAA trinucleotide repeat expansion in the frataxin gene.
  • Frataxin has been proposed to participate in at least five different capacities: 1) as an iron chaperone during cellular heme and iron-sulfur (Fc-S) cluster production; 2) as an iron-storage protein during conditions of iron overload; 3) as an aid in the repair of oxidatively damaged aconitase Fe-S clusters; 4) as a factor that controls cellular oxidative stress by moderating the concentration of reactive oxygen species (ROS); and finally 5) as an active participant in pathways involving energy conversion and oxidative phosphorylation.
  • ROS reactive oxygen species
  • pharmacological activity refers to the inherent physical properties of a peptide or polypeptide. These properties include but are not limited to half-life, solubility, and stability and other pharmacokinetic properties.
  • test compound refers to a chemical to be tested by one or more screening method(s) as a putative modulator.
  • a test compound can be any chemical, such as an inorganic chemical, an organic chemical, a protein, a peptide, a nucleic acid, a carbohydrate, a lipid, or a combination thereof.
  • various predetermined concentrations of test compounds are used for screening, such as 0.01 micromolar, 1 micromolar and 10 micromolar.
  • Test compound controls can include the measurement of a signal in the absence of the test compound or comparison to a compound known to modulate the target.
  • the terms “high,” “higher,” “increases,” “elevates,” or “elevation” refer to increases above basal levels, e.g., as compared to a control.
  • the terms “low,” “lower,” “reduces,” or “reduction” refer to decreases below basal levels, e.g., as compared to a control.
  • modulate refers to the ability of a compound to change an activity in some measurable way as compared to an appropriate control.
  • activities can increase or decrease as compared to controls in the absence of these compounds.
  • an increase in activity is at least 25%, more preferably at least 50%, most preferably at least 100% compared to the level of activity in the absence of the compound.
  • a decrease in activity is preferably at least 25%, more preferably at least 50%, most preferably at least 100% compared to the level of activity in the absence of the compound.
  • a compound that increases a known activity is an “agonist”.
  • One that decreases, or prevents, a known activity is an “antagonist”.
  • preventing refers to administering a compound prior to the onset of clinical symptoms of a disease or conditions so as to prevent a physical manifestation of aberrations associated with the disease or condition.
  • in need of treatment refers to a judgment made by a caregiver (e.g. physician, nurse, nurse practitioner, or individual in the case of humans; veterinarian in the case of animals, including non-human mammals) that a subject requires or will benefit from treatment. This judgment is made based on a variety of factors that are in the realm of a care giver's expertise, but that includes the knowledge that the subject is ill, or will be ill, as the result of a condition that is treatable by the disclosed compounds.
  • a caregiver e.g. physician, nurse, nurse practitioner, or individual in the case of humans; veterinarian in the case of animals, including non-human mammals
  • treatment and “treating” is meant the medical management of a subject with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder, particularly FDRA.
  • This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder.
  • this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.
  • palliative treatment that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder
  • preventative treatment that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder
  • supportive treatment that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.
  • the effects of treatment can be measured or assessed as described herein and as known in the art as is suitable FDRA and its associated pathologies.
  • a cell can be in vitro.
  • a cell can be in vivo and can be found in a subject.
  • a “cell” can be a cell from any organism.
  • the cells are human cells.
  • an effective amount of a compound as provided herein is meant a nontoxic but sufficient amount of the compound to provide the desired result.
  • pharmaceutically acceptable is meant a material that is not biologically or otherwise undesirable, i.e., the material can be administered to a subject along with the selected compound without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.
  • “Inhibitory biological macromolecules” include peptides, peptide/DNA complexes, siRNA, shRNA, antisense oligonucleotides, and any nucleic acid-based molecule which encoded the proteins described herein.
  • a “derivative” of a polypeptide, polynucleotide or fragments thereof means a sequence modified by varying the sequence of the construct, e.g., by manipulation of the nucleic acid encoding the protein or by altering the protein itself. “Derivatives” of a gene or nucleotide sequence refers to any isolated nucleic acid molecule that contains significant sequence similarity to the gene or nucleotide sequence or a part thereof. In addition, “derivatives” include such isolated nucleic acids containing modified nucleotides or mimetics of naturally-occurring nucleotides.
  • nucleic acid refers to any DNA or RNA molecule, either single or double stranded and, if single stranded, the molecule of its complementary sequence in either linear or circular form.
  • nucleic acid molecules a sequence or structure of a particular nucleic acid molecule may be described herein according to the normal convention of providing the sequence in the 5' to 3' direction.
  • isolated nucleic acid refers primarily to an RNA molecule encoded by an isolated DNA molecule as defined above. Alternatively, the term may refer to an RNA molecule that has been sufficiently separated from other nucleic acids with which it would be associated in its natural state (i.c., in cells or tissues). An isolated nucleic acid (cither DNA or RNA) may further represent a molecule produced directly by biological or synthetic means and separated from other components present during its production.
  • a “specific binding pair” comprises a specific binding member (sbm) and a binding partner (bp) which have a particular specificity for each other and which in normal conditions bind to each other in preference to other molecules.
  • specific binding pairs are antigens and antibodies, biotin and streptavidin, ligands and receptors and complementary nucleotide sequences.
  • FXN-TID1 would be considered a specific binding pair.
  • the term “specific binding pair” is also applicable where either or both of the specific binding member and the binding partner comprise a part of a large molecule.
  • delivery refers to the introduction of foreign molecule (i.e., miRNA encoding the polypeptide of interest) into cells.
  • administration means the introduction of a foreign molecule into a cell.
  • delivery means the introduction of a foreign molecule into a cell. The term is intended to be synonymous with the term “delivery”.
  • “reduce” or “inhibit” refers to the ability to cause an overall decrease of 50% or greater. In yet another embodiment, “reduce” or “inhibit” refers to the ability to cause an overall decrease of 75%, 85%, 90%, 95%, or greater.
  • the invention includes the peptide inhibitor sequences disclosed herein as well as sequences which are at least 80%, 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical thereto which retain the ability to treat or ameliorate symptoms associated with FDRA and other neurodegenerative diseases.
  • Aliphatic Amino Acid refers to a nonpolar amino acid having a saturated or unsaturated straight chain, branched or cyclic hydrocarbon side chain.
  • Examples of genetically encoded aliphatic amino acids include Ala, Leu, Vai, and He.
  • Examples of non-encoded aliphatic amino acids include Nle.
  • Acidic Amino Acid refers to a hydrophilic amino acid having a side chain pK value of less than 7. Acidic amino acids typically have negatively charged side chains at physiological pH due to loss of a hydrogen ion. Examples of genetically encoded acidic amino acids include aspartic acid (aspartate) and glutamic acid (glutamate).
  • Basic Amino Acid refers to a hydrophilic amino acid having a side chain pK value of greater than 7.
  • Basic amino acids typically have positively charged side chains at physiological pH due to association with hydronium ion.
  • genetically encoded basic amino acids include arginine, lysine and histidine.
  • non-genetically encoded basic amino acids include ornithine, 2,3-diaminopropionic acid, 2,4-diaminobutyric acid, and homoarginine.
  • Ionizable Amino Acid refers to an amino acid that can be charged at a physiological pH.
  • Such ionizable amino acids include acidic and basic amino acids, for example, D-aspartic acid, D-glutamic acid, D-histidine, D-arginine, D-lysine, D-hydroxylysine, D-omithine, L- aspartic acid, L-glutamic acid, L-histidine, L-arginine, L-lysine, L-hydroxylysine, or L-ornithine.
  • tyrosine has both a nonpolar aromatic ring and a polar hydroxyl group.
  • tyrosine has several characteristics that could be described as nonpolar, aromatic and polar.
  • the nonpolar ring is dominant and so tyrosine is generally considered to be nonpolar.
  • cysteine also has nonpolar character.
  • cysteine can be used to confer hydrophobicity or nonpolarity to a peptide.
  • polar amino acids contemplated by the present invention include, for example, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, histidine, homocysteine, lysine, hydroxylysine, ornithine, serine, threonine, and structurally related amino acids.
  • the polar amino is an ionizable amino acid such as arginine, aspartic acid, glutamic acid, histidine, hydroxylysine, lysine, or ornithine.
  • polar or nonpolar amino acid residues examples include, for example, alanine, valine, leucine, methionine, isoleucine, phenylalanine, tryptophan, tyrosine, and the like.
  • variant refers to a nucleic acid sequence or polypeptide comprising a sequence, which differs (by deletion, insertion, and/or substitution of a nucleic acid or amino acid, an L or D stereoisomer an amino acid, or a non-naturally occurring amino acid) in one or more nucleic acid or amino acid positions differ from that of a wild type nucleic acid or polypeptide sequence.
