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EP3906253A1 - Analogues peptidiques de conotoxine et leurs utilisations pour le traitement de la douleur et d'affections inflammatoires - Google Patents

Analogues peptidiques de conotoxine et leurs utilisations pour le traitement de la douleur et d'affections inflammatoires

Info

Publication number
EP3906253A1
EP3906253A1 EP19702500.0A EP19702500A EP3906253A1 EP 3906253 A1 EP3906253 A1 EP 3906253A1 EP 19702500 A EP19702500 A EP 19702500A EP 3906253 A1 EP3906253 A1 EP 3906253A1
Authority
EP
European Patent Office
Prior art keywords
peptide analog
conotoxin peptide
pharmaceutically acceptable
acceptable salt
pegylated
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.)
Withdrawn
Application number
EP19702500.0A
Other languages
German (de)
English (en)
Inventor
Jose Mercado
Eric J. Tarcha
Jeffrey J. POSAKONY
Shawn Iadonato
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.)
Kineta Chronic Pain LLC
Original Assignee
Kineta Chronic Pain LLC
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 Kineta Chronic Pain LLC filed Critical Kineta Chronic Pain LLC
Publication of EP3906253A1 publication Critical patent/EP3906253A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1767—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • 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

Definitions

  • alpha-conotoxin peptide analogs including alpha-conotoxin peptide analogs that are covalently attached to polyethylene glycol (PEG), and pharmaceutical compositions of such alpha-conotoxin peptide analogs.
  • PEG polyethylene glycol
  • methods of treating or preventing a condition conducive to treatment or prevention by inhibition of an a9- containing nicotinic acetylcholine receptor (nAChR) e.g., the a9a10 subtype of the nAChR
  • neuropharmacologically active peptides conotoxin peptides or conotoxins
  • conotoxin peptides or conotoxins neuropharmacologically active peptides
  • Native alpha-conotoxin peptides are highly disulfide cross-linked peptides with a C1-C3 and C2-C4 disulfide bridge pattern (Azam and McIntosh, 2009, Acta Pharmacol. Sin. 30:771-783).
  • Alpha-conotoxin peptides have generally been shown to be nicotinic acetylcholine receptor (nAChR) antagonists (Nicke et al., 2004, Eur. J. Biochem.271:2305-2319).
  • nAChRs are pentameric ligand-gated ion channels assembled from one or more a subunits (a1- a10) either alone or together with one or more non-a subunits (b1-b4) (Sine and Engel, 2006, Nature 440:448-455).
  • the a9a10 nAChR subunits are expressed in diverse tissues. In the inner ear, a9a10 nAChRs mediate synaptic transmission between efferent olivocochlear fibers and cochlear hair cells (Vetter et al., 1999, Neuron 23:93-103). The a9a10 subunits are also found in dorsal root ganglion neurons (Lips et al., 2002, Neuroscience 115:1-5), lymphocytes (Peng et al., 2004, Life. Sci.76:263-280), skin keratinocytes (Nguyen et al., 2000, Am. J.
  • Pathol.157:1377-1391 the pars tuberalis of the pituitary (Elgoyhen et al., 1994, Cell 79:705-715; Zuo et al., 1999, Proc. Natl. Acad. Sci. USA 96:14100-14105).
  • Compounds that have a pharmacological profile that includes a9a10 antagonist activity prevent or attenuate the expression of pain in several rodent models, including neuropathic pain induced by chemotherapy, traumatic nerve injury, and diabetes (see Hone and McIntosh, 2018, FEBS Lett.592:1045-1062).
  • Native conotoxin peptide RgIA has the amino acid sequence Gly-Cys-Cys-Ser-Asp- Pro-Arg-Cys-Arg-Tyr-Arg-Cys-Arg (SEQ ID NO:1) (Ellison et al., 2008, J. Mol. Biol.
  • Alpha-conotoxin peptide RgIA has been shown to block a9a10 nAChR activity (Romero et al., 2017, Proc. Natl. Acad. Sci. USA 14:E1825-E1832).
  • RgIA belongs to the a-4/3 family of a-conotoxins.
  • the native structure of RgIA is characterized by two compact intra-cysteine loops defined by two disulfide bonds formed between Cys2-Cys8 (sometimes referred CysI-CysIII) and Cys3-Cys12 (sometimes referred to CysII-CysIV) (Ellison et al, 2008, J. Mol. Biol.377:1216-1227; Armishaw, 2010, Toxins 2:1471-1499).
  • the first and second cysteine residues are always adjacent, but the number of amino acid residues between the second and third cysteine, and between the third and fourth cysteine residues can vary. This gives rise to two loops of intervening amino acids denoted loop 1 or the m-loop and loop 2 or the n-loop. In RgIA, loop 1 contains 4 amino acids and loop 2 contains 3 amino acids. The disulfide bonds and therefore the secondary structure of a-conotoxins are unstable and subject to rearrangement (Armishaw, 2010, Toxins 2:1471-1499).
  • the two disulfide bonds of the a-conopeptide are subject to rearrangement and can form alternative three-dimensional structures including a ribbon form (disulfide bonds Cys-2- Cys12 and Cys3-Cys8) and a bead form (disulfide bonds Cys2-Cys3 and Cys8-Cys12). While the native or globular form of the peptide is active on the a9a10-nAChr, neither the ribbon or bead form are thought to be active (Dekan et al., 2011, J. Am. Chem. Soc.133:15866-15869; Armishaw et al., 2006, J. Bio. Chem.281:14136-14143).
  • Hargittai et al. evaluated four lactam-bridged derivatives of the alpha-conotoxin SI, and only one of the four lactam-bridged derivatives did not lose significant activity in binding to BC3H1 cells (Hargittai et al., 2000, J. Med. Chem.43:4787-4792).
  • Bondebjerg et al. evaluated synthesized thioether mimetics of the alpha-conotoxin GI and found that the two isomers tested had significantly less pharmacologic activity than the native peptide (Bondebjerg et al., 2003, ChemBioChem 4:186-194).
  • Non-reducible dicarba analogs of alpha-ImI were evaluated and in both instances found to have an approximately 10-fold reduction in pharmacological activity (MacRaild et al., 2009, J. Med. Chem.52:755-762). Certain specific selenocysteine and cystathionine derivatives of ImI retained activity in blocking the rat a7 nAChR (Dekan et al., 2011, J. Am. Chem. Soc.133:15866-15869; Armishaw et al., 2006, J. Bio. Chem.281:14136- 14143).
  • van Lierop evaluated dicarba substitutions of the disulfide bonds of alpha-conotoxin Vc1.1 and found that the [2,8]-cis and [2,8]-trans dicarba analogs had no activity on the rat a9a10 nAChr, whereas the [3,16]-cis and [3,16]-trans isomers of Vc1.1 had greatly reduced (10- 100-fold) activity on rat a9a10 nAChR (van Lierop et al., 2013, ACS Chemical Biology 8:1815- 1821). Yu et al created N-terminal to C-terminal peptide-linked derivatives of Vc1.1 that had one or more cysteine residues replaced with His or Phe.
  • Native RgIA has approximately 300-fold lower affinity for the human than the rat a9a10 nAChR, and this difference in affinity has been mapped to variation in the amino acid sequence of the alpha 9 subunit within the RgIA binding pocket (Azam et al., 2015, Mol.
  • Conotoxin peptides and their analogs are competitive inhibitors of the a9a10 nAChR and they bind at the ACh binding site, i.e., at the interface between adjacent subunits.
  • the subunit interfaces buries to a 1.4 ⁇ probe radius, a surface area of ⁇ 1300 ⁇ , where these conotoxins interact with residues between subunits encompassing distances less than 5 ⁇
  • Non-covalent or covalent attachment to polyethylene glycol polymer(s) can change the physical and chemical properties of a biomolecule such as a peptide, such as its conformation, electrostatic binding, and hydrophobicity (Veronese and Mero, 2008, BioDrugs, 22:315-329; see also Harris et al., 2001, Clin. Pharmacokinet.40:539-551).
  • PEGylation of a therapeutic protein often leads to a loss in its binding affinity due to the steric hindrance of the PEG polymer to the drug-target binding interaction (Fishburn, 2008, J. Pharm. Sci.97:4167-4183).
  • PEGylated therapeutic proteins can lose biological activity or potency, either through steric effects or through hydrophobic-hydrophobic interactions between the PEG and hydrophobic domains within the protein (Parrott and DeSimone, 2011, Nat. Chem. 4:13-14).
  • AA is Tyr, Phe, Trp, or a D-isomer of Tyr, Phe, or Trp, and X 2
  • a A is N-Me-Gly, D-Tyr, or N-Me-Tyr;
  • C-terminus of the conotoxin peptide analog is a carboxylic acid or an amide group.
  • the triazole bridge is
  • the triazole bridge is
  • x is 1, 2, or 3.
  • x is 1.
  • y is 2 or 3.
  • y is 3.
  • x is 1, 2, or 3, and y is 2 or 3.
  • x is 1 and y is 3.
  • x is 2, and y is 3.
  • x is 2, and y is 2.
  • x is 1, and y is 3.
  • x is 2, and y is 2.
  • the triazole bridge is , wherein the single wavy line ( indicates the point of attachment of the triazole bridge to the C 1 carbon of the
  • X is X 1
  • X is X 1
  • a A is selected from the group consisting of Tyr, D-Tyr and Phe.
  • X is Tyr
  • the C-terminus of the conotoxin peptide analog is OH.
  • the C-terminus of the conotoxin peptide analog is NH 2 .
  • the conotoxin peptide analog is of Formula (Ia) (SEQ ID NO: 1
  • R 1 is OH or NH2.
  • R 1 is OH.
  • R 1 is NH2.
  • the conotoxin peptide analog is of Formula (Ig) (SEQ ID NO: 1
  • the conotoxin peptide analog is of Formula (Ih) (SEQ ID NO:
  • the conotoxin peptide analog is of Formula (Ii) (SEQ ID NO:36):
  • the conotoxin peptide analog is of Formula (Ik) (SEQ ID N).
  • the conotoxin peptide analog is of Formula (Il) (SEQ ID N).
  • the conotoxin peptide analog is of Formula (Im) (SEQ ID NO:
  • the conotoxin peptide analog is of Formula (In) (SEQ ID NO: 1]
  • the conotoxin peptide analog is of Formula (Io) (SEQ ID NO:
  • the conotoxin peptide analog is of Formula (Ip) (SEQ ID NO: 1]
  • conotoxin peptide analogs or pharmaceutically acceptable salt thereof wherein the conotoxin peptide analog is of Formula (I) (SEQ ID NO:93):
  • a A is Tyr, Phe, Trp, or a D-isomer of Tyr, Phe, or Trp, and X 2
  • a A is N-Me-Gly, D-Tyr, or N-Me-Tyr;
  • C-terminus of the conotoxin peptide analog is a carboxylic acid or an amide group
  • conotoxin peptide analog is covalently attached directly or via a linking group to one or more polyethylene glycol (PEG) polymers.
  • PEG polyethylene glycol
  • the triazole bridge is wherein the single wavy line ( ndicates the point of
  • the triazole bridge is [0059] In a specific embodiment, wherein the triazole bridge is [0060] In a specific embodiment, the triazole bridge is
  • x is 1, 2, or 3.
  • x is 1.
  • y is 2 or 3.
  • y is 3.
  • x is 1, 2, or 3
  • y is 2 or 3.
  • x is 1 and y is 3.
  • x is 2 and y is 3.
  • x is 2 and y is 2.
  • x is 1 and y is 3.
  • x is 2 and y is 2.
  • the triazole bridge is wherein the single wavy line ( ndicates the point of attachment of the triazole bridge to the C 1 carbon of the
  • X is X 1
  • X is X 1
  • AA is selected from the group consisting of Tyr, D-Tyr and Phe.
  • X is Tyr
  • the C-terminus of the conotoxin peptide analog is OH.
  • the C-terminus of the conotoxin peptide analog is NH2.
  • the conotoxin peptide analog is of Formula (Ia) (SEQ ID NO:94)
  • R 1 is OH
  • R 1 is NH2.
  • the conotoxin peptide analog is of Formula (Ig) (SEQ ID NO: 1]
  • the conotoxin peptide analog is of Formula (Ih) (SEQ ID NO:33):
  • the conotoxin peptide analog is of Formula (Ii) (SEQ ID NO:
  • the conotoxin peptide analog is of Formula (Ik) (SEQ ID N).
  • the conotoxin peptide analog is of Formula (Il) (SEQ ID NO:
  • the conotoxin peptide analog is of Formula (Im) (SEQ ID N
  • the conotoxin peptide analog is of Formula (In) (SEQ ID N).
  • the conotoxin peptide analog is of Formula (Io) (SEQ ID NO:54)
  • the conotoxin peptide analog is of Formula (Ip) (SEQ ID NO: 1]
  • the conotoxin peptide analog is covalently attached to one PEG polymer.
  • the PEG polymer is covalently attached to the N-terminus of the conotoxin peptide analog.
  • the PEG polymer is covalently attached to the C-terminus of the conotoxin peptide analog.
  • the PEG polymer is covalently attached to an amino acid residue position that is not the N-terminus or the C-terminus of the conotoxin peptide analog.
  • the PEG polymer is covalently attached to the conotoxin peptide analog via a linking group.
  • the linking group is a valerate linker having a formula of .
  • the linking group is a butylene.
  • the linking group is a carbonyl
  • the PEG polymer is a linear or branched PEG polymer.
  • the PEG polymer is a linear PEG polymer.
  • the PEG polymer has molecular weight in the range of 10 kDa and 40 kDa.
  • the PEG polymer is a linear 30 kDa PEG polymer.
  • the PEG polymer is a linear 30 kDa mPEG polymer.
  • the PEGylated conotoxin peptide analog is of Formula (IIa) (
  • the PEGylated conotoxin peptide analog is of Formula ( li
  • the PEGylated conotoxin peptide analog is of Formula ( li
  • the PEGylated conotoxin peptide analog is of Formula (IIi) (
  • the PEGylated conotoxin peptide analog is of Formula (
  • the PEGylated conotoxin peptide analog is of Formula (IIl) (SEQ ID NO:99):
  • the PEGylated conotoxin peptide analog is of Formula (
  • the PEGylated conotoxin peptide analog is of Formula (
  • the PEGylated conotoxin peptide analog is of Formula (IIo) (SEQ ID NO:102):
  • the PEGylated conotoxin peptide analog is of Formula (
  • R 2 is OH or NH 2 .
  • R 2 is OH.
  • R 2 is NH2.
