CN113966339A

CN113966339A - Conotoxin peptide analogs and uses for treating pain and inflammatory conditions

Info

Publication number: CN113966339A
Application number: CN201980093540.XA
Authority: CN
Inventors: 约瑟·梅尔卡多; 埃里克·J·塔查; 杰弗里·J·波萨科尼; 肖恩·艾度纳托
Original assignee: Kineta Three LLC
Current assignee: Kineta Three LLC
Priority date: 2019-01-04
Filing date: 2019-01-04
Publication date: 2022-01-21
Also published as: JP2022522935A; KR20210116505A; CA3125383A1; MX2021008070A; EP3906253A1; WO2020142102A1; SG11202106847SA; CO2021008845A2; BR112021013302A2; AU2019418319A1; IL284574A; CL2021001775A1; CR20210363A; WO2020142102A9; PE20211734A1

Abstract

Provided herein are alpha-conotoxin peptide analogs, including alpha-conotoxin peptide analogs covalently linked to polyethylene glycol (PEG) and pharmaceutical compositions of these alpha-conotoxin peptide analogs. Also provided herein are methods of treating or preventing a condition that facilitates treatment or prevention by inhibiting α 9-containing nicotinic acetylcholine receptors (nachrs) (e.g., the α 9 α 10 subtype of nachrs) in a subject.

Description

Conotoxin peptide analogs and uses for treating pain and inflammatory conditions

Reference to an electronically submitted sequence Listing

The present application incorporates by reference the sequence LISTING filed with the present application as a text file entitled "SEQ _ testing _ 14520-.

1. Field of the invention

Provided herein are alpha-conotoxin peptide analogs, including alpha-conotoxin peptide analogs covalently linked to polyethylene glycol (PEG) and pharmaceutical compositions of these alpha-conotoxin peptide analogs. Also provided herein are methods of treating or preventing a condition that facilitates treatment or prevention by inhibiting the alpha 9-containing nicotinic acetylcholine receptors (nachrs) (e.g., the alpha 9 alpha 10 subtype of nachrs) in a subject

2. Background of the invention

Predatory sea snails in the genus Conus have venom rich in neuropharmacological active peptides (conotoxin peptides or conotoxins) (Olivera et al, 1990, Science 249: 257-. There are approximately 500 species of conus, and among those that have been investigated so far, the conserved feature is the presence of alpha-conotoxin peptides in their venom. Native alpha-conotoxin peptides are highly disulfide-crosslinked peptides with disulfide bridge patterns of C1-C3 and C2-C4 (Azam and McIntosh,2009, Acta Pharmacol. sin.30: 771-783).

Alpha-conotoxin peptides have been shown generally as 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 alpha subunits (. alpha.1-. alpha.10) alone or together with one or more non-alpha subunits (. beta.1-. beta.4) (Sine and Engel,2006, Nature440: 448-.

The α 9 α 10nAChR subunit is expressed in different tissues. In the inner ear, the α 9 α 10nAChR mediates synaptic transmission between efferent cochlear fibers and cochlear hair cells (Vetter et al, 1999, Neuron 23: 93-103). The α 9 α 10 subunit is also present in dorsal root ganglion neurons (Lips et al, 2002, Neuroscience 115:1-5), lymphocytes (Peng et al, 2004, Life. Sci.76: 263-.

Compounds with a pharmacological profile that includes α 9 α 10 antagonist activity prevent or attenuate pain manifestations in several rodent models, including neuropathic pain caused by chemotherapy, traumatic nerve injury, and diabetes (see Hone and mcinosh, 2018, FEBS lett.592: 1045-.

The natural 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.377: 1216-1227). The α -conotoxin peptide RgIA has been shown to block α 9 α 10nAChR activity (Romero et al, 2017, proc.natl.acad.sci.usa 14: E1825-E1832).

RgIA belongs to the alpha-4/3 family of alpha-conotoxins. The natural structure of RgIA is characterized by two compact cysteine inner loops defined by two disulfide bonds formed between Cys2-Cys8 (sometimes referred to as CysI-CysIII) and Cys3-Cys12 (sometimes referred to as CysII-CysIV) (Ellison et al, 2008, J.mol.biol.377: 1216-. In alpha-conotoxin, the first and second cysteine residues are always adjacent, but the number of amino acid residues between the second and third cysteine residues and between the third and fourth cysteine residues may differ. This results in two intermediate amino acid rings, denoted as ring 1 or m-ring and ring 2 or n-ring. In RgIA, loop 1 contains 4 amino acids and loop 2 contains 3 amino acids. The disulfide bond and hence the secondary structure of alpha-conotoxin is unstable and undergoes rearrangement (Armishaw,2010, Toxins 2: 1471-.