  • linker refers to a connection between two protein coding sequences or their protein products.
  • Linkers comprise a stretch of contiguous nucleic acids or amino acids, which holds at least one cleavage site that enables separation of the genes or their products through cleavage of the linker.
  • the linker comprises a cleavage site at its 5' end and a cleavage site at its 3' end, or a cleavage site at its N-terminal end and a cleavage site at its C-terminal end.
  • the peptide may be fused to biotin, Poly-lysine, lysozyme, Green fluorescent protein (and derivatives), SUMO or other desired proteinaceous tags.
  • Production of the desired peptide sequence can be carried out in E.coli, , SF9, Pichia, etc., using existing technologies, e.g. with protein fusion tags that can either be removed or left as desired.
  • the peptide of interest may be fused via a linker.
  • the peptide can be fused to one or more cell penetrating peptides (CPP) which are useful for facilitating delivery of the peptide into target cells.
  • CPP cell penetrating peptides
  • TAT peptide is exemplified herein
  • CPPs include without limitation, penetratin (RQIKIWFQNRRMKWKK) (SEQ ID NO: 4), VP22 peptide (DAATATRGRSAASRPTER PRAPARSASRPRRVD) (SEQ ID NO: 6), MAP (KLALKLALKALKAALKLA-amidc) (SEQ ID NO: 7), Transportin (GWTLNSAGYLLGKINLKALAALAKKIL-amide) (SEQ ID NO: 8) R7 (RRRRRRR) (SEQ ID NO: 9), MPG (GALFLGWLGAAGSTMGAPKKRKV) (SEQ ID NO: 10), and Pep-1 (KETWWETWWTEWSQPKKKRKV) (SEQ ID
  • the peptide can be expressed as a fusion to larger proteins, facilitating expression at large scales, ease of purification, and ensuring quality of product.
  • Expression systems can also be leveraged to generate large sequence libraries, allowing for directed evolution for targeted properties.
  • Peptides can be produced sustainably using environmentally friendly, existing fermentation technologies.
  • codon optimization refers to changing the codons of a nucleotide sequence without altering the amino acid sequence that it encodes in order to favor expression in a specific species. Codon optimization may be used to increase the abundance of the peptide or protein that the nucleotide sequence encodes since “rare” codons are removed and replaced with abundant codons.
  • Peptides can be synthesized chemically cither in solution or on a solid phase.
  • the process involves directed and selective formation of an amide bond between an N-protected amino acid and an amino acid bearing a free amino group and protected carboxylic acid.
  • the carboxyl protecting group is linked to a polymer support. Following bond formation, the amino-protecting group of the dipeptide is removed, and the next N-protected amino-acid is coupled.
  • Solid-phase peptide synthesis is the most frequently used method of peptide synthesis due to its efficiency, simplicity, speed, and ease of parallelization.
  • SPPS involves sequential addition of amino and side-chain protected amino acid residues to an amino acid or peptide attached to an insoluble polymeric support.
  • Either an acid-labile Boc group (Boc SPPS) or base-labile Fmoc-group (Fmoc SPPS) is used for N-a-protection. After removal of this protecting group, the next protected amino acid is added using either a coupling reagent or preactivated protected amino acid derivative.
  • the C-terminal amino acid is anchored to the resin via a linker, the nature of which determines the conditions required to release the peptide from the support after chain extension.
  • Side-chain protecting groups are often chosen so as to be cleaved simultaneously with detachment of the peptide from the resin.
  • Peptides of 50 amino acids can be routinely prepared although the synthesis of proteins of over 100 amino acids are commonly reported. Longer proteins can be made by native chemical ligation of fully deprotected peptides in solution. With this method, it is possible to synthesize natural peptides that are difficult to express in bacteria, to incorporate unnatural or D-amino acids, and to generate cyclic, branched, labelled, and post-translationally modified peptides.
  • Liquid-phase peptide synthesis has been superseded by SPPS except for existing processes of large-scale synthesis of peptides for industrial purposes. Desired sequences can be developed by any one of the several commercial entities who provide this service for a fee, including Sigma Aldrich, and Avivasysbio for example.
  • peptide mimetics can be generated that mimic the blocking peptide described herein.
  • a mimetic in this context refers to a substance which has some chemical similarity to SEQ ID NO: 1, but which disrupts FXN - TID1 binding complex formation or activity.
  • a mimetic may be a carbohydrate or peptide or chemical molecule that mimics elements of secondary structure (Johnson et al., "Peptide Turn Mimetics” in Biotechnology and Pharmacy, Pezzuto et al., Eds., Chapman and Hall, New York, 1993).
  • a mimetic is designed to permit molecular interactions similar to the natural molecule.
  • Peptide or non-peptide mimetics may be useful, for example, to inhibit the deleterious effects on FXN levels mediated by FXN-TID1 complex formation.
  • the designing of mimetics to a pharmaceutically active compound is a known approach to the development of pharmaceuticals based on a "lead" compound. This might be desirable where the active compound is difficult or expensive to synthesize or where it is unsuitable for a particular method of administration.
  • Mimetic design, synthesis and testing are generally used to avoid randomly screening large numbers of molecules for a desired property.
  • the three-dimensional structure of the ligand and its binding partner are modeled. This can be especially useful where the ligand and/or binding partner change conformation on binding, allowing the model to take account of this in the design of the mimetic. Modeling can be used to generate inhibitors which interact with the linear sequence or a three-dimensional configuration.
  • a template molecule is then selected onto which chemical groups which mimic the pharmacophore can be grafted.
  • the template molecule and the chemical groups grafted onto it can conveniently be selected so that the mimetic is easy to synthesize, is likely to be pharmacologically acceptable, and does not degrade in vivo, while retaining the biological activity of the lead compound.
  • the mimetic is peptide-based
  • further stability can be achieved by cyclizing the peptide, increasing its rigidity.
  • the mimetic or mimetics found by this approach can then be screened to see whether they have the target property, or to what extent they exhibit it. Further optimization or modification can then be carried out to arrive at one or more final mimetics for in vivo or clinical testing.
  • a transgenic cell may be obtained by introducing a recombinant nucleic acid molecule that encodes a protein of this disclosure.
  • the term “recombinant nucleic acid” refers to a polynucleotide that is manipulated by human intervention.
  • a recombinant nucleic acid molecule can contain two or more nucleotide sequences that are linked in a manner such that the product is not found in a cell in nature.
  • the two or more nucleotide sequences can be operatively linked and, for example, can encode a fusion polypeptide.
  • a recombinant nucleic acid molecule also can be based on, but manipulated so as to be different, from a naturally occurring polynucleotide, for example, a polynucleotide having one or more nucleotide changes such that a first codon, which normally is found in the polynucleotide, is biased for chloroplast codon usage, or such that a sequence of interest is introduced into the polynucleotide, for example, a restriction endonuclease recognition site or a splice site, a promoter, a DNA origin of replication, or the like.
  • Any appropriate technique for introducing recombinant nucleic acid molecules into cells may be used.
  • Techniques for nuclear’ and chloroplast transformation include, without limitation, electroporation, biolistic transformation (also referred to as micro- projectile/particle bombardment), agitation in the presence of glass beads, and Agrobacteriumbased transformation.
  • the term “construct” refers to recombinant polynucleotides including, without limitation, DNA and RNA, which may be single-stranded or double- stranded and may represent the sense or the antisense strand.
  • Recombinant polynucleotides are polynucleotides formed by laboratory methods that include polynucleotide sequences derived from at least two different natural sources or they may be synthetic. Constructs thus may include new modifications to endogenous genes introduced by, for example, genome editing technologies. Constructs may also include recombinant polynucleotides created using, for example, recombinant DNA methodologies.
  • RNA interference double-stranded RNA
  • siRNA double-stranded RNA
  • dsRNA double-stranded RNA
  • ds siRNAs double-stranded small interfering RNAs
  • RNAi can be triggered by 21-nucleotide (nt) duplexes of small interfering RNA (siRNA) (Chiu et al., Mol. Cell. 10:549-561 (2002); Elbashir et al., Nature 411 :494-498 (2001)), or by micro- RNAs (miRNA), functional small-hairpin RNA (shRNA), or other dsRNAs which can be expressed in vivo using DNA templates with RNA polymerase III promoters (Zeng et al., Mol. Cell 9:1327-1333 (2002); Paddison et al., Genes Dev.
  • siRNA small interfering RNA
  • shRNA functional small-hairpin RNA
  • the inhibitory nucleic acid is an siRNA.
  • the inhibitory nucleic acid has 100% sequence identity with at least a part of the target mRNA.
  • inhibitory nucleic acids having 70%, 80% or greater than 90% or 95% sequence identity may be used.
  • sequence variations that might be expected due to genetic mutation, strain polymorphism, or evolutionary divergence can be tolerated.
  • siRNA specific for TID 1 are described herein.
  • Each strand is composed of 18-20 RNA bases and two DNA bases overhang on the 3’ terminus.
  • Dharmacon, Inc. (Lafayette, CO) provides siRNA duplexes using the 2’-ACE RNA synthesis technology.
  • Qiagen (Valencia, CA) uses TOM-chemistry to offer siRNA with high individual coupling yields (Li, et al., Nat. Med., 11(9):944-951 (2005).