  • conotoxin peptide analogs or a pharmaceutically acceptable salt thereof, wherein the conotoxin peptide analog is of Formula (Ib) (SEQ ID NO:104):
  • R 2 is OH or NH2
  • conotoxin peptide analog is covalently attached directly or via a linking group to one or more polyethylene glycol (PEG) polymers.
  • PEG polyethylene glycol
  • R 2 is OH
  • R 2 is NH2.
  • the conotoxin peptide analog is covalently attached to one PEG polymer.
  • the PEG polymer is covalently attached to the N-terminus of the conotoxin peptide analog.
  • the PEG polymer is covalently attached to the C-terminus of the conotoxin peptide analog.
  • the PEG polymer is covalently attached to an amino acid residue position that is not the N-terminus or the C-terminus of the conotoxin peptide analog.
  • the PEG polymer is covalently attached to the conotoxin peptide analog via a linking group.
  • the linking group is a valerate linker having a formula of .
  • the linking group is a butylene.
  • the linking group is a carbonyl
  • the PEG polymer is a linear or branched PEG polymer.
  • the PEG polymer is a linear PEG polymer.
  • the PEG polymer has molecular weight in the range of 10 kDa and 40 kDa.
  • the PEG polymer is a linear 30 kDa PEG polymer.
  • the PEG polymer is a linear 30 kDa mPEG polymer.
  • the PEGylated conotoxin peptide analog is of Formula (IIb) (SEQ ID NO:105):
  • conotoxin peptide analogs selected from the group consisting of conotoxin peptide analogs Ia, Ia’, Ib, Ib’, Ic, Id, Ie, If, Ig, Ih, Ii, Ij, Ik, Il, Im, In, Io, Ip, Iq, Ir, Is, It, Iu, and Iv, or a pharmaceutically acceptable salt thereof.
  • the conotoxin peptide analog is selected from the group consisting of conotoxin peptide analogs Ia, Ia’, Ib, Ib’, Ig, Ih, Ii, Ik, Il, Im, In, Io, and Ip.
  • the conotoxin peptide analog is selected from the group consisting of conotoxin peptide analogs Ia, Ia’, Ib, and Ib’.
  • compositions comprising a conotoxin peptide analog or pharmaceutically acceptable salt thereof, or a PEGylated conotoxin peptide analog or pharmaceutically acceptable salt thereof described herein, and optionally a
  • Also provided herein are methods of treating or preventing a condition conducive to treatment or prevention by inhibition of an a9-containing nicotinic acetylcholine receptor (nAChR) in a subject comprising administering to the subject a therapeutically effective amount of a conotoxin peptide analog or pharmaceutically acceptable salt thereof, or a PEGylated conotoxin peptide analog or pharmaceutically acceptable salt thereof, or pharmaceutical composition described herein.
  • nAChR nicotinic acetylcholine receptor
  • Also provided herein are methods of treating or preventing a condition associated with activation of an a9-containing nicotinic acetylcholine receptor (nAChR) in a subject comprising administering to the subject a therapeutically effective amount of a conotoxin peptide analog or pharmaceutically acceptable salt thereof, or a PEGylated conotoxin peptide analog or pharmaceutically acceptable salt thereof, or pharmaceutical composition described herein.
  • nAChR nicotinic acetylcholine receptor
  • the condition conducive to treatment or prevention by inhibition of the a9-containing nAChR is pain or inflammation.
  • the condition is pain.
  • the pain is selected from the group consisting of general pain, chronic pain, neuropathic pain, nociceptive pain, inflammatory pain, visceral pain, somatic pain, pain induced by peripheral nerve damage, pain induced by an inflammatory disorder, pain induced by a metabolic disorder, pain induced by cancer, pain induced by chemotherapy, pain induced by a surgical procedure, and pain induced by a burn.
  • the pain is cancer-related chronic pain.
  • condition conducive to treatment or prevention by inhibition of the a9-containing nAChR is an inflammatory condition.
  • the inflammatory condition is selected from the group consisting of inflammation, chronic inflammation, a rheumatic disease, sepsis, fibromyalgia, inflammatory bowel disease, sarcoidosis, endometriosis, uterine fibroids, an inflammatory skin disease, an inflammatory condition of the lungs, a disease associated with inflammation of the nervous system, periodontal disease, and cardiovascular disease.
  • the inflammatory condition is mediated by immune cells.
  • the inflammatory condition is long-term inflammation and/or peripheral neuropathy following injury.
  • the condition conducive to treatment or prevention by inhibition of the a9-containing nAChR is pain and inflammation.
  • the condition conducive to treatment or prevention by inhibition of the a9-containing nAChR is inflammation and neuropathy.
  • the condition conducive to treatment or prevention by inhibition of an a9-containing nicotinic acetylcholine receptor is a condition conducive to treatment or prevention by inhibition of an a9a10 subtype of nAChR.
  • the subject is a human.
  • Also provided herein are methods of treating or preventing pain or inflammation in a subject comprising administering to the subject a therapeutically effective amount of a conotoxin peptide analog or pharmaceutically acceptable salt thereof, or a PEGylated conotoxin peptide analog or pharmaceutically acceptable salt thereof, or pharmaceutical composition described herein.
  • conotoxin peptide analogs or pharmaceutically acceptable salt thereof or PEGylated conotoxin peptide analogs or pharmaceutically acceptable salt thereof, or pharmaceutical compositions described herein, for use in treating or preventing a condition conducive to treatment or prevention by inhibition of an a9-containing nicotinic acetylcholine receptor (nAChR) in a subject.
  • nAChR nicotinic acetylcholine receptor
  • compositions comprising a conotoxin peptide analog or pharmaceutically acceptable salt thereof, or a PEGylated conotoxin peptide analog or pharmaceutically acceptable salt thereof, for use in treating or preventing a condition conducive to treatment or prevention by inhibition of an a9-containing nicotinic acetylcholine receptor (nAChR) in a subject.
  • nAChR nicotinic acetylcholine receptor
  • conotoxin peptide analog or pharmaceutically acceptable salt thereof or a PEGylated conotoxin peptide analog or pharmaceutically acceptable salt thereof, or pharmaceutical composition described herein, in the preparation of a medicament for treating or preventing a condition conducive to treatment or prevention by inhibition of an a9-containing nicotinic acetylcholine receptor (nAChR) in a subject.
  • conotoxin peptide analogs or a salt thereof wherein the amino acid sequence of the conotoxin peptide analog is
  • AA is selected from the group consisting of (S)-propargyl glycine, (S)-azidoalanine, (S)- homopropargyl glycine, (S)-azidohomoalanine, (S)-azidonorvaline and (S)-bishomopropargyl glycine;
  • AA Citrulline
  • a A is selected from the group consisting of (S)-azidohomoalanine, (S)-homopropargyl glycine, (S)-azidonorvaline, and (S)-bishomopropargyl glycine;
  • AA is selected from the group consisting of (S)-propargyl glycine, (S)- homopropargyl glycine, and (S)-bishomopropargyl glycine, X 12
  • a A is (S)-azidohomoalanine or (S)-azidonorvaline; and when X 3
  • a A is selected from the group consisting of (S)-azidoalanine, (S)-azidohomoalanine, and (S)-azidonorvaline, X 12
  • AA is (S)-homopropargyl glycine or (S)- bishomopropargyl glycine;
  • a A is Tyr, Phe, Trp, or a D-isomer of Tyr, Phe, or Trp, and X 2
  • AA is N-Me-Gly, D-Tyr, or N-Me-Tyr
  • C-terminus of the conotoxin peptide analog is a carboxylic acid or an amide group.
  • a A is (S)-propargyl glycine or (S)-azidoalanine.
  • AA is (S)-azidonorvaline or (S)-bishomopropargyl glycine.
  • a A is (S)-propargyl glycine and X AA is (S)- azidonorvaline.
  • AA is (S)-homopropargyl glycine and X 12
  • AA is (S)- azidonorvaline.
  • a A is (S)-homopropargyl glycine and X 12
  • a A is (S)- azidohomoalanine.
  • AA is (S)-azidohomoalanine and X 12
  • AA is (S)- homopropargyl glycine. [00162] In a specific embodiment, X 3
  • AA is (S)-azidoalanine and X 12
  • AA is (S)- bishomopropargyl glycine.
  • X is X 1
  • X is X 1
  • AA is selected from the group consisting of Tyr, D-Tyr and Phe.
  • X is Tyr
  • the C-terminus of the conotoxin peptide analog is OH.
  • the C-terminus of the conotoxin peptide analog is NH 2 .
  • a A is (S)-propargyl glycine, X 12
  • a A is (S)-azidonorvaline, X is Tyr.
  • the C-terminus of the conotoxin peptide analog is a carboxylic acid group.
  • the C-terminus of the conotoxin peptide analog is an amide group.
  • a A is (S)-homopropargyl glycine, X 12
  • a A is (S)- azidonorvaline, X is Tyr; and wherein the C-terminus of the conotoxin peptide analog is a carboxylic acid group.
  • a A is (S)-homopropargyl glycine, X 12
  • a A is (S)- azidohomoalanine, X is Tyr; and wherein the C-terminus of the conotoxin peptide analog is a carboxylic acid group.
  • a A is (S)-azidohomoalanine, X 12
  • a A is (S)-homopropargyl glycine, X is Tyr; and wherein the C-terminus of the conotoxin peptide analog is a carboxylic acid group.
  • a A is (S)-azidoalanine, X 12
  • a A is (S)-bishomopropargyl glycine, X is Tyr; and wherein the C-terminus of the conotoxin peptide analog is a carboxylic acid group.
  • AA is (S)-propargyl glycine
  • AA is (S)-azidonorvaline
  • X is Phe
  • the C-terminus of the conotoxin peptide analog is a carboxylic acid group.
  • AA is (S)-propargyl glycine
  • AA is (S)-azidonorvaline, X is D-Tyr; and wherein the C-terminus of the conotoxin peptide analog is a carboxylic acid group.
  • AA is (S)-propargyl glycine
  • AA is (S)-azidonorvaline
  • X is Tyr-N-Me-Gly
  • C-terminus of the conotoxin peptide analog is a carboxylic acid group
  • AA is (S)-propargyl glycine
  • AA is (S)-azidonorvaline
  • X is Tyr-D-Tyr
  • C-terminus of the conotoxin peptide analog is a carboxylic acid group
  • a A is (S)-propargyl glycine, X 12
  • a A is (S)-azidonorvaline, X is Tyr-N-Me-Tyr; and wherein the C-terminus of the conotoxin peptide analog is a carboxylic acid group.
  • conotoxin peptide analogs or a salt thereof wherein the amino acid sequence of the conotoxin peptide analog is
  • AA is (S)-propargyl glycine
  • AA Citrulline
  • a A is 3-iodo-Tyr
  • AA is (S)-azidonorvaline
  • C-terminus of the conotoxin peptide analog is a carboxylic acid or an amide group.
  • the C-terminus of the conotoxin peptide analog is a carboxylic acid group.
  • the C-terminus of the conotoxin peptide analog is an amide group.
  • a A is Tyr, Phe, Trp, or a D-isomer of Tyr, Phe, or Trp, and X 2
  • a A is N-Me-Gly, D-Tyr, or N-Me-Tyr;
  • C-terminus of the conotoxin peptide analog of Formula (I) is a carboxylic acid or an amide group
  • AA is selected from the group consisting of (S)-propargyl glycine, (S)-azidoalanine, (S)- homopropargyl glycine, (S)-azidohomoalanine, (S)-azidonorvaline and (S)-bishomopropargyl glycine;
  • a A is Citrulline
  • AA is selected from the group consisting of (S)-azidohomoalanine, (S)-homopropargyl glycine, (S)-azidonorvaline, and (S)-bishomopropargyl glycine;
  • a A is selected from the group consisting of (S)-propargyl glycine, (S)- homopropargyl glycine, and (S)-bishomopropargyl glycine, X 12
  • AA is (S)-azidohomoalanine or (S)-azidonorvaline; when X 3
  • AA is selected from the group consisting of (S)-azidoalanine, (S)- azidohomoalanine, and (S)-azidonorvaline, X 12
  • a A is (S)-homopropargyl glycine or (S)- bishomopropargyl glycine;
  • X is as defined above for the conotoxin peptide analog of Formula (I).
  • AA is (S)-propargyl glycine
  • AA Citrulline
  • a A is 3-iodo-Tyr
  • AA is (S)-azidonorvaline
  • amino acid sequence of the conotoxin peptide analog is Gly-Cys-X 3
  • a A is selected from the group consisting of (S)-propargyl glycine, (S)-azidoalanine, (S)- homopropargyl glycine, (S)-azidohomoalanine, (S)-azidonorvaline and (S)-bishomopropargyl glycine;
  • a A is Citrulline
  • AA is selected from the group consisting of (S)-azidohomoalanine, (S)-homopropargyl glycine, (S)-azidonorvaline, and (S)-bishomopropargyl glycine;
  • a A is selected from the group consisting of (S)-propargyl glycine, (S)- homopropargyl glycine, and (S)-bishomopropargyl glycine, X 12
  • AA is (S)-azidohomoalanine or (S)-azidonorvaline; when X 3
  • AA is selected from the group consisting of (S)-azidoalanine, (S)- azidohomoalanine, and (S)-azidonorvaline, X 12
  • a A is (S)-homopropargyl glycine or (S)- bishomopropargyl glycine;
  • AA is Tyr, Phe, Trp, or a D-isomer of Tyr, Phe, or Trp, and X 2
  • a A is N-Me-Gly, D-Tyr, or N-Me-Tyr;
  • C-terminus of the conotoxin peptide analog is a carboxylic acid or an amide group.
  • amino acid sequence of the conotoxin peptide analog is Gly-Cys-X 3
  • a A is (S)-propargyl glycine
  • AA Citrulline
  • AA is 3-iodo-Tyr
  • a A is (S)-azidonorvaline
  • FIG.1A-FIG.1D Disulfide shuffling of CSP-4-NH2 in human serum.
  • FIG.1A CSP-4-NH2 was spiked into human serum and incubated at 37 °C for the specified period, after which samples were frozen, purified by precipitation and filtration and analyzed by RP-HPLC. A time-dependent conversion of main peak to secondary peak with faster retention time was observed.
  • FIG.1B Individual injections of synthetically constructed CSP-4-NH2 ribbon, bead and native isomers together with a sample of CSP-4-NH2 isolated from human serum were analyzed. Comigration of serum-derived peaks indicated conversion of the native form to the ribbon isomer .
  • FIG.1C Isolation of serum-derived compound CSP-4-NH2 followed by lyophilization and mass spectrometry analysis indicated that both the native and ribbon isomer have the same mass-to-charge ratio.
  • FIG.1D Conversion of the globular to ribbon form by glutathione reduction of CSP-4-NH2 indicated that disulfide shuffling underlies isomerization.