The two disulfide bonds of alpha-conopeptide undergo rearrangement and can form alternative steric structures, including ribbon-like forms (disulfide bonds Cys-2-Cys12 and Cys3-Cys8) and bead forms (disulfide bonds Cys2-Cys3 and Cys8-Cys 12). While native or globular forms of the peptide are active on α 9 α 10-nAChr, neither the ribbon nor bead forms are considered 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 alpha-conotoxin SI, and only one of these four lactam-bridged derivatives did not lose significant activity in binding to BC3H1 cells (Hargittai et al, 2000, J.Med.chem.43: 4787-. Bondebjerg et al evaluated synthetic thioether mimetics of the α -conotoxin GI and found that the pharmacological activity of the two isomers tested was significantly lower than that of the native peptide (Bondebjerg et al, 2003, ChemBiochem 4: 186-. Non-reducing dicarbo-heteroanalogues of alpha-ImI were evaluated and in both cases an approximately 10-fold reduction in pharmacological activity was found (MacRaild et al, 2009, J.Med.chem.52: 755-. ImI retain activity in blocking rat α 7nAChR (Dekan et al, 2011, J.Am.chem.Soc.133: 15866-15869; Armishaw et al, 2006, J.Bio.chem.281: 14136-14143).

van Lierop evaluated the two-carbon heterosubstitution of the disulfide bond of alpha-conotoxin Vc1.1 and found that [2,8] -cis and [2,8] -trans two-carbon heteroanalogs were not active against rat α 9 α 10nAChR, whereas the [3,16] -cis and [3,16] -trans isomers of Vc1.1 were greatly reduced (10-100 fold) in rat α 9 α 10nAChR activity (van Lierop et al, 2013, ACS Chemical Biology 8: 1815-1821). Yu et al produced an N-terminal to C-terminal peptide-linked derivative of vc1.1 having one or more cysteine residues replaced by His or Phe. The efficacy of these derivatives on human α 9 α 10 nachrs was significantly reduced (20 to 40-fold lower) relative to native vc1.1 (Yu et al 2015, sci. rep.5: 13264). In separate studies of the same study group, the substitution of Cys2 by His and Cys8 by Phe in Vc1.1 also resulted in a loss of activity on human α 9 α 10nAChR in the absence of N-terminal to C-terminal cyclization, although analysis confirmed retention of secondary structure (Tabassum et al 2017, ACS Omega2: 4621-one 4631).

Similar work has been carried out to replace the disulfide bond in RgIA with a two-carbon heterobond (Chhabra et al 2014, J.Med.chem.57: 993-. Although this study found that a two-carbon heteroanalog of RgIA was synthetically feasible, structural comparison of NMR-based native RgIA with a two-carbon heteroanalog demonstrated significant structural differences in the second ring of the peptide. Consistent with these structural differences and observations from related vc1.1, the [2,8] -cis and [2,8] -trans two-carbon heteroanalogs of RgIA were not active against rat α 9 α 10 nachrs, while both the [3,12] -cis and [3,12] -trans isomers reduced > 100-fold inhibition of acetylcholine-induced α 9 α 10 current. Chhabra et al did not evaluate the activity of two-carbon heterobridged RgIA analogs on human α 9 α 10 nAChR.

Knuhtsen et al (Knuhtsen et al, Chemical Science,2019, advanced Article) studied analogues of the α -conotoxin GI in which the Cys2-Cys7 or Cys3-Cys13 disulfide bond was replaced by a 1, 5-disubstituted 1,2, 3-triazole bridge.

The affinity of native RgIA for human α 9 α 10nAChR is approximately 300-fold lower than for rat α 9 α 10nAChR, and this difference in affinity has been reflected in variations in the amino acid sequence of the α 9 subunit within the binding pocket of RgIA (Azam et al, 2015, mol. Pharmacol.87: 855-.

Conus toxin peptide analogues of RgIA have been reported (WO 2008/011006; Romero et al, 2017, Proc. Natl. Acad. Sci. USA 14: E1825-E1832; WO 2016/073949).

Studies have demonstrated a greater degree of structural flexibility in loop 2 of RgIA, particularly around the carboxy terminus, and the importance of loop 2 residues in human receptor binding interactions (Clark et al, 2008, FEBS Lett.582(5): 597-. NMR spectroscopy also showed that Cys12 of RgIA can interact with Tyr10 in wild-type or native peptides (Ellison et al, 2008, J.mol.biol.377: 1216-1227; Armishaw,2010, Toxins2: 1471-1499).