  • the TID1 inhibitor is an antisense oligonucleotide.
  • An “antisense” nucleic acid sequence can include a nucleotide sequence that is complementary to a “sense” nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to the TID1 mRNA.
  • Antisense nucleic acid sequences and delivery methods are well known in the art (Goodchild , Curr. Opin. Mol.
  • An antisense nucleic acid can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art.
  • the antisense nucleic acid also can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest).
  • the TID1 inhibitor is a ribozyme specific for a TID1 protein.
  • Ribozymes are a type of RNA that can be engineered to enzymatically cleave and inactivate other RNA targets in a specific, sequence-dependent fashion. Ribozymes and methods for their delivery are well known in the art (Hendry, et al., BMC Chem. Biol., 4(1): 1 (2004); Grassi, et al., Curr. Pharm. Biotechnol., 5(4):369-386 (2004); Bagheri, et al., Curr. Mol. Med., 4(5):489-506 (2004); Kashani-Sabet M., Expert Opin. Biol.
  • Ribozymes By cleaving the target RNA, ribozymes inhibit translation, thus preventing the expression of the target gene. Ribozymes can be chemically synthesized in the laboratory and structurally modified to increase their stability and catalytic activity using methods known in the ail. Alternatively, ribozyme genes can be introduced into cells through gene-delivery mechanisms known in the art.
  • the TID1 inhibitor is a Triplex forming nucleic acid.
  • Triplex forming nucleic acid molecules are molecules that can interact with either double-stranded or singlestranded nucleic acid. When triplex molecules interact with a target region, a structure called a triplex is formed, in which there are three strands of DNA forming a complex dependent on both Watson-Crick and Hoogsteen base-pairing. Triplex molecules are preferred because they can bind target regions with high affinity and specificity. It is preferred that the triplex forming molecules bind the target molecule with a Kd less than 10-6, 10-8, 10-10, or 10-12. Examples of how to make and use triplex forming molecules to bind a variety of different target molecules are known in the art.
  • a “vector” is capable of transferring gene sequences to target cells.
  • vector construct means any nucleic acid construct capable of directing the expression of a gene of interest and which can transfer gene sequences to target cells.
  • vector transfer vector mean any nucleic acid construct capable of directing the expression of a gene of interest and which can transfer gene sequences to target cells.
  • the term includes cloning and expression vehicles, as well as integrating vectors.
  • constructs and expression cassettes provided herein may include a promoter operably linked to any one of the polynucleotides described herein but need not have a promoter and may be used for homologous recombination into the cell.
  • the constructs may include a promoter and the promoter may be a heterologous promoter or an endogenous promoter associated with the polypeptide.
  • heterologous promoter refers generally to transcriptional regulatory regions of a gene, which may be found at the 5' or 3' side of the polynucleotides described herein, or within the coding region of the polynucleotides, or within introns in the polynucleotides.
  • a promoter is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence.
  • the typical 5' promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background.
  • a transcription initiation site (conveniently defined by mapping with nuclease SI), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.
  • the disclosed polynucleotides are operably connected to the promoter.
  • a polynucleotide is “operably connected” or “operably linked” when it is placed into a functional relationship with a second polynucleotide sequence.
  • a promoter is operably linked to a polynucleotide if the promoter is connected to the polynucleotide such that it may affect transcription of the polynucleotides.
  • Heterologous promoters useful in the practice of the present invention include, but are not limited to, constitutive, inducible, temporally-regulated, developmentally regulated, chemically regulated, tissue-pref erred and tissue-specific promoters.
  • the heterologous promoter may be a plant, animal, bacterial, fungal, or synthetic promoter.
  • novel FXN-TID1 binding complex inhibitors described herein are preferably formulated such that they can cross the blood brain barrier e.g., via intracranial injection, microinfusion etc., and thereby modulate deleterious reductions in FXN levels in cells in the brain.
  • they can be formulated for enteral, parenteral, topical, or pulmonary administration.
  • the compounds can be combined with one or more pharmaceutically acceptable carriers and/or excipients that are considered safe and effective and may be administered to an individual without causing undesirable biological side effects or unwanted interactions.
  • the carrier is all components present in the pharmaceutical formulation other than the active ingredient or ingredients. See, e.g., Remington's Pharmaceutical Sciences, latest edition, by E.W. Martin Mack Pub.
  • compositions that can be used in conjunction with the preparation of formulations of the compounds described herein and which is incorporated by reference herein. These most typically would be standard carriers for administration of compositions to humans.
  • humans and non-humans including solutions such as sterile water, saline, and buffered solutions at physiological pH.
  • Other compounds will be administered according to standard procedures used by those skilled in the art.
  • compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like.
  • parenteral administration may include administration to a patient intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly, intrapro statically, intrapleurally, intratracheally, intravitreally, intratumorally, intramuscularly, subcutaneously, subconjunctivally, intravesicularly, intrapericardially, intraumbilically, by injection, and by infusion.
  • Parenteral formulations can be prepared as aqueous compositions using techniques known in the art.
  • compositions can be prepared as injectable formulations, for example, solutions or suspensions; solid forms suitable for using to prepare solutions or suspensions upon the addition of a reconstitution medium prior to injection; emulsions, such as water-in-oil (w/o) emulsions, oil-in-water (o/w) emulsions, and microemulsions thereof, liposomes, or emulsomes.
  • injectable formulations for example, solutions or suspensions
  • solid forms suitable for using to prepare solutions or suspensions upon the addition of a reconstitution medium prior to injection emulsions, such as water-in-oil (w/o) emulsions, oil-in-water (o/w) emulsions, and microemulsions thereof, liposomes, or emulsomes.
  • the compositions are packaged in solutions of sterile isotonic aqueous buffer.
  • the composition may also include a solubilizing agent.
  • the components of the composition are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or concentrated solution in a hermetically sealed container such as an ampoule or sachet indicating the amount of active agent.
  • the composition can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampoule of sterile water or saline can be provided so that the ingredients may be mixed prior to injection.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, one or more polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), oils, such as vegetable oils (e.g., peanut oil, corn oil, sesame oil, etc.), and combinations thereof.
  • polyols e.g., glycerol, propylene glycol, and liquid polyethylene glycol
  • oils such as vegetable oils (e.g., peanut oil, corn oil, sesame oil, etc.)
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and/or by the use of surfactants.
  • isotonic agents for example, sugars or sodium chloride.
  • Solutions and dispersions of the active compounds as the free acid or base or pharmacologically acceptable salts thereof can be prepared in water or another solvent or dispersing medium suitably mixed with one or more pharmaceutically acceptable excipients including, but not limited to, surfactants, dispersants, emulsifiers, pH modifying agents, viscosity modifying agents, and combination thereof.
  • Suitable surfactants may be anionic, cationic, amphoteric or nonionic surface-active agents.
  • Suitable anionic surfactants include, but are not limited to, those containing carboxylate, sulfonate and sulfate ions.
  • anionic surfactants include sodium, potassium, ammonium of long chain alkyl sulfonates and alkyl aryl sulfonates such as sodium dodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodium dodecylbenzene sulfonate; dialkyl sodium sulfosuccinates, such as sodium bis-(2-cthylthioxyl)-sulfosuccinate; and alkyl sulfates such as sodium lauryl sulfate.
  • Cationic surfactants include, but are not limited to, quaternary ammonium compounds such as benzalkonium chloride, benzethonium chloride, cetrimonium bromide, stearyl dimethylbenzyl ammonium chloride, polyoxyethylene, and coconut amine.
  • nonionic surfactants include ethylene glycol monostearate, propylene glycol myristate, glyceryl monostearate, glyceryl stearate, polyglyceryl-4-oleate, sorbitan acylate, sucrose acylate, PEG- 150 laurate, PEG-400 monolaurate, polyoxyethylene monolaurate, polysorbates, polyoxyethylene octylphenylether, PEG- 1000 cetyl ether, polyoxyethylene tridecyl ether, polypropylene glycol butyl ether, Poloxamer® 401, stearoyl monoisopropanolamide, and polyoxyethylene hydrogenated tallow amide.
  • amphoteric surfactants include sodium N-dodecyl-.beta.-alanine, sodium N-lauryl-P- iminodipropionate, myristoamphoacetate, lauryl betaine, and lauryl sulfobetaine.
  • the formulation can contain a preservative to prevent the growth of microorganisms. Suitable preservatives include, but are not limited to, parabens, chlorobutanol, phenol, sorbic acid, and thimerosal.
  • the formulation may also contain an antioxidant to prevent degradation of the active agent(s).
  • the formulation is typically buffered to a pH of 3-8 for parenteral administration upon reconstitution.
  • Suitable buffers include, but are not limited to, phosphate buffers, acetate buffers, and citrate buffers.