  • FIG.2A-FIG.2B Reverse phase HPLC analyses of conotoxin peptide analog Ia and conotoxin peptide analog CSP-4-NH2 in rat plasma.
  • FIG.2A reverse phase HPLC analyses of conotoxin peptide analog Ia at 0 h, 8 h and 24 h (black line: 0 h; dark grey line: 8 h; light grey line: 24 h)..
  • FIG.2B reverse phase HPLC analyses of conotoxin peptide analog CSP-4-NH 2 (“native” form, consisting of two disulfide bonds, one between Cys2 and Cys8, and a second between Cys3 and Cys12) in rat plasma at 0 h, 8 h and 24 h (black line: 0 h; dark grey line: 8 h; light grey line: 24 h).
  • FIG.3A-FIG.3B Concentration-response curves of conotoxin peptide analogs for inhibition of ACh-gated currents in the human a9a10 nAChR.
  • FIG.3A Concentration-response curves of conotoxin peptide analogs Ia, Ia’ and Ib’ for inhibition of ACh-gated currents in the human a9a10 nAChR ( ⁇ : Ia; ⁇ : Ia’; ⁇ : Ib’).
  • FIG.3B Concentration-response curves of conotoxin peptide analogs CSP-4-NH2 and CSP-4-OH for inhibition of ACh-gated currents in the human a9a10 nAChR ( ⁇ : CSP-4-NH2; ⁇ : CSP-4-OH).
  • FIG.4 Comparison of the amino acid sequences of RgIA (SEQ ID NO:1), ImI (SEQ ID NO:2) and Vc1.1 (SEQ ID NO:3).
  • FIG.5A-FIG.5B Concentration-response curves of conotoxin peptide analogs for inhibition of ACh-gated currents in the human a9a10 nAChR.
  • FIG.5A Concentration-response curves of conotoxin peptide analogs CSP-4-OH and CSP-4-desTyr-OH for inhibition of ACh- gated currents in the human a9a10 nAChR ( ⁇ : CSP-4-OH; ⁇ : CSP-4-desTyr-OH).
  • FIG. 5B Concentration-response curves of conotoxin peptide analogs 1a and 1q for inhibition of ACh-gated currents in the human a9a10 nAChR ( ⁇ : 1a; ⁇ : 1q).
  • FIG.6A-FIG.6B Representative traces from an oocyte injected with either the human (left) or rat (right) a9a10 nAChR. ACh-gated currents before and after exposure to compound PEGyated conotoxin peptide analog IIa.
  • FIG.6A Response to ACh of the human a9a10 nAChR.
  • FIG.6B Response to ACh of the rat a9a10 nAChR.
  • FIG.7A Pharmacokinetic profile of PEGylated conotoxin peptide analog IIa ( : 1 mg/kg IV administration; : 1 mg/kg SC administration).
  • FIG.7B Pharmacokinetic profile of conotoxin peptide analog Ia ( : 1 mg/kg IV administration; 1 mg/kg SC administration).
  • FIG.9 Analgesic efficacy and duration of effect of conotoxin peptide analog Ib’ in a rat chronic constriction injury (CCI) model ( ⁇ : 0.1 mg/kg/day of conotoxin peptide analog Ib’; ⁇ : vehicle).
  • FIG.10A-FIG.10C Analgesic efficacy and duration of effect of conotoxin peptide analog CSP-4-NH2 and PEGylated derivatives of CSP-4-NH2 in rat CIPN model.
  • FIG.10A Mechanical hyperalgesia was measured by Randall-Selitto testing, demonstrating analgesic efficacy (normalized response) of CSP-4-NH2 and PEGylated conotoxin peptides VIII and X in the rat CIPN model over 96 h post dose.
  • FIG.10B Mechanical hyperalgesia was measured by Randall-Selitto testing, demonstrating analgesic efficacy (normalized response) of CSP-4-NH2 and PEGylated conotoxin peptides VII, IX, XI and XII in the rat CIPN model over 96 h post dose.
  • FIG.10C Summary plot of duration of efficacy of CSP-4-NH2 and PEGylated derivatives of CSP-4-NH2 showed that duration of efficacy correlates with both PEGylation conjugation chemistry (branch vs. linear) and conjugated PEG polymer size.
  • FIG.11A-FIG.11C Analgesic efficacy and duration of effect of conotoxin peptide analogs Ia’, Ia, and PEGylated conotoxin peptide analog IIa in a rat chemotherapy induced peripheral neuropathy (CIPN) model.
  • FIG.11A Administration of a single 0.5 mg/kg dose of conotoxin peptide analog Ia’ to rats having oxaliplatin-induced peripheral neuropathy, 14 days following induction of neuropathy, resulted in a statistically significant reduction in mechanical hyperalgesia compared to vehicle treated control animals for up to 24 h. ( ⁇ : 0.5 mg/kg of conotoxin peptide analog Ia’; ⁇ : vehicle).
  • FIG.11B Administration of a single 0.5 mg/kg dose of Ia’ to rats with spared nerve injury on day 14 following surgical induction of the model resulted in a statistically significant reduction in mechanical hyperalgesia compared to vehicle treated control animals for up to 24 h ( ⁇ : 0.5 mg/kg of conotoxin peptide analog Ia’; ⁇ :
  • FIG.11C Administration of a single 0.5 mg/kg dose of conotoxin peptide analog Ia or PEGylated conotoxin peptide analog IIa to rats with oxaliplatin-induced peripheral neuropathy, 14 days following induction of neuropathy, resulted in a significant reduction in mechanical hyperalgesia compared to vehicle treated control animals for up to 4 h (conotoxin peptide analog Ia) or 72 h conotoxin peptide analog Ia) post dose ( vehicle; : conotoxin peptide analog Ia; PEGylated conotoxin peptide analog IIa).
  • Mechanical hyperalgesia was measured by Randall Selitto paw withdrawal threshold in grams. The endpoint for all studies was the Randall Siletto paw withdrawal threshold in grams. Two-way ANOVA, *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001. 5.
  • alpha-conotoxin peptide analogs including alpha-conotoxin peptide analogs that are covalently attached to polyethylene glycol (PEG), and pharmaceutical compositions of such alpha-conotoxin peptide analogs.
  • PEG polyethylene glycol
  • nAChR nicotinic acetylcholine receptor
  • amino acids According to the single letter or three letter codes.
  • the amino acids forming all or a part of a peptide may be from among the known 21 naturally occurring amino acids, which are referred to by both their single letter abbreviations and their common three-letter abbreviation.
  • conventional amino acid residues have their conventional meaning.
  • “Leu” is leucine
  • “Ile” is isoleucine
  • “Nle” is norleucine
  • AA is Tyr, Phe, Trp, or a D-isomer of Tyr, Phe, or Trp, and X 2
  • AA is N-Me-Gly, D-Tyr, or N-Me-Tyr;
  • C-terminus of the conotoxin peptide analog is a carboxylic acid or an amide group.
  • the triazole bridge is
  • the single wavy line ( ) indicates the point of attachment of the triazole bridge to the C 1 carbon of the conotoxin peptide analog
  • the double wavy lines ( indicate the point of attachment of the triazole bridge to the C 2 carbon of the conotoxin peptide analog
  • x is 1, 2, 3, or 4
  • y is 2, 3 or 4.
  • the triazole bridge is
  • the triazole bridge is
  • x is 1, 2, 3, or 4. In one preferred embodiment, x is 1, 2, or 3. In a specific embodiment, x is 1 or 2. In one embodiment, x is 1. In another embodiment, x is 2.
  • y is 2, 3, or 4. In a specific embodiment, y is 2 or 3. In one embodiment, y is 3. In another embodiment, y is 2.
  • x is 1, 2 or 3, and y is 2 or 3. In a specific embodiment, x is 1, 2, or 3, and y is 2. In a specific embodiment, x is 1, 2, or 3, and y is 3. In a specific embodiment, x is 1 or 2, and y is 2 or 3. In a specific embodiment, x is 1 or 3, and y is 2 or 3. In a specific embodiment, x is 2 or 3, and y is 2 or 3. In a specific embodiment, x is 1, and y is 2 or 3. In a specific embodiment, x is 2, and y is 2 or 3. In a specific embodiment, x is 3, and y is 2 or 3. In one preferred embodiment, x is 1 and y is 3. In one preferred embodiment, x is 2 and y is 3. In one preferred embodiment, x is 2 and y is 2.
  • the triazole bridge is , wherein the single wavy line ( ) indicates the point of attachment of the triazole bridge to the C 1 carbon of the conotoxin peptide analog, and the double wavy lines ( ) indicate the point of attachment of the triazole bridge to the C 2 carbon of the conotoxin peptide analog.
  • the triazole bridge is , wherein the single wavy line ( ) indicates the point of attachment of the triazole bridge to the C 1 carbon of the conotoxin peptide analog, and the double wavy lines ( ) indicate the point of attachment of the triazole bridge to the C 2 carbon of the conotoxin peptide analog.
  • X is X 1 2
  • X is X 1
  • a A is Tyr, Phe, Trp, or a D- isomer of Tyr, Phe, or Trp.
  • AA is Tyr, Phe, or Trp.
  • X 1 is Trp.
  • AA is Tyr, D- Tyr, or Phe.
  • X 1 is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-N- Tyr, or Phe.
  • AA is Tyr or D-Tyr.
  • X 1 is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-N-Tyr.
  • AA is Tyr or Phe.
  • X 1 is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethy
  • a A is D-Tyr or Phe.
  • a A is Phe.
  • AA is D-Phe.
  • X 1 is N-Phe.
  • AA is Trp.
  • X 1 is Trp.
  • AA is D-Trp.
  • X 1 is D-Trp.
  • AA is D-Tyr.
  • X 1 is D-Tyr.
  • a A is Tyr. [00213] In a specific embodiment, X 2
  • AA is N-Me-Gly, D-Tyr, or N-Me-Tyr.
  • X 2 is N-Me-Gly, D-Tyr, or N-Me-Tyr.
  • a A is N-Me-Gly or D-Tyr.
  • X 2 is N-Me-Gly or D-Tyr.
  • a A is D-Tyr or N-Me- Tyr.
  • X 2 is N-Me- Tyr.
  • a A is N-Me-Gly or N-Me-Tyr.
  • X 2 is N-Me-Gly or N-Me-Tyr.
  • AA is N-Me-Gly.
  • X 2 is N-Me-Gly.
  • AA is N-Me-Tyr.
  • X 2 is N-Me-Tyr.
  • AA is D-Tyr.
  • AA is optionally present.
  • X 2 is optionally present.
  • a A is present.
  • a A is absent.
  • X is selected from the group consisting of Tyr, Phe, D-Tyr, (Tyr)-(D-Tyr), (Tyr)-(N-Me-Gly), (Tyr)-(N-Me-Tyr), N-Me-Tyr, D-Arg, N-Me-D-Tyr, beta-Tyr, and N-Me-Arg.
  • X is selected from the group consisting of Tyr, Phe, D-Tyr, (Tyr)-(D-Tyr), (Tyr)-(N-Me-Gly), and (Tyr)-(N-Me-Tyr).
  • X is selected from the group consisting of Tyr, Phe, and D-Tyr. In a specific embodiment, X is selected from the group consisting of (Tyr)-(D-Tyr), (Tyr)-(N-Me-Gly), and (Tyr)-(N-Me-Tyr). In a specific embodiment, X is selected from the group consisting of N-Me-Tyr, D-Arg, N-Me- D-Tyr, beta-Tyr, and N-Me-Arg. In a specific embodiment, X is Tyr. In a specific embodiment, X is Phe. In a specific embodiment, X is D-Tyr.
  • X is (Tyr)-(D-Tyr). In a specific embodiment, X is (Tyr)-(N-Me-Gly). In a specific embodiment, X is (Tyr)-(N-Me- Tyr). In a specific embodiment, X is N-Me-Tyr. In a specific embodiment, X is D-Arg. In a specific embodiment, X is N-Me-D-Tyr. In a specific embodiment, X is beta-Tyr. In a specific embodiment, X is N-Me-Arg.
  • the C-terminus of the conotoxin peptide analog is a carboxylic acid or an amide group. In one preferred embodiment, the C-terminus of the conotoxin peptide analog is OH. In a specific embodiment, the C-terminus of the conotoxin peptide analog is NH 2 .
  • the conotoxin peptide analog or pharmaceutically acceptable salt of Formula (I) is of Formula (Ia) (SEQ ID NO:94):
  • R 1 is OH or NH 2 .
  • R 1 is OH.
  • R 1 is NH2.
  • the conotoxin peptide analog or pharmaceutically acceptable salt of Formula (I) is of Formula (Ig) (SEQ ID NO:30):
  • the conotoxin peptide analog or pharmaceutically acceptable salt of Formula (I) is of Formula (Ih) (SEQ ID NO:33):
  • the conotoxin peptide analog or pharmaceutically acceptable salt of Formula (I) is of Formula (Ii) (SEQ ID NO:36):
  • the conotoxin peptide analog or pharmaceutically acceptable salt of Formula (I) is of Formula (Ik) (SEQ ID NO:42):
  • the conotoxin peptide analog or pharmaceutically acceptable salt of Formula (I) is of Formula (Il) (SEQ ID NO:45):
  • the conotoxin peptide analog or pharmaceutically acceptable salt of Formula (I) is of Formula (Im) (SEQ ID NO:48):
  • the conotoxin peptide analog or pharmaceutically acceptable salt of Formula (I) is of Formula (In) (SEQ ID NO:51):
  • the conotoxin peptide analog or pharmaceutically acceptable salt of Formula (I) is of Formula (Io) (SEQ ID NO:54):
  • the conotoxin peptide analog or pharmaceutically acceptable salt of Formula (I) is of Formula (Ip) (SEQ ID NO:57):
  • conotoxin peptide analogs of Formula (Ib) (SEQ ID NO:104):
  • R 2 is OH or NH2.
  • R 2 is OH. In a specific embodiment, R 2 is NH2.
  • conotoxin peptide analogs selected from the group consisting of conotoxin peptide analogs Ia, Ia’, Ib, Ib’, Ic, Id, Ie, If, Ig, Ih, Ii, Ij, Ik, Il, Im, In, Io, Ip, Iq, Ir, Is, It, Iu, and Iv, or a pharmaceutically acceptable salt thereof.
  • the conotoxin peptide analog is selected from the group consisting of conotoxin peptide analogs Ia, Ia’, Ib, Ib’, Ig, Ih, Ii, Ik, Il, Im, In, Io, and Ip.