Conotoxin peptides and their analogs are competitive inhibitors of α 9 α 10 nachrs, and they bind at the ACh binding site, i.e., at the interface between adjacent subunits. Subunit interface is buried to

The radius and the surface area of the probe(s) are as long as

Wherein the content between the conotoxins and subunits is less than

(Hansen et al, 2005, EMBO J.,24(20): 3635-.

Amino acid comparisons with other known nAChR subunits show that α 9 α 10 nAChRs are closely related to α 7 nAChRs (Elgoyhen et al, 1994, Cell 79(4): 705:. sup. 715; Lustig et al, 2001, Genomics,73(3): 272:. sup. 283; Sgard et al, 2002, mol. Pharmacol.61(1): 150-. α 7nAChR has pleiotropic effects in the nervous system (Ellison et al, 2008, J.mol.biol.377: 1216-1227; Armishaw,2010, Toxins2: 1471-1499).

Non-covalent or covalent attachment to polyethylene glycol polymers (PEGylation) can alter the physical and chemical properties of biomolecules, such as peptides, such as their conformation, electrostatic binding and hydrophobicity (Veronese and Mero,2008, BioDrugs,22: 315-; see also Harris et al, 2001, Clin. Pharmacokinet.40: 539-.

PEGylation of therapeutic proteins typically results in a loss of their binding affinity due to steric hindrance of the PEG polymer to drug-target binding interactions (Fishburn,2008, J.Pharm. Sci.97: 4167-4183). Pegylated therapeutic proteins can lose biological activity or potency by steric effects or by hydrophobic-hydrophobic interactions between PEG and hydrophobic domains within the protein (Parrott and DeSimone,2011, nat. chem.4: 13-14).

Studies have shown that antagonists of α 9-containing nAChRs exhibit analgesic activity in animal models of neuropathic pain (Hone et al, 2018, British Journal of Pharmacology,175: 1915-1927). Studies have also shown that inhibition of α 9 α 10 nachrs in rodents prevents chemotherapy-induced neuropathic pain (Romero et al, 2018, proc.natl.acad.sci.usa 114(10): E1825-E1832). See also, Mohammadi and Christie,2014, Molecular Pain,10: 64-72; simard et al, 2013, Immunology and Cell Biology,91: 195-200.

There is an unmet medical need for conotoxin peptide analogs with improved metabolic stability while maintaining sufficient antagonistic activity against α 9-containing nachrs. The conotoxin peptide analogs of the present invention meet these needs.

Citation of references herein shall not be construed as an admission that such references are prior art to the present disclosure.

3. Summary of the invention

The invention discloses a conotoxin peptide analogue (SEQ ID NO: 93) shown in a chemical formula (I):

or a pharmaceutically acceptable salt thereof,

wherein

X is X_AA ¹Or X_AA ¹X_AA ²(ii) a Wherein X_AA ¹Is Tyr, Phe, Trp, or the D-isomer of Tyr, Phe or Trp, and X_AA ²Is N-Me-Gly, D-Tyr or N-Me-Tyr;

Wherein the C-terminal of the conotoxin peptide analogue is carboxylic acid or amido.

In a specific embodiment, the triazole bridge is

Wherein the single wave line

C representing triazole bridge and conotoxin peptide analogues¹Carbon tie points, and double wave lines

C representing triazole bridge and conotoxin peptide analogues²A point of attachment for carbon; and wherein x is 1, 2, 3 or 4; and y is 2, 3 or 4.

In a specific embodiment, wherein the triazole bridge is

In a specific embodiment, the triazole bridge is

In particular embodiments, x is 1, 2, or 3.

In a specific embodiment, x is 1.

In particular embodiments, y is 2 or 3.

In a specific embodiment, y is 3.

In particular embodiments, x is 1, 2 or 3, and y is 2 or 3.

In a specific embodiment, x is 1 and y is 3.

In a specific embodiment, x is 2 and y is 3.

In a specific embodiment, x is 2 and y is 2.

In a specific embodiment, x is 1 and y is 3.

In a specific embodiment, x is 2 and y is 2.

In a specific embodiment, the triazole bridge is

Wherein the single wave line

C representing triazole bridge and conotoxin peptide analogues²The point of attachment of the carbon.

In a specific embodiment, X is X_AA ¹。

In a specific embodiment, X is X_AA ¹X_AA ²。

In a specific embodiment, X_AA ¹Selected from Tyr, D-Tyr and Phe.

In a specific embodiment, X is Tyr.