  • Water-soluble polymers are often used in formulations for parenteral administration. Suitable water-soluble polymers include, but are not limited to, polyvinylpyrrolidone, dextran, carboxymethylcellulose, and polyethylene glycol.
  • Sterile injectable solutions can be prepared by incorporating the active compounds in the required amount in the appropriate solvent or dispersion medium with one or more of the excipients listed above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those listed above.
  • the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the powders can be prepared in such a manner that the particles are porous in nature, which can increase dissolution of the particles. Methods for making porous particles arc well known in the art.
  • parenteral formulations described herein can be formulated for controlled release including immediate release, delayed release, extended release, pulsatile release, and combinations thereof.
  • the one or more compounds, and optional one or more additional active agents can be incorporated into microparticles, nanoparticles, or combinations thereof that provide controlled release of the compounds and/or one or more additional active agents.
  • the formulations contain two or more drugs
  • the drugs can be formulated for the same type of controlled release (e.g., delayed, extended, immediate, or pulsatile) or the drugs can be independently formulated for different types of release (e.g., immediate and delayed, immediate and extended, delayed and extended, delayed and pulsatile, etc.).
  • Polymers which are slowly soluble and form a gel in an aqueous environment, such as hydroxypropyl methylcellulose or polyethylene oxide, can also be suitable as materials for drug containing microparticles.
  • Other polymers include, but are not limited to, poly anhydrides, poly(ester anhydrides), polyhydroxy acids, such as polylactide (PLA), polyglycolide (PGA), poly(lactide-co-glycolide) (PLGA), poly-3-hydroxybutyrate (PHB) and copolymers thereof, poly-4-hydroxybutyrate (P4HB) and copolymers thereof, polycaprolactone and copolymers thereof, and combinations thereof.
  • PLA polylactide
  • PGA polyglycolide
  • PLGA poly(lactide-co-glycolide)
  • PHB poly-4-hydroxybutyrate
  • P4HB polycaprolactone and copolymers thereof, and combinations thereof.
  • the drug(s) can be incorporated into microparticles prepared from materials which are insoluble in aqueous solution or slowly soluble in aqueous solution, but are capable of degrading within the GI tract by means including enzymatic degradation, surfactant action of bile acids, and/or mechanical erosion.
  • slowly soluble in water refers to materials that are not dissolved in water within a period of 30 minutes. Preferred examples include fats, fatty substances, waxes, wax-like substances and mixtures thereof.
  • Suitable fats and fatty substances include fatty alcohols (such as lauryl, myristyl stearyl, cetyl or cetostearyl alcohol), fatty acids and derivatives, including but not limited to fatty acid esters, fatty acid glycerides (mono-, di- and tri-glycerides), and hydrogenated fats.
  • fatty alcohols such as lauryl, myristyl stearyl, cetyl or cetostearyl alcohol
  • fatty acids and derivatives including but not limited to fatty acid esters, fatty acid glycerides (mono-, di- and tri-glycerides), and hydrogenated fats.
  • Specific examples include, but are not limited to hydrogenated vegetable oil, hydrogenated cottonseed oil, hydrogenated castor oil, hydrogenated oils available under the trade name Sterotex®, stearic acid, cocoa butter, and stearyl alcohol.
  • Suitable waxes and wax-like materials include natural or synthetic waxes, hydrocarbons, and normal wax
  • waxes include beeswax, glycowax, castor wax, carnauba wax, paraffins and candelilla wax.
  • a wax-like material is defined as any material, which is normally solid at room temperature and has a melting point of from about 30 to 300°C.
  • rate-controlling (wicking) agents can be formulated along with the fats or waxes listed above.
  • rate-controlling materials include certain starch derivatives (e.g., waxy maltodextrin and drum dried corn starch), cellulose derivatives (e.g., hydroxypropylmethyl-cellulose, hydroxypropylcellulose, methylcellulose, and carboxymethylcellulose), alginic acid, lactose and talc.
  • a pharmaceutically acceptable surfactant for example, lecithin may be added to facilitate the degradation of such microparticles.
  • Proteins which are water insoluble, such as zein, can also be used as materials for the formation of drug containing microparticles. Additionally, proteins, polysaccharides and combinations thereof, which are water-soluble, can be formulated with drug into microparticles and subsequently cross-linked to form an insoluble network. For example, cyclodextrins can be complexed with individual drug molecules and subsequently cross-linked.
  • Encapsulation or incorporation of the peptide mimetic or inhibitory oligonucleotide into carrier materials to produce inhibitor-containing microparticles can be achieved through known pharmaceutical formulation techniques.
  • the carrier material is typically heated above its melting temperature and the mimetic or drug is added to form a mixture comprising drug particles suspended in the carrier material, drug dissolved in the carrier material, or a mixture thereof.
  • Microparticles can be subsequently formulated through several methods including, but not limited to, the processes of congealing, extrusion, spray chilling or aqueous dispersion.
  • wax is heated above its melting temperature, drug is added, and the molten wax-drug mixture is congealed under constant stirring as the mixture cools.
  • the molten wax-drug mixture can be extruded and spheronized to form pellets or beads.
  • a solvent evaporation technique to produce drug-containing microparticles.
  • drug and carrier material are co-dissolved in a mutual solvent and microparticles can subsequently be produced by several techniques including, but not limited to, forming an emulsion in water or other appropriate media, spray drying or by evaporating off the solvent from the bulk solution and milling the resulting material.
  • the particles can also be coated with one or more modified release coatings.
  • Solid esters of fatty acids which are hydrolyzed by lipases, can be spray coated onto microparticles or drug particles.
  • Zein is an example of a naturally water-insoluble protein. It can be coated onto drug containing microparticles or drug particles by spray coating or by wet granulation techniques.
  • some substrates of digestive enzymes can be treated with cross-linking procedures, resulting in the formation of non-soluble networks.
  • Many methods of cross-linking proteins initiated by both chemical and physical means, have been reported. One of the most common methods to obtain cross-linking is the use of chemical crosslinking agents.
  • cross-linking agents examples include aldehydes (gluteraldehyde and formaldehyde), epoxy compounds, carbodiimides, and genipin.
  • aldehydes gluteraldehyde and formaldehyde
  • epoxy compounds carbodiimides
  • genipin examples include aldehydes (gluteraldehyde and formaldehyde), epoxy compounds, carbodiimides, and genipin.
  • oxidized and native sugars have been used to cross-link gelatin.
  • Cross-linking can also be accomplished using enzymatic means; for example, transglutaminase has been approved as a GRAS substance for cross-linking seafood products.
  • cross-linking can be initiated by physical means such as thermal treatment, UV irradiation and gamma irradiation.
  • a water-soluble protein can be spray coated onto the microparticles and subsequently cross-linked by the one of the methods described above.
  • drug-containing microparticles can be microencapsulated within protein by coacervation-phase separation (for example, by the addition of salts) and subsequently crosslinked.
  • suitable proteins for this purpose include gelatin, albumin, casein, and gluten.
  • Polysaccharides can also be cross-linked to form a water-insoluble network. For many polysaccharides, this can be accomplished by reaction with calcium salts or multivalent cations, which cross-link the main polymer chains. Pectin, alginate, dextran, amylose and guar’ gum are subject to cross-linking in the presence of multivalent cations. Complexes between oppositely charged polysaccharides can also be formed; pectin and chitosan, for example, can be complexed via electrostatic interactions.
  • the peptide mimetics or inhibitory nucleic acids described herein can be incorporated into inj ectable/implan table solid or semi-solid implants, such as polymeric implants.
  • the compounds are incorporated into a polymer that is a liquid or paste at room temperature, but upon contact with aqueous medium, such as physiological fluids, exhibits an increase in viscosity to form a semi-solid or solid material.
  • Exemplary polymers include, but are not limited to, hydroxyalkanoic acid polyesters derived from the copolymerization of at least one unsaturated hydroxy fatty acid copolymerized with hydroxyalkanoic acids. The polymer can be melted, mixed with the active substance and cast or injection molded into a device.
  • melt fabrication requires polymers having a melting point that is below the temperature at which the substance to be delivered and polymer degrade or become reactive.
  • the device can also be prepared by solvent casting where the polymer is dissolved in a solvent and the drug dissolved or dispersed in the polymer solution and the solvent is then evaporated. Solvent processes require that the polymer be soluble in organic solvents.
  • Another method is compression molding of a mixed powder of the polymer and the drug or polymer particles loaded with the active agent.
  • the compounds can be incorporated into a polymer matrix and molded, compressed, or extruded into a device that is a solid at room temperature.
  • the compounds can be incorporated into a biodegradable polymer, such as polyanhydrides, polyhydroalkanoic acids (PHAs), PLA, PGA, PLGA, polycaprolactone, polyesters, polyamides, poly orthoesters, polyphosphazenes, proteins and polysaccharides such as collagen, hyaluronic acid, albumin and gelatin, and combinations thereof and compressed into solid device, such as disks, or extruded into a device, such as rods.
  • PHAs polyhydroalkanoic acids