  • the conotoxin peptide analog is selected from the group consisting of conotoxin peptide analogs Ia, Ia’, Ib, and Ib’. In one preferred embodiment, the conotoxin peptide analog is conotoxin peptide analog Ia or Ia’. In one preferred embodiment, the conotoxin peptide analog is conotoxin peptide analog Ia. 5.3. PEGylated Conotoxin Peptide RgIA Analogs
  • the conotoxin peptide analogs of the invention are PEGylated, in particular, covalently attached to one or more PEG polymers.
  • a conotoxin peptide analog is covalently attached to one PEG polymer. In a specific embodiment, a conotoxin peptide analog is covalently attached to more than one PEG polymers. In a specific embodiment, a conotoxin peptide analog is covalently attached to two PEG polymers. In a specific embodiment, a conotoxin peptide analog is covalently attached to three PEG polymers. [00233] In a preferred embodiment, a PEG polymer is covalently attached to the N-terminus of a conotoxin peptide analog, most preferably one PEG polymer is attached only to the N- terminus.
  • a PEG polymer is covalently attached to the C-terminus of a conotoxin peptide analog. In a specific embodiment, a PEG polymer is covalently attached to an amino acid residue position that is not the N-terminus or the C-terminus of the conotoxin peptide analog.
  • a PEG polymer is covalently attached to the conotoxin peptide analog via a linking group. In a specific embodiment, a PEG polymer is covalently attached to the conotoxin peptide analog directly.
  • the linking group is a valerate linker having a formula
  • the linking group is a butylene.
  • the linking group is a butylene.
  • the linking group is a carbonyl.
  • the PEG polymer is a linear or branched PEG polymer. In a specific embodiment, the PEG polymer is a branched PEG polymer. In one preferred embodiment, the PEG polymer is a linear PEG polymer.
  • the PEG polymer has a molecular weight in the range of 10 kDa and 40 kDa. In one preferred embodiment, the PEG polymer is a 30 kDa PEG polymer. In specific embodiments, the PEG polymer is a linear 30 kDa PEG polymer. In specific
  • the PEG polymer is a linear 30 kDa mPEG polymer.
  • one PEG polymer is attached at the amino terminus of a conotoxin peptide analog, and the PEG polymer is attached via a linking group to the conotoxin peptide analog, and the linking group is a valerate linker having a formula o
  • the PEG polymer is a linear 30 kDa mPEG polymer.
  • a PEGylated conotoxin peptide analog or pharmaceutically acceptable salt thereof wherein the conotoxin peptide analog is of Formula (I) (SEQ ID NO:93):
  • AA is Tyr, Phe, Trp, or a D-isomer of Tyr, Phe, or Trp, and X 2
  • AA is N-Me-Gly, D-Tyr, or N-Me-Tyr;
  • C-terminus of the conotoxin peptide analog is a carboxylic acid or an amide group
  • conotoxin peptide analog is covalently attached directly or via a linking group to one or more polyethylene glycol (PEG) polymers.
  • PEG polyethylene glycol
  • the triazole bridge is
  • the single wavy line ( ) indicates the point of attachment of the triazole bridge to the C 1 carbon of the conotoxin peptide analog
  • the double wavy lines ( ) indicate the point of attachment of the triazole bridge to the C 2 carbon of the conotoxin peptide analog
  • x is 1, 2, 3, or 4
  • y is 2, 3 or 4.
  • the triazole bridge is
  • the triazole bridge is
  • x is 1, 2, 3, or 4. In one preferred embodiment, x is 1, 2, or 3. In a specific embodiment, x is 1 or 2. In one embodiment, x is 1. In another embodiment, x is 2.
  • y is 2, 3, or 4. In a specific embodiment, y is 2 or 3. In one embodiment, y is 3. In another embodiment, y is 2. [00243] In a specific embodiment, x is 1, 2 or 3, and y is 2 or 3. In a specific embodiment, x is 1, 2, or 3, and y is 2. In a specific embodiment, x is 1, 2, or 3, and y is 3. In a specific embodiment, x is 1 or 2, and y is 2 or 3. In a specific embodiment, x is 1 or 3, and y is 2 or 3. In a specific embodiment, x is 2 or 3, and y is 2 or 3. In a specific embodiment, x is 1, and y is 2 or 3. In a specific embodiment, x is 1, and y is 2 or 3.
  • x is 2, and y is 2 or 3. In a specific embodiment, x is 3, and y is 2 or 3. In one preferred embodiment, x is 1 and y is 3. In one preferred embodiment, x is 2 and y is 3. In one preferred embodiment, x is 2 and y is 2.
  • the triazole bridge is , wherein the single wavy line ( indicates the point of attachment of the triazole bridge to the C 1 carbon of the
  • the triazole bridge is , wherein the single wavy line ( indicates the point of attachment of the triazole bridge to the C 1 carbon of the
  • X is X 1
  • X is X 1 2
  • a A is Tyr, Phe, Trp, or a D- isomer of Tyr, Phe, or Trp.
  • AA is Tyr, Phe, or Trp.
  • X 1 is Trp.
  • AA is Tyr, D- Tyr, or Phe.
  • X 1 is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-N- Tyr, or Phe.
  • a A is Tyr or D-Tyr.
  • a A is Tyr or Phe.
  • a A is D-Tyr or Phe.
  • a A is Phe.
  • AA is D-Phe.
  • X 1 is N-Phe.
  • AA is Trp.
  • X 1 is Trp.
  • AA is D-Trp.
  • X 1 is D-Trp.
  • AA is D-Tyr.
  • X 1 is D-Tyr.
  • a A is Tyr.
  • AA is N-Me-Gly, D-Tyr, or N-Me-Tyr.
  • X 2 is N-Me-Gly, D-Tyr, or N-Me-Tyr.
  • AA is N-Me-Gly or D-Tyr.
  • X 2 is N-Me-Gly or D-Tyr.
  • AA is D-Tyr or N-Me- Tyr.
  • X 2 is N-Me- Tyr.
  • a A is N-Me-Gly or N-Me-Tyr.
  • X 2 is N-Me-Gly or N-Me-Tyr.
  • AA is N-Me-Gly.
  • X 2 is N-Me-Gly.
  • AA is N-Me-Tyr.
  • X 2 is N-Me-Tyr.
  • a A is D-Tyr.
  • a A is optionally present.
  • X 2 is optionally present.
  • a A is present.
  • a A is absent.
  • X is selected from the group consisting of Tyr, Phe, D-Tyr, (Tyr)-(D-Tyr), (Tyr)-(N-Me-Gly), (Tyr)-(N-Me-Tyr), N-Me-Tyr, D-Arg, N-Me-D-Tyr, beta-Tyr, and N-Me-Arg.
  • X is selected from the group consisting of Tyr, Phe, D-Tyr, (Tyr)-(D-Tyr), (Tyr)-(N-Me-Gly), and (Tyr)-(N-Me-Tyr).
  • X is selected from the group consisting of Tyr, Phe, and D-Tyr. In a specific embodiment, X is selected from the group consisting of (Tyr)-(D-Tyr), (Tyr)-(N-Me-Gly), and (Tyr)-(N-Me-Tyr). In a specific embodiment, X is selected from the group consisting of N-Me-Tyr, D-Arg, N-Me- D-Tyr, beta-Tyr, and N-Me-Arg. In a specific embodiment, X is Tyr. In a specific embodiment, X is Phe. In a specific embodiment, X is D-Tyr.
  • X is (Tyr)-(D-Tyr). In a specific embodiment, X is (Tyr)-(N-Me-Gly). In a specific embodiment, X is (Tyr)-(N-Me- Tyr). In a specific embodiment, X is N-Me-Tyr. In a specific embodiment, X is D-Arg. In a specific embodiment, X is N-Me-D-Tyr. In a specific embodiment, X is beta-Tyr. In a specific embodiment, X is N-Me-Arg.
  • the C-terminus of the conotoxin peptide analog is a carboxylic acid or an amide group. In one preferred embodiment, the C-terminus of the conotoxin peptide analog is OH. In a specific embodiment, the C-terminus of the conotoxin peptide analog is NH2.
  • the conotoxin peptide analog or pharmaceutically acceptable salt of Formula (I) is of Formula (Ia) (SEQ ID NO:94):
  • R 1 is OH or NH2. In a specific embodiment, R 1 is OH. In a specific embodiment, R 1 is NH 2 .
  • the conotoxin peptide analog or pharmaceutically acceptable salt of Formula (I) is of Formula (Ig) (SEQ ID NO:30):
  • the conotoxin peptide analog or pharmaceutically acceptable salt of Formula (I) is of Formula (Ih) (SEQ ID NO:33):
  • the conotoxin peptide analog or pharmaceutically acceptable salt of Formula (I) is of Formula (Ii) (SEQ ID NO:36):
  • the conotoxin peptide analog or pharmaceutically acceptable salt of Formula (I) is of Formula (Ik) (SEQ ID NO:42):
  • the conotoxin peptide analog or pharmaceutically acceptable salt of Formula (I) is of Formula (Il) (SEQ ID NO:45):
  • the conotoxin peptide analog or pharmaceutically acceptable salt of Formula (I) is of Formula (Im) (SEQ ID NO:48):
  • the conotoxin peptide analog or pharmaceutically acceptable salt of Formula (I) is of Formula (In) (SEQ ID NO:51):
  • the conotoxin peptide analog or pharmaceutically acceptable salt of Formula (I) is of Formula (Io) (SEQ ID NO:54):
  • the conotoxin peptide analog or pharmaceutically acceptable salt of Formula (I) is of Formula (Ip) (SEQ ID NO:57):
  • the PEGylated conotoxin peptide analog is of Formula (IIa) (
  • the PEGylated conotoxin peptide analog is of Formula (IIg) (
  • the PEGylated conotoxin peptide analog is of Formula (IIh) (SEQ ID NO:96):
  • the PEGylated conotoxin peptide analog is of Formula (IIi) (
  • the PEGylated conotoxin peptide analog is of Formula (IIk) (
  • the PEGylated conotoxin peptide analog is of Formula (IIl) (SEQ ID NO:99):
  • the PEGylated conotoxin peptide analog is of Formula (IIm) (
  • the PEGylated conotoxin peptide analog is of Formula (IIn) (
  • the PEGylated conotoxin peptide analog is of Formula (IIo) (SEQ ID NO:102):
  • the PEGylated conotoxin peptide analog is of Formula (IIp) (
  • PEGylated conotoxin peptide analogs or a pharmaceutically acceptable salt thereof wherein the conotoxin peptide analog is of Formula (Ib) (SEQ ID NO:104):
  • conotoxin peptide analog is covalently attached directly or via a linking group to one or more polyethylene glycol (PEG) polymers.
  • PEG polyethylene glycol
  • R 2 is OH. In a specific embodiment of Formula (Ib), R 2 is NH 2 .
  • the PEGylated conotoxin peptide analog is of Formula (IIb) (SEQ ID NO:105):
  • compositions which comprise a conotoxin peptide analog provided herein or a PEGylated conotoxin peptide analog provided herein, and one or more pharmaceutically acceptable carriers.
  • a conotoxin peptide analog is present in a therapeutically effective amount.
  • a conotoxin peptide analog is present in a prophylactically effective amount.
  • the pharmaceutical compositions can be used in accordance with the methods and uses provided herein. Thus, for example, the pharmaceutical compositions can be administered to a subject in order to practice the treatment or prevention methods and uses provided herein.
  • Pharmaceutical compositions provided herein can be formulated to be compatible with the intended method or route of administration; exemplary routes of administration are set forth herein.
  • compositions typically comprise a therapeutically effective amount of at least one of the conotoxin peptide analogs or PEGylated conotoxin peptide analogs provided herein, and a pharmaceutically acceptable carrier.
  • suitable pharmaceutically acceptable carriers include, but are not limited to, antioxidants (e.g., ascorbic acid), preservatives (e.g., benzyl alcohol, methyl parabens, p-hydroxybenzoate), emulsifying agents, suspending agents, dispersing agents, solvents, buffers, lubricants, fillers, and/or diluents.
  • a suitable vehicle may be physiological saline solution.
  • Typical buffers that can be used include, but are not limited to pharmaceutically acceptable weak acids, weak bases, or mixtures thereof.
  • Buffer components can also include water soluble reagents such as phosphoric acid, tartaric acids, succinic acid, citric acid, acetic acid, and salts thereof.
  • a vehicle may contain other pharmaceutically acceptable excipients for modifying or maintaining the pH, osmolarity, viscosity, or stability of the pharmaceutical composition.
  • the vehicle is an aqueous buffer.
  • a vehicle comprises, for example, sodium chloride.
  • compositions provided herein may contain still other
  • formulation agents for modifying or maintaining the rate of release of a conotoxin peptide analog described herein.
  • formulation agents include, for example, those substances known to those skilled in the art in preparing sustained-release or controlled release formulations.
  • pharmaceutically acceptable formulation agents see, for example, Remington’s Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, Pa.18042) pages 1435-1712, and The Merck Index, 12th Ed. (1996, Merck Publishing Group, Whitehouse, NJ).
  • a pharmaceutical composition is provided in a sterile vial as a solution, suspension, gel, emulsion, or dehydrated or lyophilized powder.
  • compositions may, for example, be stored either in a ready to use form, a lyophilized form requiring reconstitution prior to use, or a liquid form requiring dilution prior to use.
  • a pharmaceutical composition is provided in a single-use container (e.g., a single- use vial, ampoule, syringe, or autoinjector).
  • a pharmaceutical composition is provided in a multi-use container (e.g., a multi-use vial or cartridge). Any drug delivery apparatus may be used to deliver a conotoxin peptide analog or a PEGylated conotoxin peptide analog or pharmaceutical composition described herein, including implants (e.g., implantable pumps) and catheters.
  • depot injections can be utilized to release a conotoxin peptide analog or a PEGylated conotoxin peptide analog or pharmaceutical composition described herein over a defined period of time. Depot injections are generally administered subcutaneously or intramuscularly.
  • a pharmaceutical composition can be formulated to be compatible with its intended route of administration as described herein.
  • compositions may be in the form of a sterile injectable aqueous suspension.
  • This suspension may be formulated using suitable dispersing or wetting agents and suspending agents known to those skilled in the art.
  • the sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent.
  • the sterile injectable solution can, for example, be a solution in 1,3- butane diol.
  • Acceptable diluents, solvents or dispersion media include water, Ringer’s solution, saline, Cremophor ELTM, phosphate buffered saline (PBS), ethanol, polyol (e.g., glycerol or liquid polyethylene glycol), and mixtures thereof.
  • Prolonged absorption of particular injectable formulations can be achieved by including an agent that delays absorption (e.g., aluminum monostearate or gelatin).
  • compositions can also include carriers to protect the composition against degradation or elimination from the body.