In a specific 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。

In a specific embodiment, the conotoxin peptide analog is of formula (Ia) (SEQ ID NO: 94)

Wherein R is¹Is OH or NH₂。

In a specific embodiment, R¹Is OH.

In a specific embodiment, R¹Is NH₂。

In a specific embodiment, the conotoxin peptide analog is of formula (Ig) (SEQ ID NO: 30):

in a specific embodiment, the conotoxin peptide analog is of formula (Ih) (SEQ ID NO: 33):

in a specific embodiment, the conotoxin peptide analog is of formula (Ii) (SEQ ID NO: 36):

in a specific embodiment, the conotoxin peptide analog is of formula (Ik) (SEQ ID NO: 42):

in a specific embodiment, the conotoxin peptide analog is of formula (Il) (SEQ ID NO: 45):

in a specific embodiment, the conotoxin peptide analog is of formula (Im) (SEQ ID NO: 48)

In a specific embodiment, the conotoxin peptide analog is of formula (In) (SEQ ID NO: 51)

In a specific embodiment, the conotoxin peptide analog is of formula (Io) (SEQ ID NO: 54)

In a specific embodiment, the conotoxin peptide analog is of formula (Ip) (SEQ ID NO: 57)

Also provided herein are pegylated conotoxin peptide analogs, or pharmaceutically acceptable salts thereof, wherein the conotoxin peptide analogs have formula (I) (SEQ ID NO: 93):

wherein

wherein the C-terminal of the conotoxin peptide analog is carboxylic acid or amido; and

wherein the conotoxin peptide analog is covalently attached, directly or through a linking group, to one or more polyethylene glycol (PEG) polymers.

In a specific embodiment, the triazole bridge is

Wherein the single wave line

In a specific embodiment, wherein the triazole bridge is

In a specific embodiment, the triazole bridge is

In particular embodiments, x is 1, 2 or 3.

In a specific embodiment, x is 1.

In particular embodiments, y is 2 or 3.

In a specific embodiment, y is 3.

In a specific embodiment, x is 1, 2 or 3 and y is 2 or 3.

In a specific embodiment, x is 1 and y is 3.

In a specific embodiment, x is 2 and y is 3.

In a specific embodiment, x is 2 and y is 2.

In a specific embodiment, x is 1 and y is 3.

In a specific embodiment, x is 2 and y is 2.

In a specific embodiment, the triazole bridge is

Wherein the single wave line

In a specific embodiment, X is X_AA ¹。

In a specific embodiment, X is X_AA ¹X_AA ²。

In a specific embodiment, X_AA ¹Selected from Tyr, D-Tyr and Phe.

In a specific embodiment, X is Tyr.

In a specific 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。

In a specific embodiment, R¹Is OH.

In a specific embodiment, R¹Is NH₂。

In particular embodiments, the conotoxin peptide analog is covalently attached to a PEG polymer.

In a specific embodiment, the PEG polymer is covalently attached to the N-terminus of the conotoxin peptide analog.

In a specific embodiment, the PEG polymer is covalently attached to the C-terminus of the conotoxin peptide analog.

In a specific embodiment, the PEG polymer is covalently attached to the non-conotoxin peptide analog at an amino acid residue position at the N-terminus or C-terminus.

In a specific embodiment, the PEG polymer is covalently attached to the conotoxin peptide analog via a linking group.

In a specific embodiment, the linking group is a valerate linker having the formula:

in a specific embodiment, the linking group is butylene.

In a specific embodiment, the linking group is a carbonyl group.

In a specific embodiment, the PEG polymer is a linear or branched PEG polymer.

In a specific embodiment, the PEG polymer is a linear PEG polymer.

In a specific embodiment, the PEG polymer has a molecular weight in the range of 10kDa to 40 kDa.

In a specific embodiment, the PEG polymer is a linear 30kDa PEG polymer.

In a specific embodiment, the PEG polymer is a linear 30kDa mPEG polymer.

In a specific embodiment, the PEGylated conotoxin peptide analog has the formula (IIa) (SEQ ID NO: 83):

in a specific embodiment, the PEGylated conotoxin peptide analog has formula (IIg) (SEQ ID NO: 95):

in a specific embodiment, the PEGylated conotoxin peptide analog has formula (IIh) (SEQ ID NO: 96):

in a specific embodiment, the PEGylated conotoxin peptide analog has formula (IIi) (SEQ ID NO: 97):

in a specific embodiment, the PEGylated conotoxin peptide analog has formula (IIk) (SEQ ID NO: 98):

in a specific embodiment, the PEGylated conotoxin peptide analog has formula (IIl) (SEQ ID NO: 99):

in a specific embodiment, the PEGylated conotoxin peptide analog has formula (IIm) (SEQ ID NO: 100):

in a specific embodiment, the PEGylated conotoxin peptide analog has formula (IIn) (SEQ ID NO: 101):

in a specific embodiment, the PEGylated conotoxin peptide analog has the formula (IIo) (SEQ ID NO: 102):

in a specific embodiment, the PEGylated conotoxin peptide analog has formula (IIp) (SEQ ID NO: 103):

Also provided herein are conotoxin peptide analogs of formula (Ib) (SEQ ID NO: 104):

or a pharmaceutically acceptable salt thereof,

wherein R is²Is OH or NH₂。

In a specific embodiment, R²Is OH.