  • PLA polyhydroalkanoic acids
  • PGA PGA
  • PLGA polycaprolactone
  • polyesters polyamides
  • poly orthoesters polyphosphazenes
  • proteins and polysaccharides such as collagen, hyaluronic acid, albumin and gelatin
  • the release of the one or more compounds from the implant can be varied by selection of the polymer, the molecular weight of the polymer, and/or modification of the polymer to increase degradation, such as the formation of pores and/or incorporation of hydrolyzable linkages.
  • Methods for modifying the properties of biodegradable polymers to vary the release profile of the compounds from the implant are well known in the ait.
  • Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, sodium saccharine, starch, magnesium stearate, cellulose, magnesium carbonate, etc.
  • Such compositions will contain a therapeutically effective amount of the compound and/or antibiotic together with a suitable amount of carrier so as to provide the proper form to the patient based on the mode of administration to be used.
  • Suitable oral dosage forms include tablets, capsules, solutions, suspensions, syrups, and lozenges. Tablets can be made using compression or molding techniques well known in the ail. Gelatin or non-gelatin capsules can prepared as hard or soft capsule shells, which can encapsulate liquid, solid, and semi-solid fill materials, using techniques well known in the art.
  • Formulations may be prepared using a pharmaceutically acceptable carrier.
  • carrier includes, but is not limited to, diluents, preservatives, binders, lubricants, disintegrators, swelling agents, fillers, stabilizers, and combinations thereof.
  • Carrier also includes all components of the coating composition, which may include plasticizers, pigments, colorants, stabilizing agents, and glidants.
  • suitable coating materials include, but are not limited to, cellulose polymers such as cellulose acetate phthalate, hydroxypropyl cellulose, hydroxypropyl mcthylccllulosc, hydroxypropyl methylcellulose phthalate and hydroxypropyl methylcellulose acetate succinate; polyvinyl acetate phthalate, acrylic acid polymers and copolymers, and methacrylic resins that are commercially available under the trade name EUDRAGIT® (Roth Pharma, Westerstadt, Germany), zein, shellac, and polysaccharides.
  • EUDRAGIT® Roth Pharma, Westerstadt, Germany
  • the coating material may contain conventional carriers such as plasticizers, pigments, colorants, glidants, stabilization agents, pore formers and surfactants.
  • “Diluents”, also referred to as “fillers,” are typically necessary to increase the bulk of a solid dosage form so that a practical size is provided for compression of tablets or formation of beads and granules.
  • Suitable diluents include, but are not limited to, dicalcium phosphate dihydrate, calcium sulfate, lactose, sucrose, mannitol, sorbitol, cellulose, microcrystalline cellulose, kaolin, sodium chloride, dry starch, hydrolyzed starches, pregelatinized starch, silicone dioxide, titanium oxide, magnesium aluminum silicate and powdered sugar.
  • Binders are used to impart cohesive qualities to a solid dosage formulation, and thus ensure that a tablet or bead or granule remains intact after the formation of the dosage forms.
  • Suitable binder materials include, but are not limited to, starch, pregelatinized starch, gelatin, sugars (including sucrose, glucose, dextrose, lactose and sorbitol), polyethylene glycol, waxes, natural and synthetic gums such as acacia, tragacanth, sodium alginate, cellulose, including hydroxypropylmethylcellulose, hydroxypropylcellulose, ethylcellulose, and veegum, and synthetic polymers such as acrylic acid and methacrylic acid copolymers, methacrylic acid copolymers, methyl methacrylate copolymers, aminoalkyl methacrylate copolymers, poly aery lie acid/poly methacrylic acid and polyvinylpyrrolidone.
  • Lubricants are used to facilitate tablet manufacture.
  • suitable lubricants include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, glycerol behenate, polyethylene glycol, talc, and mineral oil.
  • Disintegrants are used to facilitate dosage form disintegration or "breakup" after administration, and generally include, but are not limited to, starch, sodium starch glycolate, sodium carboxymethyl starch, sodium carboxymethylcellulose, hydroxypropyl cellulose, pregelatinized starch, clays, cellulose, alginine, gums or cross linked polymers, such as crosslinked PVP (Polyplasdone® XL from GAF Chemical Corp).
  • Stabilizers are used to inhibit or retard drug decomposition reactions, which include, by way of example, oxidative reactions.
  • Suitable stabilizers include, but arc not limited to, antioxidants, butylated hydroxytoluene (BHT); ascorbic acid, its salts and esters; Vitamin E, tocopherol and its salts; sulfites such as sodium metabisulphite; cysteine and its derivatives; citric acid; propyl gallate, and butylated hydroxyanisole (BHA).
  • BHT butylated hydroxytoluene
  • ascorbic acid its salts and esters
  • Vitamin E tocopherol and its salts
  • sulfites such as sodium metabisulphite
  • cysteine and its derivatives citric acid
  • propyl gallate propyl gallate
  • BHA butylated hydroxyanisole
  • Oral dosage forms such as capsules, tablets, solutions, and suspensions, can for formulated for controlled release.
  • the one or more compounds and optional one or more additional active agents can be formulated into nanoparticles, microparticles, and combinations thereof, and encapsulated in a soft or hard gelatin or non-gelatin capsule or dispersed in a dispersing medium to form an oral suspension or syrup.
  • the particles can be formed of the drug and a controlled release polymer or matrix.
  • the drug particles can be coated with one or more controlled release coatings prior to incorporation in to the finished dosage form.
  • the one or more compounds and optional one or more additional active agents are dispersed in a matrix material, which gels or emulsifies upon contact with an aqueous medium, such as physiological fluids.
  • aqueous medium such as physiological fluids.
  • the matrix swells entrapping the active agents, which are released slowly over time by diffusion and/or degradation of the matrix material.
  • Such matrices can be formulated as tablets or as fill materials for hard and soft capsules.
  • the one or more compounds, and optional one or more additional active agents are formulated into a sold oral dosage form, such as a tablet or capsule, and the solid dosage form is coated with one or more controlled release coatings, such as a delayed release coatings or extended release coatings.
  • the coating or coatings may also contain the compounds and/or additional active agents.
  • the extended-release formulations are generally prepared as diffusion or osmotic systems, which are known in the ail.
  • a diffusion system typically consists of two types of devices, a reservoir and a matrix, and is well known and described in the art.
  • the matrix devices are generally prepared by compressing the drug with a slowly dissolving polymer carrier into a tablet form.
  • the three major types of materials used in the preparation of matrix devices are insoluble plastics, hydrophilic polymers, and fatty compounds.
  • Plastic matrices include, but arc not limited to, methyl acrylate-methyl methacrylate, polyvinyl chloride, and polyethylene.
  • Hydrophilic polymers include, but are not limited to, cellulosic polymers such as methyl and ethyl cellulose, hydroxyalkylcelluloses such as hydroxypropyl-cellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and Carbopol® 934, polyethylene oxides and mixtures thereof.
  • Fatty compounds include, but are not limited to, various waxes such as carnauba wax and glyceryl tristearate and wax-type substances including hydrogenated castor oil or hydrogenated vegetable oil, or mixtures thereof.
  • the plastic material is a pharmaceutically acceptable acrylic polymer, including but not limited to, acrylic acid and methacrylic acid copolymers, methyl methacrylate, methyl methacrylate copolymers, ethoxy ethyl methacrylates, cyanoethyl methacrylate, aminoalkyl methacrylate copolymer, poly(acrylic acid), poly(methacrylic acid), methacrylic acid alkylamine copolymer poly(methyl methacrylate), poly(methacrylic acid)(anhydride), polymethacrylate, polyacrylamide, poly(methacrylic acid anhydride), and glycidyl methacrylate copolymers.
  • acrylic acid and methacrylic acid copolymers including but not limited to, acrylic acid and methacrylic acid copolymers, methyl methacrylate, methyl methacrylate copolymers, ethoxy ethyl methacrylates, cyanoethyl methacrylate, aminoalky
  • the acrylic polymer is comprised of one or more ammonio methacrylate copolymers.
  • Ammonio methacrylate copolymers are well known in the art, as fully polymerized copolymers of acrylic and methacrylic acid esters with a low content of quaternary ammonium groups.
  • the acrylic polymer is an acrylic resin lacquer such as that which is commercially available from Rohm Pharma under the tradename EUDRAGIT®.
  • the acrylic polymer comprises a mixture of two acrylic resin lacquers commercially available from Rohm Pharma under the tradenames EUDRAGIT® RL30D and EUDRAGIT ® RS30D, respectively.
  • EUDRAGIT® RL30D and EUDRAGIT ® RS30D are copolymers of acrylic and methacrylic esters with a low content of quaternary ammonium groups, the molar ratio of ammonium groups to the remaining neutral (meth)acrylic esters being 1:20 in EUDRAGIT ® RL30D and 1:40 in EUDRAGIT® RS30D.
  • the mean molecular weight is about 150,000.
  • EUDRAGIT ® S-100 and EUDRAGIT ® L-100 are also preferred.
  • the code designations RL (high permeability) and RS (low permeability) refer to the permeability properties of these agents.
  • EUDRAGIT ® RL/RS mixtures are insoluble in water and in digestive fluids. However, multiparticulate systems formed to include the same are swellable and permeable in aqueous solutions and digestive fluids.
  • the polymers described above such as EUDRAGIT ® RL/RS may be mixed together in any desired ratio in order to ultimately obtain a sustained-release formulation having a desirable dissolution profile. Desirable sustained-release multiparticulate systems may be obtained, for instance, from 100% EUDRAGIT® RL, 50% EUDRAGIT® RL and 50% EUDRAGIT t® RS, and 10% EUDRAGIT® RL and 90% EUDRAGIT® RS.