  • Various antibacterial and antifungal agents for example, parabens, chlorobutanol, ascorbic acid, thimerosal, can be included in the
  • nAChR nicotinic acetylcholine receptor
  • the condition conducive to treatment or prevention by inhibition of an a9-containing nicotinic acetylcholine receptor is a condition conducive to treatment or prevention by inhibition of the a9a10 subtype of nAChR.
  • the condition conducive to treatment or prevention by inhibition of an a9-containing nAChR is pain or inflammation.
  • an a9-containing nAChR e. g., the a9a10 subtype of nAChR
  • the method provided herein is a method of treating a condition conducive to treatment by inhibition of an a9-containing nicotinic acetylcholine receptor (nAChR) comprising administering to a subject a therapeutically effective amount of a conotoxin peptide analog as described herein or a pharmaceutically acceptable salt thereof, or a PEGylated conotoxin peptide analog as described herein or pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described herein.
  • nAChR nicotinic acetylcholine receptor
  • the condition conducive to treatment by inhibition of an a9-containing nicotinic acetylcholine receptor is a condition conducive to treatment by inhibition of the a9a10 subtype of nAChR.
  • the condition conducive to treatment by inhibition of an a9-containing nAChR is pain or inflammation.
  • the method provided herein is a method of preventing a condition conducive to prevention by inhibition of an a9-containing nicotinic acetylcholine receptor (nAChR) comprising administering to a subject a therapeutically effective amount of a conotoxin peptide analog as described herein or a pharmaceutically acceptable salt thereof, or a PEGylated conotoxin peptide analog as described herein or pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described herein.
  • nAChR nicotinic acetylcholine receptor
  • the condition conducive to prevention by inhibition of an a9-containing nicotinic acetylcholine receptor is a condition conducive to treatment by inhibition of the a9a10 subtype of nAChR.
  • the condition conducive to prevention by inhibition of an a9-containing nAChR is pain or inflammation.
  • the condition conducive to treatment or prevention by inhibition of an a9-containing nicotinic acetylcholine receptor is pain.
  • the pain is selected from the group consisting of general pain, chronic pain, neuropathic pain, nociceptive pain, inflammatory pain, pain induced by peripheral nerve damage, pain induced by an inflammatory disorder, pain induced by a metabolic disorder, pain induced by cancer, pain induced by chemotherapy, pain induced by a surgical procedure, and pain induced by a burn.
  • the pain is cancer- related chronic pain.
  • the pain is pain associated with neuropathy, which, by way of example, can be drug (e.g., cancer chemotherapeutic)-induced neuropathy or infection-induced neuropathy.
  • the pain is neuralgia, including but not limited to postherpetic neuralgia, for example, postherpetic neuralgia due to shingles.
  • the condition conducive to treatment or prevention by inhibition of an a9-containing nicotinic acetylcholine receptor is an inflammatory condition.
  • the inflammatory condition is selected from the group consisting of is inflammation, chronic inflammation, a rheumatic disease, sepsis, fibromyalgia, inflammatory bowel disease, sarcoidosis, endometriosis, uterine fibroids, an inflammatory skin disease, an inflammatory condition of the lungs, a disease associated with inflammation of the nervous system, periodontal disease, and cardiovascular disease.
  • the inflammatory condition is mediated by immune cells.
  • the inflammatory condition is long-term inflammation and/or peripheral neuropathy following injury.
  • the condition associated with the a9-containing nicotinic acetylcholine receptor e. g., the a9a10 subtype of nAChR
  • the condition associated with the a9a10 subtype of nAChR condition is inflammation and neuropathy.
  • a method of treating or preventing a condition associated with activation of an a9-containing nAChR, e. g., the a9a10 subtype of nAChR, in a subject comprising administering to the subject a therapeutically effective amount of a conotoxin peptide analog as described herein or pharmaceutically acceptable salt thereof, or a PEGylated conotoxin peptide analog as described herein or pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described herein.
  • a method of treating or preventing pain or inflammation in a subject comprising administering to the subject a therapeutically effective amount of a conotoxin peptide analog as described herein or pharmaceutically acceptable salt thereof, or a PEGylated conotoxin peptide analog as described herein or pharmaceutically acceptable salt thereof, or pharmaceutical composition as described herein.
  • the pain is pain associated with neuropathy, which, by way of example, can be drug (e.g., cancer chemotherapeutic)-induced neuropathy or infection-induced neuropathy.
  • the pain is neuralgia, including but not limited to postherpetic neuralgia, for example, postherpetic neuralgia due to shingles.
  • the method provided herein is a method of treating pain or inflammation. In a specific embodiment, the method provided herein is a method of treating pain. In a specific embodiment, the method provided herein is a method of treating
  • the method provided herein is a method of preventing pain or inflammation. In a specific embodiment, the method provided herein is a method of preventing pain. In a specific embodiment, the method provided herein is a method of preventing inflammation.
  • the method of preventing pain is to prevent pain associated with neuropathy.
  • the pain associated with neuropathy is neuralgia.
  • the neuralgia is postherpetic neuralgia.
  • the method of preventing pain is to prevent pain induced by cancer chemotherapy (and thus a conotoxin peptide analog or pharmaceutically acceptable salt thereof is administered to the patient prior to the patient being administered cancer chemotherapy).
  • the method of preventing pain is to prevent pain induced by a surgical procedure; in a specific embodiment of such method, a conotoxin peptide analog or pharmaceutically acceptable salt thereof is administered prior to and/or concurrently with the surgical procedure, in particular, where the surgical procedure is reasonably expected to result in pain and/or inflammation.
  • nAChR nicotinic acetylcholine receptor
  • a method of inhibiting an a9-containing nicotinic acetylcholine receptor (nAChR), e.g., the a9a10 subtype of nAChR comprising administering to the subject a therapeutically effective amount of a conotoxin peptide analog as described herein or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described herein.
  • nAChR nicotinic acetylcholine receptor
  • “treat”,“treating”, treatment” and the like refer to an action (such as administering a conotoxin peptide analog or pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising a conotoxin peptide analog or pharmaceutically acceptable salt thereof) initiated after a disease, disorder or condition, or a symptom thereof, has been diagnosed, observed, and the like so as to eliminate, reduce, suppress, mitigate, or ameliorate, either temporarily or permanently, at least one of the underlying causes of a disease, disorder, or condition afflicting a subject, or at least one of the symptoms associated with a disease, disorder, condition afflicting a subject.
  • treatment can include inhibiting (i.e., arresting the development or further development of the disease, disorder or condition or clinical symptoms associated therewith) an active disease.
  • the prevention need not be complete; in a specific embodiment, the prevention is partial inhibition or partial prevention or a reduction in the disease, condition or disorder relative to that which would otherwise be expected to occur absent the administration of the conotoxin peptide analog or pharmaceutically acceptable salt thereof.
  • the administering can be by injecting or otherwise physically delivering a conotoxin peptide analog into a patient, such as by mucosal, intradermal, intravenous, intramuscular delivery and/or any other method of physical delivery described herein or known in the art.
  • a disease, disorder or condition, or a symptom thereof is being treated, administration of the conotoxin peptide analog or pharmaceutically acceptable salt thereof typically occurs after the onset of disease, disorder or condition or symptom thereof.
  • administration of the conotoxin peptide analog or pharmaceutically acceptable salt thereof typically occurs before the onset of the disease, disorder or condition or symptom thereof.
  • a conotoxin peptide analog or pharmaceutically acceptable salt thereof is administered prior to and after the onset of pain and/or inflammation to both prevent and treat the pain and/or inflammation.
  • a conotoxin peptide analog including but not limited to a PEGylated conotoxin peptide analog, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition, as described herein, is used in any of the methods provided herein.
  • a conotoxin peptide analog including but not limited to a PEGylated conotoxin peptide analog, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition, provided herein can be used in a method for treatment or prevention of a disease or disorder or condition described herein.
  • a conotoxin peptide analog including but not limited to a PEGylated conotoxin peptide analog, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition, provided herein can be for use as a medicament.
  • a conotoxin peptide analog including but not limited to a PEGylated conotoxin peptide analog, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition, provided herein can be for use in a method for the treatment or prevention of a condition conducive to treatment or prevention by inhibition of an a9-containing nicotinic acetylcholine receptor (nAChR), e.g. the a9a10 subtype of nAChR.
  • nAChR nicotinic acetylcholine receptor
  • a conotoxin peptide analog including but not limited to a PEGylated conotoxin peptide analog, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition, provided herein can be for use in a method for the treatment or prevention of a condition conducive to treatment or prevention by inhibition of an a9-containing nicotinic acetylcholine receptor (nAChR), e.g. the a9a10 subtype of nAChR.
  • nAChR nicotinic acetylcholine receptor
  • a conotoxin peptide analog including but not limited to a PEGylated conotoxin peptide analog, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition, provided herein can be for use in a method for the treatment or prevention of pain or inflammation.
  • a conotoxin peptide analog including but not limited to a PEGylated conotoxin peptide analog, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition, described herein may be administered to a patient by any of a variety of routes. These include, but are not limited to, parenteral, intra-articular, intranasal, intratracheal, oral, intradermal, topical, intramuscular, intraperitoneal, transdermal, intravenous, intratumoral, conjunctival, subcutaneous, and pulmonary routes.
  • a conotoxin peptide analog including but not limited to a PEGylated conotoxin peptide analog, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition, described herein is administered via subcutaneous administration.
  • a conotoxin peptide analog including but not limited to a PEGylated conotoxin peptide analog, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition, described herein is administered via intravenous administration.
  • a conotoxin peptide analog including but not limited to a PEGylated conotoxin peptide analog, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition, described herein is administered via intra-articular administration.
  • a conotoxin peptide analog including but not limited to a PEGylated conotoxin peptide analog, or a pharmaceutically acceptable salt thereof, to be administered to the patient will depend on the nature of the disease and the condition of the patient, and can be determined by standard clinical techniques and the knowledge of the physician.
  • the precise dose and regime to be employed in a composition will also depend on the route of administration, and the seriousness of the disease, and should be decided according to the judgment of the physician and each patient's circumstance. Determination of the proper dosage can be determined by one skilled in the medical arts.
  • the total daily dosage may be divided and administered in portions throughout the day or by means providing continuous delivery.
  • a conotoxin peptide analog or pharmaceutically acceptable salt thereof is administered to a human subject at a dosage of between 0.01 to about 50 mg/kg of body weight.
  • a conotoxin peptide analog e.g., a PEGylated conotoxin peptide analog, or a pharmaceutically acceptable salt thereof, is administered to a human subject at a dosage of about 0.5 mg/kg of body weight.
  • the human dose is from 1 to 1000 mg/day.
  • the human daily dose is from 1 to 750 mg/day; or from 10 to 500 mg/day.
  • a conotoxin peptide analog e.g., a PEGylated conotoxin peptide analog, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition, disclosed herein can be administered at once, or multiple times at intervals of time. It is understood that the precise dosage and duration of treatment may vary with the age, weight, and condition of the patient being treated, and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test or diagnostic data. It is further understood that for any particular individual, specific dosage regimens may be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the formulations. 5.7. Patients
  • the patient referred to in this disclosure can be, but is not limited to, a human or non- human vertebrate such as a wild, domestic or farm animal.
  • the patient is a mammal, e.g., a human, a cow, a dog, a cat, a goat, a horse, a sheep, a pig, a rabbit, a rat, or a mouse.
  • the patient is a human patient.
  • the human patient is an adult (at least age 16). In another specific embodiment, the human patient is an adolescent (age 12-15). In another specific embodiment, the patient is a child (under age 12). 5.8. Methods of Making Conotoxin Peptide Analogs [00311] This disclosure provides methods of making conotoxin peptide analogs, in which intermediate conotoxin peptide analogs (prior to triazole bridge formation) are subjected to triazole formation conditions to form the conotoxin peptide analogs of the invention. 5.8.1. Intermediate Conotoxin Peptide Analogs
  • This disclosure provides syntheses of intermediate conotoxin peptide analogs for synthesizing conotoxin peptide analogs with a triazole bridge.
  • An intermediate conotoxin peptide analog can, for example, be synthesized using solid phase peptide synthesis.
  • necessary amino acids with appropriate reaction groups for example, an azide group or an acetylene group
  • necessary amino acids can be introduced into the peptide (e.g., a conotoxin peptide of RgIA).
  • the necessary amino acids can be introduced into the 3- and 12- position of the conotoxin peptide analog of RgIA.
  • an amino acid residue bearing an acetylene group e.g., (S)-propargyl glycine, (S)-homopropargyl glycine, or (S)- bishomopropargyl glycine
  • an amino acid bearing an azide group e.g., (S)-azidoalanine, (S)-azidohomoalanine, (S)-azidonorvaline
  • the acetylene group and the azide group within the same intermediate conotoxin peptide can form a triazole ring when the intermediate conotoxin peptide is subjected to triazole formation conditions, thereby forming a triazole bridge in the resulting conotoxin peptide analog.
  • the necessary amino acids for forming the triazole bridge are, for example, selected from the group of (S)-propargyl glycine, (S)-azidoalanine, (S)- homopropargyl glycine, (S)-azidohomoalanine, (S)-azidonorvaline, and (S)-bishomopropargyl glycine.
  • AA is selected from the group consisting of (S)-propargyl glycine, (S)-azidoalanine, (S)- homopropargyl glycine, (S)-azidohomoalanine, (S)-azidonorvaline and (S)-bishomopropargyl glycine;
  • AA Citrulline
  • a A is selected from the group consisting of (S)-azidohomoalanine, (S)-homopropargyl glycine, (S)-azidonorvaline, and (S)-bishomopropargyl glycine;
  • AA is selected from the group consisting of (S)-propargyl glycine, (S)- homopropargyl glycine, and (S)-bishomopropargyl glycine, X 12
  • a A is (S)-azidohomoalanine or (S)-azidonorvaline; and when X 3
  • a A is selected from the group consisting of (S)-azidoalanine, (S)-azidohomoalanine, and (S)-azidonorvaline, X 12
  • AA is (S)-homopropargyl glycine or (S)- bishomopropargyl glycine;
  • a A is Tyr, Phe, Trp, or a D-isomer of Tyr, Phe, or Trp, and X 2
  • AA is N-Me-Gly, D-Tyr, or N-Me-Tyr
  • AA is (S)-propargyl glycine or (S)-azidoalanine.
  • a A is (S)-azidonorvaline or (S)-bishomopropargyl glycine.
  • X 3 is (S)-azidonorvaline or (S)-bishomopropargyl glycine.
  • a A is (S)-propargyl glycine and X 12
  • a A is (S)-azidonorvaline.