In a specific embodiment, R²Is NH₂。

Also provided herein are pegylated conotoxin peptide analogs, or pharmaceutically acceptable salts thereof, wherein the conotoxin peptide analogs have formula (Ib) (SEQ ID NO: 104):

wherein R is²Is OH or NH₂(ii) a And

In a specific embodiment, R²Is OH.

In a specific embodiment, R²Is NH₂。

in a specific embodiment, the linking group is butylene.

In a specific embodiment, the linking group is a carbonyl group.

In a specific embodiment, the PEG polymer is a linear or branched PEG polymer.

In a specific embodiment, the PEG polymer is a linear PEG polymer.

In a specific embodiment, the PEG polymer is a linear 30kDa PEG polymer.

In a specific embodiment, the PEG polymer is a linear 30kDa mPEG polymer.

In a specific embodiment, the PEGylated conotoxin peptide analog has formula (IIb) (SEQ ID NO: 105):

also provided herein are conotoxin peptide analogs selected from conotoxin peptide analogs Ia, Ia ', Ib', Ic, Id, Ie, If, Ig, Ih, Ii, Ij, Ik, Il, Im, In, Io, Ip, Iq, Ir, Is, It, Iu and Iv, or pharmaceutically acceptable salts thereof.

In a particular embodiment, the conotoxin peptide analog is selected from the group consisting of conotoxin peptide analogs Ia, Ia ', Ib', Ig, Ih, Ii, Ik, Il, Im, In, Io and Ip.

In a particular embodiment, the conotoxin peptide analog is selected from conotoxin peptide analogs Ia, Ia ', Ib and Ib'.

Also provided herein are pharmaceutical compositions comprising a conotoxin peptide analog or pharmaceutically acceptable salt thereof or a pegylated conotoxin peptide analog or pharmaceutically acceptable salt thereof as described herein and optionally a pharmaceutically acceptable carrier.

Also provided herein are methods of treating or preventing a condition by inhibiting the treatment or prevention of α 9-containing nicotinic acetylcholine receptors (nachrs) in a subject, comprising administering to the subject a therapeutically effective amount of a conotoxin peptide analog, or a pharmaceutically acceptable salt thereof, or a pegylated conotoxin peptide analog, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein.

Also provided herein are methods of treating or preventing a condition associated with activation of α 9-containing nicotinic acetylcholine receptors (nachrs) in a subject comprising administering to the subject a therapeutically effective amount of a conotoxin peptide analog or a pharmaceutically acceptable salt thereof, or a pegylated conotoxin peptide analog or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein.

In a specific embodiment, the condition that contributes to treatment or prevention by inhibiting α 9-containing nachrs is pain or inflammation.

In a specific embodiment, the inclusion is pain.

In particular embodiments, the pain is selected from the group consisting of general pain, chronic pain, neuropathic pain, nociceptive pain, inflammatory pain, visceral pain, somatic pain, pain caused by peripheral nerve injury, pain caused by inflammatory disorders, pain caused by metabolic disorders, pain caused by cancer, pain caused by chemotherapy, pain caused by surgical procedures, and pain caused by burns.

In a specific embodiment, the pain is cancer-related chronic pain.

In a particular embodiment, the condition that contributes to treatment or prevention by inhibiting α 9-containing nachrs is an inflammatory condition.

In particular embodiments, the inflammatory condition is selected from the group consisting of inflammation, chronic inflammation, rheumatic diseases, sepsis, fibromyalgia, inflammatory bowel disease, sarcoidosis, endometriosis, uterine fibroids, inflammatory skin diseases, inflammatory lung diseases, diseases associated with nervous system inflammation, periodontal disease, and cardiovascular disease.

In particular embodiments, the inflammatory condition is mediated by immune cells.

In particular embodiments, the inflammatory condition is long-term inflammation and/or peripheral neuropathy after injury.

In particular embodiments, conditions that contribute to treatment or prevention by inhibition of α 9-containing nachrs are pain and inflammation.