  • acrylic polymers may also be used, such as, for example, EUDRAGIT® L.
  • extended release formulations can be prepared using osmotic systems or by applying a semi-permeable coating to the dosage form.
  • the desired drug release profile can be achieved by combining low permeable and high permeable coating materials in suitable proportion.
  • the devices with different drug release mechanisms described above can be combined in a final dosage form comprising single or multiple units.
  • multiple units include, but are not limited to, multilayer tablets and capsules containing tablets, beads, or granules
  • An immediate release portion can be added to the extended release system by means of either applying an immediate release layer on top of the extended release core using a coating or compression process or in a multiple unit system such as a capsule containing extended and immediate release beads.
  • the present inhibitors can be delivered locally to the respiratory system, for example to the nose, sinus cavities, sinus membranes or lungs.
  • the present peptide inhibitors, and inhibitory nucleic acids or pharmaceutical compositions containing the same can be delivered to the respiratory system in any suitable manner, such as by inhalation via the mouth or intranasally.
  • the present compositions can be dispensed as a powdered or liquid nasal spray, suspension, nose drops, a gel or ointment, through a tube or catheter, by syringe, by packtail, by pledget, or by submucosal infusion.
  • the compounds of the preferred embodiments of the present invention may be conveniently delivered in the form of an aerosol spray using a pressurized pack or a nebulizer and a suitable propellent, e.g., without limitation, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane or carbon dioxide.
  • a pressurized aerosol the dosage unit may be controlled by providing a valve to deliver a metered amount.
  • Capsules and cartridges of, for example, gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
  • a propellant for an aerosol formulation may include compressed air, nitrogen, carbon dioxide, or a hydrocarbon based low boiling solvent.
  • the compound of compounds of the present invention can be delivered in the form of an aerosol spray presentation from a nebulizer or the like.
  • the active ingredients are suitably micronized so as to permit inhalation of substantially all of the active ingredients into the lungs upon administration of the dry powder formulation, thus the active ingredients will have a particle size of less than 100 microns, desirably less than 20 microns, and preferably in the range of 1 to 10 microns.
  • C57BL/6 mice (stock no: 000664) were purchased from Jackson Laboratory.
  • Doxycycline inducible frataxin knockdown (FRDAkd) mice were originally obtained from Drs. Geschwind and Chandran at University of California (Los Angeles, CA), then bred with C57BL/6 mice (The Jackson Laboratory Stock No: 000664) to generate wild type (WT) and transgenic (TG) mice.
  • FRDAkd Doxycycline inducible frataxin knockdown mice
  • mice All mice were housed in an environment of 12 h light/dark cycle, temperature of 25 ⁇ 2 °C, 55% humidity, with ad libitum standard diet and water; treated according to the protocols approved by the Children's Hospital of Philadelphia Institutional Animal Care and Use Committee (IACUC; protocol 16-250); and genotyped at weaning by commercial vendor (Transnetyx, Cordova, TN). Wildtype and transgenic mice were fed Dox- compounded chow diet (200PPM Doxycycline, Animal Specialties and Provisions, LLC., Quakertown, PA) to induce frataxin knockdown in the transgenic mice. Mice were harvested at 4 weeks after doxycycline treatment.
  • Dox- compounded chow diet 200PPM Doxycycline, Animal Specialties and Provisions, LLC., Quakertown, PA
  • mice were deeply anesthetized and euthanized by decapitation.
  • the cerebellum, heart and skeletal muscle were collected and homogenized with a Potter-Elvehjem homogenizer (Thermo Fisher Scientific Inc., Hampton, NH) in RIPA buffer (100 mM Tris-HCL, 150 mM NaCl, 1% IGEPAL, 1 mM EDTA, pH 7.4) containing a protease inhibitor cocktail (1 :500 dilution; Calbiochem, Darmstadt, Germany).
  • the homogenates were spun down at 13,000 rpm for 15 minutes (min). The supernatant was frozen and kept at -80°C until it was used for Western blot or Co-Immunoprecipitation.
  • Primary rat cortical neurons were derived from embryonic day 17 Sprague Dawley rat embryos, as described previously (Dong et al., 2019). Cells were used after at least 14 days in vitro.
  • HEK293 cells were cultured and transfected as described previously (Dong et al., 2019). Briefly, plasmid DNAs containing wild-type frataxin fused to a C-terminal HA tag, frataxin G130V mutant fused to a C-terminal HA tag (Clark et al., 2017), wild-type TID1L, wild-type TID1S, TID1L H121Q, TID1S H121Q (Both wildtype and mutants are from Addgene, Cambridge, MA), TIDIS-Flag (Genscript, Piscataway, NJ) were transfected into HEK 293 cells using LipofectamineTM 2000 reagent (Thermo Fisher Scientific, Hampton, NH) for 24 hours. Cells were then collected and subjected to Western blotting or immunofluorescence.
  • PBMCs isolation whole blood was diluted with PBS at 1:1 ratio before added to the top of 8 ml RT Ficoll-Paque (GE Healthcare, Chicago, IL). Blood samples were then spun at 1600 rpm for 25 min (room temperature, no brake) in swing bucket centrifuge. PBMCs layer was transferred to a new tube and diluted with PBS (30-50ml volume). After 15 min centrifugation at 1000 rpm (room temperature, high brake), the supernatant was aspirated out and PBMCs were resuspened and washed in PBS twice before lysed in RIPA buffer (see above) for future use.
  • PBS room temperature, no brake
  • proteins were transferred to nitrocellulose, blocked with 3% dry milk, and incubated with antibodies against: frataxin (Abeam, Waltham, Boston), TID1L/S (Santa Cruz Biotechnology, Dallas, TX), TID1L (Abeam, Waltham, Boston), actin (Sigma, St. Louis, MO), HA (Cell Signaling, Danvers, MA), actin (Cell signaling, Danvers, MA), Aconitase 2 (Abeam, Waltham, Boston), GRP75 (Abeam, Waltham, Boston), TOM20 (Abeam, Waltham, Boston). Blots were then incubated with appropriate horseradish peroxidase-conjugated secondary antibodies and developed using enhanced chemiluminescence (Pierce, Rockford, IL).
  • Mitochondria were isolated from HEK293 cells transfected with frataxin-HA, TID1L or TID1S plasmid DNAs using mitochondria isolation kit (Thermo Fisher Scientific, Hampton, NH) and then lysed in RIPA buffer (See above) before Western blot analysis.
  • MitotrackerTM Red CMXRos (Thermo Fisher Scientific, Waltham, MA) was loaded to human skin fibroblasts for 45 minutes before immunofluorescence was performed.
  • the following antibodies were used: HA (Cell Signaling, Danvers, MA), Flag (Sigma, St. Louis, MO), TID1L (Abeam, Waltham, Boston), TID1L/S (Santa Cruz Biotechnology, Dallas, TX), GFP (Neuromab, Davis, CA at 1/200).
  • TIDlS448-453Tat KRSTGNYGRKKRRQRRR; SEQ ID NO: 2
  • TIDlS448-453Tat SNRTKGYGRKKRRQRRR; SEQ ID NO: 3
  • Western blot analysis or Immunofluorescence.
  • Glutathione-S-Transferase (Sigma, St. Louis, MO) or recombinant human frataxin fused to an N-terminal GST tag (GST-Frataxin) (Novus biologicals Littleton, CO) were bound to glutathione beads in RIPA buffer (100 mM Tris-HCL, 150 mM NaCl, 1% IGEPAL, 0.5% Sodium deoxycholate, 1 mM EDTA, pH 7.4) overnight at 4°C.
  • GST Glutathione-S-Transferase
  • GST-Frataxin Novus biologicals Littleton, CO
  • TID1 physically interacts with frataxin both in mouse brain cortex and neuronal cells
  • Frataxin was immunoprecipitated using an anti-frataxin antibody in mouse cortical homogenates, followed by mass spectrometry analysis in order to identify frataxain binding partners in the brain (Dong et al., 2022).
  • TID1 a 480 amino acid protein (M.W. 52.41 kDa; Accession No. Q99M87) was identified as a potential frataxin binding partner with three peptide fragments precipitated.
  • TID1L C- terminus an antibody to the TID1L C- terminus (amino acids 450-480) was utilized in the Co-IP assay.
  • TID IL antibody also pulled down frataxin in mouse cortical homogenates, but to a much lesser extent than TIDL/S antibody ( Figure 1C).
  • Figure ID The same outcome was seen in HEK293 cells ( Figure ID), suggesting that TID IS may be the main form interacting with frataxin.
  • Immunofluorescence performed in HEK293 cells transfected with frataxin-HA and TIDIS-Flag plasmids also revealed the colocalization of TID IS and frataxin ( Figure IF). Similar results were found for frataxin and endogenously expressed TTD1L, further supporting the presence of physical interactions between TTD1 and frataxin.
  • an in vitro binding assay was performed using purified glutathione S-transferase (GST)-frataxin fusion proteins and TID1L fused to a C-terminal C-Myc tag. GST-frataxin but not GST bound to TID1L in the in vitro binding assay ( Figure IE), indicating that TID1L directly interacts with frataxin.
  • GST glutathione S-transferase
  • No change in TID1L was detected in PBMCs ( Figure 3E and 3F).
  • TID1 overexpression increases frataxin precursor but decreases intermediate and mature frataxin
  • TID1 protein was overexpressed in HEK293 cells along with frataxin followed by Western blot analysis. Both TID1 precursor and mature form were detected when overexpressed in HEK293 cells.
  • TID1 is a cochaperone of GRP75 (Trentin et al, 2001)
  • TID1L H121Q and TID1S H121Q mutants which functionally inactivate TID1 by allowing them to bind to, but not activate, Hsp70 chaperone proteins (Skyen et al., 1999), to rule out the potential that GRP75 mediates the impact of TID1 on frataxin.
  • TID1L and TID1S overexpression also had no effect on GRP75 levels ( Figure 4C).
  • TID1 regulates frataxin independently of its ability to activate GRP75.
  • TID1 overexpression increases levels of frataxin precursor but decreases levels of intermediate and mature frataxin, suggesting that frataxin precursor is cither not imported or is degraded.
  • subcellular fractionation was performed in HEK293 cells followed by Western blot analysis. As shown in Figure 5 A, increased frataxin precursor caused by TID1L or TID1S overexpression were predominantly localized in the mitochondrial fraction whereas a small portion was localized in the cytosolic fraction, suggesting that decreased intermediate and mature frataxin are not caused by impaired mitochondrial import.