  • AA is (S)-homopropargyl glycine and X 12
  • AA is (S)-azidonorvaline.
  • X 3 is (S)-azidonorvaline.
  • AA is (S)-homopropargyl glycine and X 12
  • AA is (S)-azidohomoalanine.
  • X 3 is (S)-azidohomoalanine.
  • a A is (S)-azidohomoalanine and X 12
  • a A is (S)-homopropargyl glycine.
  • X 3 is (S)-homopropargyl glycine.
  • AA is (S)-azidohomoalanine and X 12
  • AA is (S)-homopropargyl glycine.
  • X is X 1
  • X is X 1 2
  • a A is Tyr, Phe, Trp, or a D- isomer of Tyr, Phe, or Trp.
  • AA is Tyr, Phe, or Trp.
  • X 1 is Trp.
  • AA is Tyr, D- Tyr, or Phe.
  • X 1 is N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-aminoethyl-N-(2-aminoethyl)-2-N- Tyr, or Phe.
  • a A is Tyr or D-Tyr.
  • a A is Tyr or Phe.
  • a A is D-Tyr or Phe.
  • a A is Phe.
  • AA is D-Phe.
  • X 1 is N-Phe.
  • AA is Trp.
  • X 1 is Trp.
  • AA is D-Trp.
  • X 1 is D-Trp.
  • AA is D-Tyr.
  • X 1 is D-Tyr.
  • a A is Tyr.
  • AA is N-Me-Gly, D-Tyr, or N-Me-Tyr.
  • X 2 is N-Me-Gly, D-Tyr, or N-Me-Tyr.
  • AA is N-Me-Gly or D-Tyr.
  • X 2 is N-Me-Gly or D-Tyr.
  • AA is D-Tyr or N-Me- Tyr.
  • X 2 is N-Me- Tyr.
  • a A is N-Me-Gly or N-Me-Tyr.
  • X 2 2 is N-Me-Gly or N-Me-Tyr.
  • AA is N-Me-Gly. In a specific embodiment, XAA is N-Me-Tyr. In a specific embodiment, X 2
  • AA is D-Tyr.
  • AA is optionally present.
  • X 2 is optionally present.
  • a A is present.
  • a A is absent.
  • X is selected from the group consisting of Tyr, Phe, D-Tyr, (Tyr)-(D-Tyr), (Tyr)-(N-Me-Gly), (Tyr)-(N-Me-Tyr), N-Me-Tyr, D-Arg, N-Me-D-Tyr, beta-Tyr, and N-Me-Arg.
  • X is selected from the group consisting of Tyr, Phe, D-Tyr, (Tyr)-(D-Tyr), (Tyr)-(N-Me-Gly), and (Tyr)-(N-Me-Tyr).
  • X is selected from the group consisting of Tyr, Phe, and D-Tyr. In a specific embodiment, X is selected from the group consisting of (Tyr)-(D-Tyr), (Tyr)-(N-Me-Gly), and (Tyr)-(N-Me-Tyr). In a specific embodiment, X is selected from the group consisting of N-Me-Tyr, D-Arg, N-Me- D-Tyr, beta-Tyr, and N-Me-Arg. In a specific embodiment, X is Tyr. In a specific embodiment, X is Phe. In a specific embodiment, X is D-Tyr.
  • X is (Tyr)-(D-Tyr). In a specific embodiment, X is (Tyr)-(N-Me-Gly). In a specific embodiment, X is (Tyr)-(N-Me- Tyr). In a specific embodiment, X is N-Me-Tyr. In a specific embodiment, X is D-Arg. In a specific embodiment, X is N-Me-D-Tyr. In a specific embodiment, X is beta-Tyr. In a specific embodiment, X is N-Me-Arg.
  • the C-terminus of the conotoxin peptide analog is a carboxylic acid or an amide group. In one preferred embodiment, the C-terminus of the conotoxin peptide analog is OH. In a specific embodiment, the C-terminus of the conotoxin peptide analog is NH 2 .
  • AA is (S)-propargyl glycine
  • AA is (S)-azidonorvaline
  • X is Tyr.
  • the C-terminus of the conotoxin peptide analog is a carboxylic acid group. In another embodiment, the C-terminus of the conotoxin peptide analog is an amide group.
  • AA is (S)-homopropargyl glycine, X 12
  • AA is (S)- azidonorvaline, X is Tyr; and wherein the C-terminus of the conotoxin peptide analog is a carboxylic acid group.
  • AA is (S)-homopropargyl glycine, X 12
  • AA is (S)- azidohomoalanine, X is Tyr; and wherein the C-terminus of the conotoxin peptide analog is a carboxylic acid group.
  • AA is (S)-azidohomoalanine, X 12
  • AA is (S)-homopropargyl glycine, X is Tyr; and wherein the C-terminus of the conotoxin peptide analog is a carboxylic acid group.
  • AA is (S)-azidoalanine, X 12
  • AA is (S)-bishomopropargyl glycine, X is Tyr; and wherein the C-terminus of the conotoxin peptide analog is a carboxylic acid group.
  • AA is (S)-propargyl glycine
  • AA is (S)-azidonorvaline, X is Phe; and wherein the C-terminus of the conotoxin peptide analog is a carboxylic acid group.
  • a A is (S)-propargyl glycine, X 12
  • a A is (S)-azidonorvaline, X is D-Tyr; and wherein the C-terminus of the conotoxin peptide analog is a carboxylic acid group.
  • a A is (S)-propargyl glycine, X 12
  • a A is (S)-azidonorvaline, X is Tyr-N-Me-Gly; and wherein the C-terminus of the conotoxin peptide analog is a carboxylic acid group.
  • a A is (S)-propargyl glycine, X 12
  • a A is (S)-azidonorvaline, X is Tyr-D-Tyr; and wherein the C-terminus of the conotoxin peptide analog is a carboxylic acid group.
  • a A is (S)-propargyl glycine, X 12
  • a A is (S)-azidonorvaline, X is Tyr-N-Me-Tyr; and wherein the C-terminus of the conotoxin peptide analog is a carboxylic acid group.
  • a A is (S)-propargyl glycine
  • AA Citrulline
  • AA is 3-iodo-Tyr
  • a A is (S)-azidonorvaline
  • C-terminus of the intermediate conotoxin peptide analog is a carboxylic acid or an amide group.
  • the C-terminus of the intermediate conotoxin peptide analog is a carboxylic acid group.
  • the C-terminus of the intermediate conotoxin peptide analog is an amide group. 5.8.2. Triazole Bridge Formation
  • a A is Tyr, Phe, Trp, or a D-isomer of Tyr, Phe, or Trp, and X 2
  • a A is N-Me-Gly, D-Tyr, or N-Me-Tyr;
  • C-terminus of the conotoxin peptide analog of Formula (I) is a carboxylic acid or an amide group
  • AA is selected from the group consisting of (S)-propargyl glycine, (S)-azidoalanine, (S)- homopropargyl glycine, (S)-azidohomoalanine, (S)-azidonorvaline and (S)-bishomopropargyl glycine;
  • a A is Citrulline
  • AA is selected from the group consisting of (S)-azidohomoalanine, (S)-homopropargyl glycine, (S)-azidonorvaline, and (S)-bishomopropargyl glycine;
  • a A is selected from the group consisting of (S)-propargyl glycine, (S)- homopropargyl glycine, and (S)-bishomopropargyl glycine, X 12
  • AA is (S)-azidohomoalanine or (S)-azidonorvaline; when X 3
  • AA is selected from the group consisting of (S)-azidoalanine, (S)- azidohomoalanine, and (S)-azidonorvaline, X 12
  • a A is (S)-homopropargyl glycine or (S)- bishomopropargyl glycine;
  • X is as defined above for the conotoxin peptide analog of Formula (I).
  • a A is (S)-propargyl glycine
  • a A is Citrulline
  • AA is 3-iodo-Tyr
  • AA is (S)-azidonorvaline; and wherein the C-terminus of the intermediate conotoxin peptide analog is as defined above for the conotoxin peptide analog of Formula (Ib); and
  • a A reacts with X AA to form a triazole bridge as depicted in the conotoxin peptide analog of Formula (Ib).
  • the triazole formation conditions are conditions for“Click Chemistry” (i.e., 1,3-dipolar cycloaddition of azides and terminal alkynes) described in Hein et al. Pharm. Res.2008, 25(10): 2216-2230.
  • the triazole formation conditions include presence of a copper catalyst.
  • the triazole formation conditions include presence of a copper catalyst and a reducing reagent.
  • the copper catalyst is a Cu(II) salt.
  • the copper catalyst is CuSO4.
  • the reducing reagent is L-ascorbic acid.
  • the reducing reagent is sodium ascorbate.
  • the triazole formation conditions include presence of a ruthenium catalyst. In one embodiment, the ruthenium catalyst is
  • the triazole formation conditions are catalyst-free conditions.
  • a salt of the conotoxin peptide analog of Formula (I) or (Ib) can be subjected to salt exchange steps to afford a pharmaceutically acceptable salt.
  • a TFA salt of a conotoxin peptide analog of Formula (I) reacts with a base in an aqueous solution (pH 7.0-8.0) and then reacts with an appropriate acid to afford a pharmaceutically acceptable salt of the conotoxin peptide analog of Formula (I).
  • the base is NH4HCO3 (aq.).
  • the conotoxin peptide analog reacts with acetic acid to afford an acetate salt of the conotoxin peptide analog. 5.8.3.
  • the synthesized conotoxin peptide analogs can further be covalently conjugated to one or more polyethylene glycol (PEG) polymers.
  • PEG polyethylene glycol
  • the conotoxin peptide analogs can, for example, be attached directly or via a linking group to one or more PEG polymers .
  • a method of making a PEGylated conotoxin peptide analog or a pharmaceutically acceptable salt thereof comprising contacting under reaction conditions a conotoxin peptide analog or a salt thereof, with one or more reactive polyethylene glycol (PEG) polymers to form a PEGylated conotoxin peptide analog, wherein the reactive PEG polymers each comprise a reactive group covalently linked, optionally via a linking group, to a PEG polymer, and wherein each reactive group reacts under the reaction conditions to form a covalent bond with the conotoxin peptide analog whereby the conotoxin peptide analog is directly or via a linking group to the one or more PEG polymers.
  • the conotoxin peptide analog is the conotoxin peptide analog is of Formula (I) (SEQ ID NO:93):
  • a A is Tyr, Phe, Trp, or a D-isomer of Tyr, Phe, or Trp, and X 2
  • a A is N-Me-Gly, D-Tyr, or N-Me-Tyr;
  • C-terminus of the conotoxin peptide analog is a carboxylic acid or an amide group
  • conotoxin peptide analog is covalently attached directly or via a linking group to one or more polyethylene glycol (PEG) polymers.
  • PEG polyethylene glycol
  • a method of making a PEGylated conotoxin peptide analog or a pharmaceutically acceptable salt thereof comprising contacting under reaction conditions a conotoxin peptide analog or a salt thereof, with one or more reactive polyethylene glycol (PEG) polymers to form a PEGylated conotoxin peptide analog, wherein the reactive PEG polymers each comprise a reactive group covalently linked, optionally via a linking group, to a PEG polymer, and wherein each reactive group reacts under the reaction conditions to form a covalent bond with the conotoxin peptide analog whereby the conotoxin peptide analog is covalently attached directly or via a linking group to the one or more PEG polymers, wherein the conotoxin peptide analog is the conotoxin peptide analog is of Formula (Ib) (SEQ ID NO:104):
  • R 2 is OH or NH 2 .
  • a method of making a PEGylated conotoxin peptide analog or a pharmaceutically acceptable salt thereof comprising contacting under reaction conditions a conotoxin peptide analog or a salt thereof, with one or more reactive polyethylene glycol (PEG) polymers to form a PEGylated conotoxin peptide analog, wherein the reactive PEG polymers each comprise a reactive group covalently linked, optionally via a linking group, to a PEG polymer, and wherein each reactive group reacts under the reaction conditions to form a covalent bond with the conotoxin peptide analog whereby the conotoxin peptide analog is covalently attached directly or via a linking group to the one or more PEG polymers, wherein the amino acid sequence of the conotoxin peptide analog is Gly-Cys-X 3
  • AA is selected from the group consisting of (S)-propargyl glycine, (S)-azidoalanine, (S)- homopropargyl glycine, (S)-azidohomoalanine, (S)-azidonorvaline and (S)-bishomopropargyl glycine;
  • AA Citrulline
  • AA is selected from the group consisting of (S)-azidohomoalanine, (S)-homopropargyl glycine, (S)-azidonorvaline, and (S)-bishomopropargyl glycine; wherein when X 3
  • AA is selected from the group consisting of (S)-propargyl glycine, (S)- homopropargyl glycine, and (S)-bishomopropargyl glycine, X 12
  • a A is (S)-azidohomoalanine or (S)-azidonorvaline; when X 3
  • a A is selected from the group consisting of (S)-azidoalanine, (S)- azidohomoalanine, and (S)-azidonorvaline, X 12
  • AA is (S)-homopropargyl glycine or (S)- bishomopropargyl glycine;
  • a A is Tyr, Phe, Trp, or a D-isomer of Tyr, Phe, or Trp, and X 2
  • AA is N-Me-Gly, D-Tyr, or N-Me-Tyr
  • C-terminus of the conotoxin peptide analog is a carboxylic acid or an amide group.
  • a method of making a PEGylated conotoxin peptide analog or a pharmaceutically acceptable salt thereof comprising contacting under reaction conditions a conotoxin peptide analog or a salt thereof, with one or more reactive polyethylene glycol (PEG) polymers to form a PEGylated conotoxin peptide analog, wherein the reactive PEG polymers each comprise a reactive group covalently linked, optionally via a linking group, to a PEG polymer, and wherein each reactive group reacts under the reaction conditions to form a covalent bond with the conotoxin peptide analog whereby the conotoxin peptide analog is covalently attached directly or via a linking group to the one or more PEG polymers, wherein the amino acid sequence of the conotoxin peptide analog is Gly-Cys-X 3
  • AA is (S)-propargyl glycine
  • AA Citrulline
  • a A is 3-iodo-Tyr
  • a A is (S)-azidonorvaline
  • C-terminus of the conotoxin peptide analog is a carboxylic acid or an amide group.
  • the reaction conditions are standard PEGylation conditions.
  • the reaction conditions are conditions are standard amide formation conditions.
  • Certain standard amide formation conditions are described in Valeur and Bradley, Chemical Society Reviews, 2009, 38:606-631.