In particular embodiments, conditions that contribute to treatment or prevention by inhibition of α 9-containing nachrs are inflammation and neuropathy.

In particular embodiments, a condition that contributes to treatment or prevention by inhibiting α 9-containing nicotinic acetylcholine receptors (nachrs) is one that contributes to treatment or prevention by inhibiting the α 9 α 10 subtype of nachrs.

In a specific embodiment, 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 a pharmaceutically acceptable salt thereof, or a pegylated conotoxin peptide analog or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein.

Also provided herein is a conotoxin peptide analog or a pharmaceutically acceptable salt thereof, or a pegylated conotoxin peptide analog or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein for use in the treatment or prevention of a condition that contributes to the treatment or prevention by inhibiting α 9-containing nicotinic acetylcholine receptors (nachrs) in a subject.

Also provided herein is the use of a pharmaceutical composition comprising a conotoxin peptide analog or a pharmaceutically acceptable salt thereof, or a pegylated conotoxin peptide analog or a pharmaceutically acceptable salt thereof, for the treatment or prevention of a condition that contributes to the treatment or prevention by inhibiting α 9-containing nicotinic acetylcholine receptors (nachrs) in a subject.

Also provided herein is the use of a conotoxin peptide analog or a pharmaceutically acceptable salt thereof, or a pegylated conotoxin peptide analog or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein, in the manufacture of a medicament for the treatment or prevention of a condition that contributes to the treatment or prevention by inhibiting the nicotinic acetylcholine receptor (nAChR) containing α 9 in a subject.

Also provided herein are conotoxin peptide analogs or salts thereof, wherein the amino acid sequence of the conotoxin peptide analogs is

Gly-Cys-X_AA ³-Thr-Asp-Pro-Arg-Cys-X_AA ⁹-Trp-Gln-X_AA ¹²-X (SEQ ID NO: 106), wherein

X_AA ³Selected from the group consisting of (S) -propargylglycine, (S) -azidoalanine, (S) -homopropargylglycine, (S) -azidohomoalanine, (S) -azidovaline and (S) -bishomopropargylglycine;

X_AA ⁹is citrulline;

X_AA ¹²selected from the group consisting of (S) -azidohomoalanine, (S) -homopropargylglycine, (S) -azidovaline and (S) -bishomopropargylglycine;

Wherein when X is_AA ³Selected from (S) -propargylglycine, (S) -homopropargylglycine and (S) -bishomopropargylglycine, X_AA ¹²Is (S) -azidohomoalanine or (S) -azidovaline; and when X_AA ³Selected from (S) -azidoalanine, (S) -azidohomoalanine and (S) -azidovaline, X_AA ¹²Is (S) -homopropargylglycine or (S) -bishomopropargylglycine;

x is X_AA ¹Or X_AA ¹X_AA ²(ii) a Wherein X_AA ¹Is Tyr, Phe, Trp, or the D-isomer of Tyr, Phe or Trp, and X_AA ²Is N-Me-Gly, D-Tyr or N-Me-Tyr; and

In a specific embodiment, X_AA ³Is (S) -propargyl glycine or (S) -azido alanine.

In a specific embodiment, X_AA ¹²Is (S) -azidovaline or (S) -bishomopropargyl glycine.

In a specific embodiment, X_AA ³Is (S) -propargylglycine and X_AA ¹²Is (S) -azidovaline.

In a specific embodiment, X_AA ³Is (S) -homopropargylglycine and X_AA ¹²Is (S) -azidovaline.

In a specific embodiment, X_AA ³Is (S) -homopropargylglycine and X_AA ¹²Is (S) -azidohomoalanine.

In a specific embodiment, X_AA ³Is (S) -azidohomoalanine and X _AA ¹²Is (S) -homopropargyl glycine.

In a specific embodiment, X_AA ³Is (S) -azidoalanine and X_AA ¹²Is (S) -di-homopropargyl glycine.

In a specific embodiment, X is X_AA ¹。

In a specific embodiment, X is X_AA ¹X_AA ²。

In a specific embodiment, X_AA ¹Selected from Tyr, D-Tyr and Phe.

In a specific embodiment, X is Tyr.

In a specific embodiment, the C-terminus of the conotoxin peptide analog is OH.

In a specific embodiment, X_AA ³Is (S) -propargylglycine, X_AA ¹²Is (S) -azidovaline and X is Tyr.

In a specific embodiment, the C-terminus of the conotoxin peptide analog is a carboxyl group.

In a specific embodiment, the C-terminus of the conotoxin peptide analog is an amide group.