  • TID1S overexpression decreases mature frataxin and ATP levels in human skin-derived fibroblasts
  • TID1 overexpression affects frataxin levels in primary cultured cells
  • lentivirus carrying pHAGE-TIDlS gene or vector control was transduced into human skin fibroblasts for 5 days followed by Western blot analysis.
  • TID1S overexpression also decreased ATP levels, a marker of cell viability (Figure 6C, 26% decrease, n-4, P ⁇ 0.05) and increased mitochondrial fragmentation in fibroblasts ( Figure 6D).
  • TID1L and TID1S have distinct roles in regulating frataxin in fibroblasts.
  • a peptide targeting TID1S rescues frataxin deficiency and mitochondrial phenotype in FRDA patient-derived skin fibroblasts
  • TID1S and TID1L only differ in the C-terminus.
  • TID1L has 33 amino acids unique to its C-terminus while TID1S contains 6 amino acids (Lu 2006).
  • KRSTGN amino acids 448-453
  • TAT transactivator of transcription
  • TID1 is a novel binding partner and regulator of frataxin.
  • overexpression of TID1S lowers the amounts of mature frataxin.
  • the effect of TID1S on frataxin is mediated by the last 6 amino acids of TID1S, as a competing peptide made from these six amino acids rescues frataxin deficiency and mitochondrial abnormalities in the FRDA cellular model, thus identifying TID1S as a negative posttranslational regulator of frataxin and a new therapeutic target for FRDA.
  • TID1L and TID1S differ only at their extreme carboxyl termini but have distinct role in regulating frataxin. While both TID1L and TID1S increase frataxin precursor and decrease mature frataxin in HEK293 cells, only TID1S overexpression decreases mature frataxin in primary cultured fibroblasts. The lack of effect of TID1L in primary cultured fibroblasts could be ascribed to both the altered protein interactome and its limited binding affinity for frataxin ( Figure 1C and ID).
  • TID1L has a longer residency time in the cytosol due to its interaction with the cytosolic chaperone Hsc70 and its cytosolic substrates such as STAT1 and STAT3, (Lu et al., 2006), whereas TID1S predominates in the mitochondria where it is more likely to interact with and control frataxin. Since TID1S overexpression has no effect on the mitochondrial localization of frataxin precursor and a protcasomal inhibitor, MG 132, docs not restore intermediate and mature frataxin in HEK293 cells, the TID1S overexpression-generated decrease in mature frataxin is neither due to compromised mitochondrial import nor to proteasomal degradation.
  • TID1S directs frataxin precursor to mitochondrial proteases for destruction. Additionally, unlike GRP75, which facilitates the maturation of frataxin by MPP, TID1S does not interact with MPP. The binding of TID1S to frataxin alone may not only decrease the efficiency of MPP-mediated frataxin maturation but also make the frataxin precursor more accessible to other methods of destruction.
  • TID 1 protein levels are noted in some neurodegenerative diseases including Alzheimer’s disease (AD), in which TID1 protein levels are upregulated in the hippocampus of AD patients and Tg2576 mice.
  • AD Alzheimer’s disease
  • TID1 protein levels are upregulated in the hippocampus of AD patients and Tg2576 mice.
  • Ap42 Zhou C 2020.
  • TID IL and TID IS are increased in the affected tissues of frataxin knockdown mice upon subacute frataxin deficiency.
  • TID IL is widely elevated whereas TID IS is only increased in specific tissues, including the heart and skeletal muscle, suggesting tissue specific effects of frataxin deficiency on TID1 splice variants levels.
  • TID IS elevation could result in a further decrease in frataxin levels leading to exacerbation of pathological changes in FRDA, forming a vicious cycle.
  • TID IL overexpression has no effect on mature frataxin in primary cultured fibroblasts, it causes mitochondrial fragmentation, just like TID1S.
  • TIDlL-caused mitochondrial fragmentation is Dynamin-related protein 1 (Drpl) dependent and associated with reduced cell viability (Elwi et al., 2012).
  • Consistent with increased TID IL in the cerebellum of frataxin knockdown mice increased activation of Drpl and structural changes in mitochondria were observed in the cerebellum of one FRDA mouse model (Mercado-Ayon, et al., 2022).
  • TID IL decreases also cause mitochondrial fragmentation in fibroblasts (data not shown), suggesting that both TID IL elevation and decrease can contribute to pathological changes in FRDA.
  • An optimal concentration of TID 1 may be needed for its physiological effects.
  • the decrease in TID1L levels may reflect decreased mitochondrial numbers (Huang et al., 2013 and Vasquez-Trincado et ah, 2022) as multiple proteins including GRP75 are lowered in FRDA patient cells (Dong et al., 2019). The easy accessibility of such patient cells suggests that TID1L decrease could serve as a biomarker for FRDA.
  • compositions and methods described can be used to ameliorate symptoms associated with Friedreich’s ataxia and other disorders related to frataxin deficiency and, or, mitochondrial fragmentation or dysfunction.
  • the patient is assessed, monitored, or diagnosed by a method comprising: (i) measuring one or more clinical symptoms or signs of FDRA and mitochondrial dysfunction in a subject, (ii) combining the measurements obtained into a single composite measurement, and (iii) assessing the overall severity of, or change in, the mitochondrial dysfunction or mitochondrial disease in the subject by comparing the composite measurement to a reference value or another composite measurement in the same subject.
  • the (i) one or more composite measurements are employed to measure the clinical effect on the subject of a diagnostic, therapeutic or other type of medical intervention; (ii) for each of the measurements tested, the subject is classified as: (a) a responder or a non-responder, (b) a member of a clinical category, or (c) a member of a metric range, based on the change in said one or more clinical symptoms as measured using the particular clinical symptom or metabolic pathway assessed; and (iii) the measurements obtained are combined into a single composite measurement, by either: (a) separately assessing the change in each measurement obtained from each assay conducted prior to combining each measurement into a single composite measurement, or (b) combining measurements obtained from a first time point and generating a single composite measurement for said first time point and then comparing the single composite measurement for the first time point to a single composite measurement generated from the same assay for a second time point.
  • Important clinical assessments for FDRA and associated mitochondrial dysfunction include one or more of Friedreich's Ataxia Rating Scale (FARS), Scale for the Rating and Assessment of Ataxia (SARA), Movement Disorders Childhood Rating Scale (CRS), Motor Function Measure, Dynamometry Measures, Cardiopulmonary exercise Test (CPET), Creatine- CEST MRI measures of post-exercise creatine and phosphocreatine levels recovery in muscle, Twelve Minute Walk Test, Six Minute Walk Test Total Distance Walked, Six Minute Walk Distance Per Minute Distance Walked, Two Minute Walk Test, Modified Fatigue Impact Scale, Neuropathy Impairment Score, or combinations thereof.
  • FARS Friedreich's Ataxia Rating Scale
  • SARA Scale for the Rating and Assessment of Ataxia
  • CRS Movement Disorders Childhood Rating Scale
  • Motor Function Measure Dynamometry Measures
  • CPET Cardiopulmonary exercise Test
  • Creatine- CEST MRI measures of post-exercise creatine and phosphocreatine levels recovery in muscle Twelve Minute Walk Test, Six Minute Walk Test Total Distance
  • a preferred assessment includes, for example, Friedreich's Ataxia Rating Scale. In some embodiments, two, three, four, five or more of the assessments listed above are performed.
  • the formulations described above or pharmaceutically acceptable salt thereof can be administered at a dose of about .01 mg, .02 mg, .05 mg, 1 mg, 2 mg, about 10 mg, about 100 mg, about 250 mg, or about 500 mg as determined by a clinician familiar with the treatment of FDRA.
  • the formulation or pharmaceutically acceptable salt thereof can be administered as a liquid formulation.
  • the liquid formulation is preferably administered via intracranial or intracerebro-ventricular injection or microinfusion.
  • the formulation can be administered by a route selected from the group consisting of oral, enteral, gastrostomy tube, jejunostomy tube, orogastric tube, nasogastric tube, parenteral, intravenous, subcutaneous, intramuscular, intraperitoneal, intracistemal, intraarticular, intracerebral, intraparenchymal, intrathecal, nasal, vaginal, sublingual, intraocular’, intravitreal, rectal, topical, transdermal and inhalation.
  • the formulation can be administered on a continuous dosing schedule. In some embodiments, it can be administered four times per day, three times per day, twice per day, once per day, once every two days, once every three days, once every four days, once every five days, once every six days, once a week, once every two weeks or once per month.
  • the therapeutic formulation is administered initially in a loading dose that is higher than a subsequent dose.
  • the therapeutic formulation can be administered with one or more additional pharmaceutical agents that may complement one another or act synergistically. For example, Omaveloxolone has been identified as a useful therapeutic for FDRA.
  • Tidl isoforms are mitochondrial DnaJ- like chaperones with unique carboxyl termini that determine cytosolic fate. J Biol Chem 281:13150-13158. DOI: 10.1074/jbc.M509179200
  • TID1 a human homolog of the Drosophila tumor suppressor l(2)tid, encodes two mitochondrial modulators of apoptosis with opposing functions. Proc Natl Acad Sci U S A. 96(15):8499-504. DOI: 10.1073/pnas.96.15.8499
  • a mouse homologue of the Drosophila tumor suppressor 1 (2) tid gene defines a novel Ras GTPase- activating protein (Ras GAP) -binding protein. J Biol Chem 276, 13087-13095.
  • Beta- Amyloid Increases the Expression Levels of Tidl responsible for Neuronal Cell Death an Amyloid Beta Production. Mol Neurobiol, 57:1099-1114. DOI: 10.1007/sl2035-019-01807-2