  • the amide formation conditions include presence of a coupling reagent such as dicyclohexylcarbodiimide (DCC), 1- hydroxy-1H-benzotriazole (HOBt), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), or 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid
  • DCC dicyclohexylcarbodiimide
  • HOBt 1- hydroxy-1H-benzotriazole
  • EDC 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
  • reaction conditions are conditions are standard amine formation conditions. In one embodiment, the amine formation conditions are reductive amination conditions. 6.
  • RgIA conotoxin peptide analogs CSP-4-OH and CSP-4-NH2 have an amino acid sequence of Gly-Cys-Cys-Thr-Asp-Pro-Arg-Cys-(Cit)-(3-iodo-Tyr)-Gln-Cys-Tyr; where CSP-4- OH (SEQ ID NO:4) has a carboxylic acid group at the C-terminus and CSP-4-NH2 has an amide group at the C-terminus.
  • CSP-4-desTyr-OH has an amino acid sequence of Gly-Cys- Cys-Thr-Asp-Pro-Arg-Cys-(Cit)-(3-iodo-Tyr)-Gln-Cys (SEQ ID NO:5), with a carboxylic acid at the C-terminus.
  • CSP-4-NH2 The active conformation of CSP-4-NH2 consist of two disulfide bonds, one between Cys2 and Cys8, and a second between Cys3 and Cys12.
  • the Cys2-8, Cys3-12“native” form of CSP-4-NH2 is active on both the human and rat a9a10 nAChR.
  • a significant portion of the native form of CSP-4-NH2 underwent isomerization into a“ribbon” form, containing alternative disulfide bonds Cys2-Cys12 and Cys3- Cys8 and a“bead” form containing alternative Cys2-Cys3 and Cys8-Cys12 disulfide bonds.
  • CSP-4-NH2 principally isomerized between the native and ribbon forms, with very little bead formation (Fig 1A, B). Neither the ribbon nor bead form were active on the human or rat a9a10 nAChR, suggesting that disulfide shuffling contributes to a reduction in potency of the molecule. Furthermore, the presence of a C-terminal amide in
  • CSP-4-NH2 did not reduce isomerization relative to a C-terminal carboxylic acid in CSP-4–OH.
  • disulfide isomerization also occurred in vivo following intravenous and subcutaneous injection of a 1 mg/kg dose of compound 6 to Sprague Dawley rats.
  • At 1 h following injection only 40– 50 % of the recovered peptide from blood remained in the native form by HPLC- MS/MS analysis, indicating that disulfide isomerization occurs in vivo similar to observations in vitro.
  • Disulfide shuffling of conotoxin peptide analogs CSP-4–OH and CSP-4-NH2 was demonstrated by incubating the peptides at a final concentration of 0.1– 0.5 mg/mL in serum or plasma from Sprague Dawley rat and human in vitro at 37 °C up to 24 h. Plasma samples were treated with anticoagulants including citrate, K2EDTA and heparin. Samples of the incubations were removed at several timepoints and for each timepoint, the resulting peptide isomers were extracted from the matrix by protein precipitation (3 volumes of methanol) followed by centrifugation of the precipitated sample at 10,000 x g through a Millipore Ultrafree-MC GV centrifugal filter.
  • each CSP-4–OH and CSP-4-NH2 isomer was determined using LC-MS analysis on an Orbitrap Elite (FIG.1C). Aliquots of non-reduced sample were diluted 1:100 in water:acetonitrile:formic acid (98:2:0.1%) to an estimated concentration of 1.2-1.5 pmol/ ⁇ L. A 10 ⁇ L injection was made by an Easy-nLC II HPLC system (Thermo Scientific) onto a 75 mm i.d. PicoTipTM 25 cm long fused silica nano-column (New Objective) coupled to a 2 cm long, 100 mm i.d.
  • IntegraFritTMtrap (New Objective).
  • the column and the trap were configured in a vented configuration (Licklider, et al., Analytical Chemistry, 2002, 74, 3076-3082), with the column packed with 5mm size Magic C18 AQ reverse-phased media (100 ⁇ pore size, Michrom Bioresources, Auburn, CA) and the trap packed with the same material (200 ⁇ pore size, Michrom).
  • the mass spectrometer was operated in the data dependent mode over the range of 400-1800 m/z. For each cycle of the instrument, the top 3 most abundant ions were selected from a precursor scan (with the orbitrap resolution set at 120k in the profile mode). MS/MS data were collected in the centroid mode using the same settings as listed above, with the exception that the isolation width was set to 2.0 m/z.
  • the dynamic exclusion settings used were as follows: repeat count of 1, a 15 second count duration, an exclusion list size of 500 and an exclusion duration of 30 seconds.
  • the native and ribbon isomers had the same mass-to-charge ratio as determined using mass spectrometry, indicating that the two HPLC peaks have the same identity (FIG.1C).
  • Isolated ribbon isomer of CSP-4-NH2 was found to have no blocking activity on the human a9a10 nAChR.
  • CSP-4-NH2 was incubated in human serum for one hour as described above, followed by preparative HPLC and collection of the peak corresponding to the ribbon isomer.
  • the ribbon isomer was also chemically synthesized with the Cys2-Cys12 and Cys3- Cys8 disulfide connectivity. Chemically synthesized ribbon isomer and isomer isolated following incubation in human serum were tested for activity on the human a9a10 nAChR using two electrode voltage clamp electrophysiology.
  • Example 1.1 Conotoxin Peptide Analog Ia (L-tyrosine, glycyl-L-cysteinyl-L- alanyl-L-threonyl-L-a-aspartyl-L-prolyl-L-arginyl-L-cysteinyl-N5-(aminocarbonyl)-L-ornithyl- L-tryptophyl-L-glutaminyl-L-norvalyl-cyclic (2®8)-disulfide-cyclic 3 3 ,12 5 -(1H-1,2,3-triazole- 4,1-diyl)) (SEQ ID NO:6):
  • 2-Chlorotrityl resin loaded with FMOC-Tyr(OtBu)-OH (1): 2-Chlorotrityl chloride resin (MFCD00040399, 15.0 g, 18 mmol) was added to a reaction vessel and was washed with CH 2 Cl 2 (100 mL) and then swelled in CH 2 Cl 2 (100 mL) for 10 min. After draining the reaction vessel, a solution of FMOC-Tyr(OtBu)-OH (2.5 g, 5.4 mmol) in CH2Cl2 (150 mL) was added, followed by dropwise addition of DIPEA (1.8 mL, 10.8 mmol). The reaction vessel was then shaken at room temperature for 1.5 h.
  • the FMOC group was removed from the tethered Tyr group by treatment with 1:4 piperidine/DMF (2x100 mL, 15 min each), and then the resin was washed with DMF (6x100 mL). Then, HATU (3.80 g, 10 mmol), HOAt (1.35 g, 10 mmol), and DIPEA (2.5 mL, 15 mmol) were added with FMOC-5-azido-Nva-OH (CAS# 1097192-04-5, 3.80 g, 10 mmol) in DMF (100 mL). The reaction vessel was shaken for a minimum of 2.5 h, followed by washing with DMF (6x100 mL). Reaction completeness was determined by the Ninhydrin test. If negative
  • FMOC-Gln(Trt)-OH (CAS#132327-80-1, 6.1 g, 10 mmol), FMOC-Trp(Boc)-OH (CAS#143824-78-6, 5.2 g, 10 mmol), FMOC-Cit-OH (CAS# 133174-15-9, 3.9 g, 10 mmol), FMOC-Cys(Trt)-OH (CAS# 103213-32-7, 5.8 g, 10 mmol), FMOC-Arg(Pbf)-OH (CAS# 154445-77-9, 6.4 g, 10 mmol), FMOC-Pro-OH (CAS# 71989-31-6, 3.3 g, 10 mmol), FMOC- Asp(OtBu)-OH (CAS# 71989-14-5, 4.1 g, 10 mmol), FMOC-Thr(tBu)-OH (CAS# 71989-35-0, 3.9 g, 10 mmol), FMOC-Pra
  • Example 1.2 Conotoxin Peptide Analog Ia’ (L-tyrosinamide, glycyl-L- cysteinyl-L-alanyl-L-threonyl-L-a-aspartyl-L-prolyl-L-arginyl-L-cysteinyl-N5-(aminocarbonyl)- L-ornithyl-L-tryptophyl-L-glutaminyl-L-norvalyl-cyclic (2®8)-disulfide-cyclic 3 3 ,12 5 -(1H- 1,2,3-triazole-4,1-diyl)) (SEQ ID NO:9):
  • Conotoxin peptide analog Ia’ was synthesized by employing the same procedure described for Conotoxin peptide analog Ia’ using intermediate conotoxin peptide analog Za’ in lieu of compound 4.
  • LC-MS single quad ESI
  • m/z 1617.5 (M+1H)/ + (calculated MW: 1616.5); HPLC method E; retention time: 14.0 min; purity 96.9%.
  • Example 1.3 Conotoxin Peptide Analog Ib (L-tyrosine, glycyl-L-cysteinyl-L- alanyl-L-threonyl-L-a-aspartyl-L-prolyl-L-arginyl-L-cysteinyl-N5-(aminocarbonyl)-L-ornithyl- L-3-iodotyrosyl-L-glutaminyl-L-norvalyl-cyclic (2®8)-disulfide-cyclic 3 3 ,12 5 -(1H-1,2,3- triazole-4,1-diyl)) (SEQ ID NO:12):
  • Conotoxin peptide analog Ib was synthesized by employing the same procedure described for conotoxin peptide analog Ia using intermediate conotoxin peptide analog Zb in lieu of compound 4.
  • Example 1.4 Conotoxin Peptide Analog Ib’ (L-tyrosinamide, glycyl-L- cysteinyl-L-alanyl-L-threonyl-L-a-aspartyl-L-prolyl-L-arginyl-L-cysteinyl-N5-(aminocarbonyl)- L-ornithyl-L-3-iodotyrosyl-L-glutaminyl-L-norvalyl-cyclic (2®8)-disulfide-cyclic 3 3 ,12 5 -(1H- 1,2,3-triazole-4,1-diyl)) (SEQ ID NO:15):
  • Intermediate conotoxin peptide analog Yb’ H-Gly-Cys-Pra-Thr-Asp-Pro-Arg- Cys-Cit-3-I-Tyr-Gln-5-azidoNVa-Tyr-NH2 (SEQ ID NO:16): Intermediate conotoxin peptide analog Yb’ was synthesized by employing the same procedure described for intermediate conotoxin peptide analog Yb using a Rink Amide resin MFCD00677976 in lieu of 2-Chlorotrityl chloride resin MFCD00040399 in the solid phase peptide sythesis.
  • Conotoxin peptide analog Ib’ was synthesized by employing the same procedure described for conotoxin peptide analog Ia using intermediate conotoxin peptide analog Zb’ in lieu of compound 4.
  • Example 1.4 Conotoxin Peptide Analog Ic (L-tyrosinamide, glycyl-L-alanyl- L-cysteinyl-L-threonyl-L- a -aspartyl-L-prolyl-L-arginyl-L-norvalyl-N5-(aminocarbonyl)-L- ornithyl-L-3-iodotyrosyl-L-glutaminyl-L-cysteinyl-cyclic 2 3 ,8 5 -(1H-1,2,3-triazole-4,1-diyl)- cyclic -(3®12)-disulfide) (SEQ ID NO:18):
  • Intermediate conotoxin peptide analog Yc (H-Gly-Pra-Cys-Thr-Asp-Pro-Arg-5- azidoNVa-Cit-3-I-Tyr-Gln-Cys-Tyr-NH2) (SEQ ID NO:19): Intermediate conotoxin peptide analog Yc was synthesized by employing the same procedure described for intermediate conotoxin peptide analog Yb’ using FMOC-Cys(Trt)-OH in lieu of FMOC-5-azido-Nva-OH (the 12-position residue), FMOC-5-azido-Nva-OH in lieu of FMOC-Cys(Trt)-OH (the 8-position residue), FMOC-Cys(Trt)-OH in lieu of FMOC-Pra-OH (the 3-position residue), and FMOC- Pra-OH in lieu of FMOC-Cys(Trt)-OH (the 2-position residue) in the solid phase peptid
  • Conotoxin peptide analog Ic was synthesized by employing the same procedure described for conotoxin peptide analog Ib’ using intermediate conotoxin peptide analog Zc in lieu of Intermediate conotoxin peptide analog Zb’.
  • LC-MS (ESI-TOF) m/z: 574.2 [M+3H]/3 + , 860.7 [M+2H]/2 + (calculated MW: 1719.55); HPLC method B; retention time: 10.2 min; Purity 98.3%.
  • Example 1.5 Conotoxin Peptide Analog Id (L-tyrosinamide, glycyl-L- norvalyl-L-cysteinyl-L-threonyl-L-a-aspartyl-L-prolyl-L-arginyl-L-alanyl-N5-(aminocarbonyl)- L-ornithyl-L-3-iodotyrosyl-L-glutaminyl-L-cysteinyl-cyclic 2 5 ,8 3 -(1H-1,2,3-triazole-1,4-diyl)- cyclic -(3®12)-disulfide) (SEQ ID NO:21):
  • Intermediate conotoxin peptide analog Yd H-Gly-5-azidoNVa-Cys-Thr-Asp-Pro- Arg-Pra-Cit-3-I-Tyr-Gln-Cys-Tyr-NH2) (SEQ ID NO:22): Intermediate conotoxin peptide analog Yd was synthesized by employing the same procedure described for intermediate conotoxin peptide analog Yb’ using FMOC-Cys(Trt)-OH in lieu of FMOC-5-azido-Nva-OH (the 12-position residue), FMOC-Pra-OH in lieu of FMOC-Cys(Trt)-OH (the 8-position residue), FMOC-Cys(Trt)-OH in lieu of FMOC-Pra-OH (the 3-position residue), and FMOC-5-azido- Nva-OH in lieu of FMOC-Cys(Trt)-OH (the 2-position residue) in the
  • Intermediate conotoxin peptide analog Zd H-Gly-5-azidoNVa-Cys-Thr-Asp-Pro- Arg-Pra-Cit-3-I-Tyr-Gln-Cys-Tyr-NH2, (Cys3®Cys12) disulfide bridge) (SEQ ID NO:23): Intermediate conotoxin peptide analog Zd was synthesized by employing the same procedure described for intermediate conotoxin peptide analog Zb’ using intermediate conotoxin peptide analog Yd in lieu of intermediate conotoxin peptide analog Yb’.
  • Conotoxin peptide analog Id was synthesized by employing the same procedure described for conotoxin peptide analog Ib’ using intermediate conotoxin peptide analog Zd in lieu of intermediate conotoxin peptide analog Zb’.