In a specific embodiment, X_AA ³Is (S) -homopropargylGlycine, X_AA ¹²Is (S) -azidovaline, X is Tyr; and wherein the C-terminus of the conotoxin peptide analog is a carboxyl group.

In a specific embodiment, X_AA ³Is (S) -homopropargylglycine, X_AA ¹²Is (S) -azidohomoalanine, X is Tyr; and wherein the C-terminus of the conotoxin peptide analog is a carboxyl group.

In a specific embodiment, X_AA ³Is (S) -azidohomoalanine, X_AA ¹²Is (S) -homopropargyl glycine, and X is Tyr; and wherein the C-terminus of the conotoxin peptide analog is a carboxyl group.

In a specific embodiment, X_AA ³Is (S) -azidoalanine, X_AA ¹²Is (S) -di-homopropargyl glycine, and X is Tyr; and wherein the C-terminus of the conotoxin peptide analog is a carboxyl group.

In a specific embodiment, X_AA ³Is (S) -propargylglycine, X_AA ¹²Is (S) -azidovaline, X is Phe; and wherein the C-terminus of the conotoxin peptide analog is a carboxyl group.

In a specific embodiment, X_AA ³Is (S) -propargylglycine, X_AA ¹²Is (S) -azidovaline, and X is D-Tyr; and wherein the C-terminus of the conotoxin peptide analog is a carboxyl group.

In a specific embodiment, X_AA ³Is (S) -propargylglycine, X_AA ¹²Is (S) -azidovaline, and X is Tyr-N-Me-Gly; and wherein the C-terminus of the conotoxin peptide analog is a carboxyl group.

In a specific embodiment, X_AA ³Is (S) -propargylglycine, X_AA ¹²Is (S) -azidovaline, and X is Tyr-D-Tyr; and wherein the C-terminus of the conotoxin peptide analog is a carboxyl group.

In a specific embodiment, X_AA ³Is (S) -propargylglycine, X _AA ¹²Is (S) -azidovalineAcid, X is Tyr-N-Me-Tyr; and wherein the C-terminus of the conotoxin peptide analog is a carboxyl group.

Gly-Cys-X_AA ³-Thr-Asp-Pro-Arg-Cys-X_AA ⁹-X_AA ¹⁰-Gln-X_AA ¹²-Tyr(SEQ ID NO：107)，

Wherein

X_AA ³Is (S) -propargylglycine;

X_AA ⁹is citrulline;

X_AA ¹⁰is 3-iodo-Tyr;

X_AA ¹²is (S) -azidovaline;

Also provided herein is a method for preparing a conotoxin peptide analog of formula (I) (SEQ ID NO: 93) or a pharmaceutically acceptable salt thereof,

wherein

wherein the C-terminal of the conotoxin peptide analogue shown in the chemical formula (I) is carboxylic acid or amido;

which comprises subjecting an intermediate conotoxin peptide analogue or salt thereof to triazole-forming conditions,wherein the amino acid sequence of the intermediate conotoxin peptide analogue is Gly-Cys-X _AA ³-Thr-Asp-Pro-Arg-Cys-X_AA ⁹-Trp-Gln-X_AA ¹²-X(SEQ ID NO：106)，

Wherein

X_AA ⁹is citrulline;

x is as defined above for the conotoxin peptide analogue of formula (I); and

wherein the C-terminus of the intermediate conotoxin peptide analog is as defined above for the conotoxin peptide analog of formula (I); and

wherein under the triazole forming conditions, X_AA ³And X_AA ¹²Reacting to form a triazole bridge in the conotoxin peptide analogue of formula (I).

Also provided herein is a method for preparing a conotoxin peptide analog of formula (Ib) (SEQ ID NO: 104) or a pharmaceutically acceptable salt thereof,

Wherein the C-terminal of the conotoxin peptide analogue shown in the chemical formula (Ib) is carboxylic acid or amido,

which comprises subjecting an intermediate conotoxin peptide analog or a salt thereof to triazole formation conditions, wherein the amino acid sequence of the intermediate conotoxin peptide analog is Gly-Cys-X_AA ³-Thr-Asp-Pro-Arg-Cys-X_AA ⁹-X_AA ¹⁰-Gln-X_AA ¹²-Tyr(SEQ ID NO：107)，

Wherein

X_AA ³Is (S) -propargylglycine;

X_AA ⁹is citrulline;

X_AA ¹⁰is 3-iodo-Tyr;

X_AA ¹²is (S) -azidovaline; and

wherein the C-terminus of the intermediate conotoxin peptide analog is as defined above for the conotoxin peptide analog of formula (Ib); and

wherein under the triazole forming conditions, X_AA ³And X_AA ¹²Reacting to form a triazole bridge as shown in conotoxin peptide analogues of formula (Ib).