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Psychology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Neurology (AREA)
  • Neurosurgery (AREA)
  • Zoology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne des compositions et des méthodes de gestion et de traitement de l'ataxie de Friedreich et d'autres troubles associés à la fragmentation mitochondriale.
PCT/US2024/055696 2023-11-13 2024-11-13 Compositions et méthodes pour le traitement de l'ataxie de friedreich Pending WO2025106527A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202363598256P 2023-11-13 2023-11-13
US63/598,256 2023-11-13

Publications (1)

Publication Number Publication Date
WO2025106527A1 true WO2025106527A1 (fr) 2025-05-22

Family

ID=95743327

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2024/055696 Pending WO2025106527A1 (fr) 2023-11-13 2024-11-13 Compositions et méthodes pour le traitement de l'ataxie de friedreich

Country Status (1)

Country Link
WO (1) WO2025106527A1 (fr)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020086844A1 (en) * 2000-07-19 2002-07-04 Joshua Syken Methods and reagents to regulate apoptosis
WO2023039476A1 (fr) * 2021-09-08 2023-03-16 The Broad Institute, Inc. Compositions modifiées pour le ciblage du système nerveux central et des muscles

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020086844A1 (en) * 2000-07-19 2002-07-04 Joshua Syken Methods and reagents to regulate apoptosis
WO2023039476A1 (fr) * 2021-09-08 2023-03-16 The Broad Institute, Inc. Compositions modifiées pour le ciblage du système nerveux central et des muscles

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SYNOFZIK ET AL.: "Autosomal recessive cerebellar ataxias: paving the way toward targeted molecular therapies", NEURON, vol. 101, no. 4, 20 February 2019 (2019-02-20), pages 560 - 583, XP085608968, DOI: 10.1016/j.neuron.2019.01.049 *

Similar Documents

Publication Publication Date Title
US11248023B2 (en) Agonist of spexin-based galanin type 2 receptor and use thereof
US20100105625A1 (en) Product and Methods for Diagnosis and Therapy for Cardiac and Skeletal Muscle Disorders
US20240216466A1 (en) COMPOSITIONS FOR TREATING HEART DISEASE BY INHIBITING THE ACTION OF mAKAP-beta
US10907153B2 (en) Treatment of heart disease by inhibtion of the action of muscle A-kinase anchoring protein (mAKAP)
TW201729833A (zh) 用於治療澱粉樣沈著症之方法及組合物
US8927498B2 (en) Compositions and methods useful in enhancement of memory
US20200138921A1 (en) Methods of preventing and treating diseases characterized by synaptic dysfunction and neurodegeneration including alzheimer's disease
WO2025106527A1 (fr) Compositions et méthodes pour le traitement de l'ataxie de friedreich
CN106659911B (zh) 治疗或预防神经退行性病症的组合物和方法
US20200131495A1 (en) Proteins derived from clpb and uses thereof
JP2018507853A (ja) 代謝障害の治療薬としてのデルマトポンチン
WO2023246847A1 (fr) Peptide bloquant b2m-glun1, composition pharmaceutique et son utilisation
CN107592866A (zh) Mcj激动剂及其用途

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: 24892133

Country of ref document: EP

Kind code of ref document: A1