  • Example 1.6 Conotoxin Peptide Analog Ie (L-tyrosinamide, glycyl-L- cysteinyl-L-norvalyl-L-threonyl-L-a-aspartyl-L-prolyl-L-arginyl-L-cysteinyl-N5- (aminocarbonyl)-L-ornithyl-L-3-iodotyrosyl-L-glutaminyl-L-alanyl-cyclic (2®8)-disulfide- cyclic 3 5 ,12 3 -(1H-1,2,3-triazole-1,4-diyl)) (SEQ ID NO:24):
  • Intermediate conotoxin peptide analog Ye (H-Gly-Cys-5-azidoNVa-Thr-Asp-Pro- Arg-Cys-Cit-3-I-Tyr-Gln-Pra-Tyr-NH2) (SEQ ID NO:25): Intermediate conotoxin peptide analog Ye was synthesized by employing the same procedure described for intermediate conotoxin peptide analog Yb’ using FMOC-Pra-OH in lieu of FMOC-5-azido-Nva-OH (the 12- position residue) and FMOC-5-azido-Nva-OH in lieu of FMOC-Pra-OH (the 3-position residue) in the solid phase peptide synthesis.
  • Conotoxin peptide analog Ie was synthesized by employing the same procedure described for conotoxin peptide analog Ib’ using intermediate conotoxin peptide analog Ze in lieu of intermediate conotoxin peptide analog Zb’.
  • Intermediate conotoxin peptide analog Yf H-Gly-Cys-Pra-Thr-Asp-Pro-Arg-Cys- Cit-3-I-Tyr-Gln-3-azidoAla-Tyr-NH2) (SEQ ID NO:28): Intermediate conotoxin peptide analog Yf was synthesized by employing the same procedure described for intermediate conotoxin peptide analog Yb’ using FMOC-3-azido-Ala-OH in lieu of FMOC-Pra-OH (the 12- position residue) and FMOC-Pra-OH in lieu of FMOC-5-azido-Nva-OH (the 3-position residue) in the solid phase peptide synthesis.
  • Intermediate conotoxin peptide analog Zf H-Gly-Cys-Pra-Thr-Asp-Pro-Arg-Cys- Cit-3-I-Tyr-Gln-3-azidoAla-Tyr-NH2, (Cys2®Cys8) disulfide bridge) (SEQ ID NO:29): Intermediate conotoxin peptide analog Zf was synthesized by employing the same procedure described for intermediate conotoxin peptide analog Zb’ using intermediate conotoxin peptide analog Yf in lieu of intermediate conotoxin peptide analog Yb’.
  • Conotoxin peptide analog If was synthesized by employing the same procedure described for conotoxin peptide analog Ib’ using intermediate conotoxin peptide analog Zf in lieu of intermediate conotoxin peptide analog Zb’.
  • Example 1.8 Conotoxin Peptide Analog Ig (L-tyrosine, glycyl-L-cysteinyl- (2S)-2-aminobutanoyl-L-threonyl-L-a-aspartyl-L-prolyl-L-arginyl-L-cysteinyl-N5- (aminocarbonyl)-L-ornithyl-L-tryptophyl-L-glutaminyl-L-norvalyl-cyclic (2®8)-disulfide-cyclic 3 4 ,12 5 -(1H-1,2,3-triazole-4,1-diyl)) (SEQ ID NO:30):
  • Intermediate conotoxin peptide analog Yg H-Gly-Cys-homoPra-Thr-Asp-Pro- Arg-Cys-Cit-Trp-Gln-5-azidoNva-Tyr-OH
  • SEQ ID NO:31 Intermediate conotoxin peptide analog Yg was synthesized by employing the same procedure described for compound 3 using FMOC-homopropargyl glycine in lieu of FMOC-Pra-OH (the 3-position residue) in the solid phase peptide synthesis.
  • Conotoxin peptide analog Ig was synthesized by employing the same procedure described for conotoxin peptide analog Ia using intermediate conotoxin peptide analog Zg in lieu of compound 4.
  • LC-MS single quad ESI
  • m/z 545.0 [M+3H]/3 + , 817.3 [M+2H]/2 + (calculated MW: 1631.67); HPLC method A; retention time: 12.5 min; purity 98.2%.
  • Example 1.9 Conotoxin Peptide Analog Ih (L-tyrosine, glycyl-L-cysteinyl- (2S)-2-aminobutanoyl-L-threonyl-L-a-aspartyl-L-prolyl-L-arginyl-L-cysteinyl-N5- (aminocarbonyl)-L-ornithyl-L-tryptophyl-L-glutaminyl-L-(2S)-2-aminobutanoyl-cyclic (2®8)- disulfide-cyclic 3 4 ,12 4 -(1H-1,2,3-triazole-4,1-diyl)) (SEQ ID NO:33):
  • Intermediate conotoxin peptide analog Yh H-Gly-Cys-homoPra-Thr-Asp-Pro- Arg-Cys-Cit-Trp-Gln-azidohomoAla-Tyr-OH
  • SEQ ID NO:34 Intermediate conotoxin peptide analog Yh was synthesized by employing the same procedure described for compound 3 using FMOC-homopropargyl glycine in lieu of FMOC-Pra-OH (the 3-position residue) and FMOC-g-azido-homoalanine in lieu of FMOC-5-azido-Nva-OH (the 12-position residue) in the solid phase peptide synthesis.
  • Conotoxin peptide analog Ih was synthesized by employing the same procedure described for conotoxin peptide analog Ia using intermediate conotoxin peptide analog Zh in lieu of compound 4.
  • LC-MS single quad ESI
  • m/z 540.6 [M+3H]/3 + , 809.9 [M+2H]/2 + (calculated MW: 1617.66); HPLC method A; retention time: 12.4 min; purity 83.9%.
  • Example 1.10 Conotoxin Peptide Analog Ii (L-tyrosine, glycyl-L-cysteinyl- (2S)-2-aminobutanoyl-L-threonyl-L-a-aspartyl-L-prolyl-L-arginyl-L-cysteinyl-N5- (aminocarbonyl)-L-ornithyl-L-tryptophyl-L-glutaminyl-L-(2S)-2-aminobutanoyl-cyclic (2®8)- disulfide-cyclic 3 4 ,12 4 -(1H-1,2,3-triazole-1,4-diyl)) (SEQ ID NO:36):
  • Conotoxin peptide analog Ii was synthesized by employing the same procedure described for conotoxin peptide analog Ia using intermediate conotoxin peptide analog Zi in lieu of compound 4.
  • LC-MS single quad ESI
  • m/z 540.7 [M+3H]/3 + , 810.5 [M+2H]/2 + (calculated MW: 1617.66); HPLC method A; retention time: 12.3 min; purity 85.8%.
  • Example 1.11 Conotoxin Peptide Analog Ij (L-tyrosine, glycyl-L-cysteinyl- (2S)-2-aminopentanoyl-L-alanyl-L-threonyl-L-a-aspartyl-L-prolyl-L-arginyl-L-cysteinyl-N5- (aminocarbonyl)-L-ornithyl-L-3-iodotyrosyl-L-glutaminyl-L-alanyl-cyclic (2®8)-disulfide- cyclic 3 5 ,12 3 -(1H-1,2,3-triazole-4,1-diyl)) (SEQ ID NO:39):
  • Intermediate conotoxin peptide analog Yj H-Gly-Cys-bishomoPra-Thr-Asp-Pro- Arg-Cys-Cit-Trp-Gln-3-azidoAla-Tyr-OH
  • SEQ ID NO:40 Intermediate conotoxin peptide analog Yj was synthesized by employing the same procedure described for compound 3 using FMOC-bis-homopropargyl glycine in lieu of FMOC-Pra-OH (the 3-position residue) and FMOC-3-azido-Ala-OH in lieu of FMOC-5-azido-Nva-OH (the 12-position residue) in the solid phase peptide synthesis.
  • Conotoxin peptide analog Ij was synthesized by employing the same procedure described for conotoxin peptide analog Ia using intermediate conotoxin peptide analog Zj in lieu of compound 4.
  • LC-MS single quad ESI
  • m/z 540.5 [M+3H]/3 + , 810.1 [M+2H]/2 + (calculated MW: 1617.66); HPLC method A; retention time: 12.04 min; purity 97.6%.
  • Example 1.12 Conotoxin Peptide Analog Ik (L-tyrosine, glycyl-L-cysteinyl-L- alanyl-L-threonyl-L-a-aspartyl-L-prolyl-L-arginyl-L-cysteinyl-N5-(aminocarbonyl)-L-ornithyl- L-tryptophyl-L-glutaminyl-L-(2S)-2-aminopentanoyl-cyclic (2®8)-disulfide-cyclic 3 3 ,12 5 -(1H- 1,2,3-triazole-1,4-diyl)) (SEQ ID NO:42):
  • Intermediate conotoxin peptide analog Yk (H-Gly-Cys-3-azidoAla-Thr-Asp-Pro- Arg-Cys-Cit-Trp-Gln-bishomoPra-Tyr-OH) (SEQ ID NO:43): Intermediate conotoxin peptide analog Yk was synthesized by employing the same procedure described for compound 3 using FMOC-3-azido-Ala-OH in lieu of FMOC-Pra-OH (the 3-position residue) and FMOC-bis- homopropargyl glycine in lieu of FMOC-5-azido-Nva-OH (the 12-position residue) in the solid phase peptide synthesis.
  • Conotoxin peptide analog Ik was synthesized by employing the same procedure described for conotoxin peptide analog Ia using intermediate conotoxin peptide analog Zk in lieu of compound 4.
  • LC-MS single quad ESI
  • m/z 540.6 [M+3H]/3 + , 810.0 [M+2H]/2 + (calculated MW: 1617.66); HPLC method A; retention time: 12.24 min; purity 97.6%.
  • Example 1.13 Conotoxin Peptide Analog Il (L-phenylalanine, glycyl-L- cysteinyl-L-alanyl-L-threonyl-L-a-aspartyl-L-prolyl-L-arginyl-L-cysteinyl-N5-(aminocarbonyl)- L-ornithyl-L-tryptophyl-L-glutaminyl-L-norvalyl-cyclic (2®8)-disulfide-cyclic 3 3 ,12 5 -(1H- 1,2,3-triazole-4,1-diyl)) (SEQ ID NO:45):
  • Intermediate conotoxin peptide analog Yl (H-Gly-Cys-Pra-Thr-Asp-Pro-Arg-Cys- Cit-Trp-Gln-5-azidoNVa-Phe-OH) (SEQ ID NO:46): Intermediate conotoxin peptide analog Yl was synthesized by employing the same procedure described for compound 3 using FMOC- Phe(OtBu)-OH-loaded 2-chlorotrityl resin loaded FMOC-Phe(OtBu)-OH (5) in lieu of the FMOC-Tyr(OtBu)-loaded 2-chlorotrityl resin (1) as the C-terminus start in the solid phase peptide synthesis.
  • Conotoxin peptide analog Il was synthesized by employing the same procedure described for conotoxin peptide analog Ia using intermediate conotoxin peptide analog Zl in lieu of compound 4.
  • LC-MS single quad ESI
  • m/z 535.2 [M+3H]/3 + , 802.2 [M+2H]/2 + (calculated MW: 1601.66); HPLC method A; retention time: 13.47 min; purity 95.6%.
  • Example 1.14 Conotoxin Peptide Analog Im (D-tyrosine, glycyl-L-cysteinyl-L- alanyl-L-threonyl-L-a-aspartyl-L-prolyl-L-arginyl-L-cysteinyl-N5-(aminocarbonyl)-L-ornithyl- L-tryptophyl-L-glutaminyl-L-norvalyl-cyclic (2®8)-disulfide-cyclic 3 3 ,12 5 -(1H-1,2,3-triazole- 4,1-diyl)) (SEQ ID NO:48):
  • 2-Chlorotrityl resin loaded FMOC-D-Tyr(OtBu)-OH was synthesized by employing the same procedure described for 2- Chlorotrityl resin 1 using FMOC-D-Tyr(OtBu)-OH in lieu of FMOC-Tyr(OtBu)-OH.
  • Intermediate conotoxin peptide analog Ym H-Gly-Cys-Pra-Thr-Asp-Pro-Arg-Cys- Cit-Trp-Gln-5-azidoNVa-D-Tyr-OH
  • SEQ ID NO:49 Intermediate conotoxin peptide analog Ym was synthesized by employing the same procedure described for compound 3 using FMOC- D-Tyr(OtBu)-OH-loaded 2-chlorotrityl resin (6) in lieu of the FMOC-Tyr(OtBu)-loaded 2- chlorotrityl resin (1) as the C-terminus start in the solid phase peptide synthesis.
  • Conotoxin peptide analog Im was synthesized by employing the same procedure described for conotoxin peptide analog Ia using intermediate conotoxin peptide analog Zm in lieu of compound 4.
  • LC-MS single quad ESI
  • m/z 540.6 [M+3H]/3 + , 810.0 [M+2H]/2 + (calculated MW: 1617.66); HPLC method A; retention time: 12.07 min; purity 86.6%.
  • Example 1.15 Conotoxin Peptide Analog In (glycine, glycyl-L-cysteinyl-L- alanyl-L-threonyl-L-a-aspartyl-L-prolyl-L-arginyl-L-cysteinyl-N5-(aminocarbonyl)-L-ornithyl- L-tryptophyl-L-glutaminyl-L-norvalyl-L-tyrosyl-N-methyl-cyclic (2®8)-disulfide-cyclic 3 3 ,12 5 - (1H-1,2,3-triazole-4,1-diyl)) (SEQ ID NO:51):

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Abstract

La présente invention concerne des analogues peptidiques d'alpha-conotoxine, notamment des analogues peptidiques d'alpha-conotoxine qui sont liés de manière covalente au polyéthylène glycol (PEG), et des compositions pharmaceutiques de tels analogues peptidiques d'alpha-conotoxine. L'invention concerne également des méthodes de traitement ou de prévention d'une affection favorable au traitement ou à la prévention par inhibition d'un récepteur nicotinique de l'acétylcholine (nAChR) contenant a9 (par exemple, le sous-type α9α10 du nAChR) chez un sujet.
EP19702500.0A 2019-01-04 2019-01-04 Analogues peptidiques de conotoxine et leurs utilisations pour le traitement de la douleur et d'affections inflammatoires Withdrawn EP3906253A1 (fr)

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US20090326192A1 (en) * 2008-04-08 2009-12-31 Aileron Therapeutics, Inc. Biologically active peptidomimetic macrocycles
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KR20210116505A (ko) 2021-09-27
CN113966339A (zh) 2022-01-21
CR20210363A (es) 2021-12-10
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JP2022522935A (ja) 2022-04-21
CL2021001775A1 (es) 2022-05-27
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CO2021008845A2 (es) 2021-10-29
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