Also provided herein are methods of making a pegylated conotoxin peptide analog or pharmaceutically acceptable salt thereof, comprising contacting a conotoxin peptide analog or salt thereof with one or more reactive polyethylene glycol (PEG) polymers under reaction conditions to form a pegylated conotoxin peptide analog, wherein the reactive PEG polymers each comprise a reactive group that is covalently attached, 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, either directly or via a linking group, to one or more PEG polymers,

Wherein the amino acid sequence of the conotoxin peptide analogue is Gly-Cys-X_AA ³-Thr-Asp-Pro-Arg-Cys-X_AA ⁹-Trp-Gln-X_AA ¹²-X(SEQ ID NO：106)，

Wherein

X_AA ⁹is citrulline;

Wherein the amino acid sequence of the conotoxin peptide analogue is Gly-Cys-X_AA ³-Thr-Asp-Pro-Arg-Cys-X_AA ⁹-X_AA ¹⁰-Gln-X_AA ¹²-Tyr(SEQ ID NO：107)，

Wherein

X_AA ³Is (S) -propargylglycine;

X_AA ⁹is citrulline;

X_AA ¹⁰is 3-iodo-Tyr;

X_AA ¹²is (S) -azidovaline;

4. Description of the drawings

Fig. 1A to fig. 1D: CSP-4-NH in human serum₂Shuffling of disulfide bonds. FIG. 1A: CSP-4-NH₂Samples were loaded into human serum and incubated at 37 ℃ for the indicated time periods, then the samples were frozen, purified by precipitation and filtration and analyzed by RP-HPLC. A time-dependent conversion of the main peak to a secondary peak with a faster retention time was observed. FIG. 1B: the synthetically constructed CSP-4-NH was analyzed₂Bands, beads and natural isomers and CSP-4-NH isolated from human serum₂Individual injections of the samples. Co-migration of serum-derived peaks indicates conversion of the native form to a ribbon isomer. FIG. 1C: serum-derived compound CSP-4-NH₂Followed by lyophilization and mass spectrometry showed that both native and ribbon isomers had the same mass to charge ratio. FIG. 1D: by CSP-4-NH₂Glutathione reduction of (a) converts the globular shape to a ribbon-like form, indicating that disulfide bond shuffling is the basis for isomerization.

Fig. 2A-2B: conotoxin peptide analogue Ia and conotoxin peptide analogue CSP-4-NH in rat plasma₂Reverse phase HPLC analysis of (1). FIG. 2A: reverse phase HPLC analysis of conotoxin peptide analog Ia at 0h, 8h and 24h (black line: 0 h; dark grey line: 8 h; light grey line: 24 h). FIG. 2B: conotoxin peptide analogue CSP-4-NH in rat plasma at 0h, 8h and 24h₂("Natural" shape)Formula (xxxvii) consisting of two disulfide bonds, one between Cys2 and Cys8 and the second between Cys3 and Cys12 (black line: 0 h; dark gray line: 8 h; light gray line: 24h) in that respect

Fig. 3A-3B: concentration-response curves for conotoxin peptide analogs to inhibit ACh-gated current in human α 9 α 10 nachrs. FIG. 3A: conotoxin peptide analogs Ia, Ia 'and Ib' inhibit ACh-gated current in human α 9 α 10 nachrs in the concentration-response curve (●: Ia;. a: Ia '; □: Ib'). FIG. 3B: conotoxin peptide analog CSP-4-NH₂And concentration-response Curve of CSP-4-OH inhibition of ACh-gated Current in human α 9 α 10nAChR (●: CSP-4-NH)₂；▲：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-5B: concentration-response curves for conotoxin peptide analogs to inhibit ACh-gated current in human α 9 α 10 nachrs. FIG. 5A: concentration-response curve of conotoxin peptide analogues CSP-4-OH and CSP-4-desTyr-OH for inhibiting ACh-gated current in human alpha 9 alpha 10nAChR (tangle-solidup: CSP-4-OH; ■: CSP-4-desTyr-OH). FIG. 5B: conotoxin peptide analogs 1a and 1q inhibit the concentration-response curve of ACh-gated current in human α 9 α 10 nachrs (●: 1 a; ■: 1 q).

Fig. 6A-6B: representative traces from oocytes injected with human (left) or rat (right) α 9 α 10 nachrs. ACh-gated current before and after exposure to the compound pegylated conotoxin peptide analog IIa. FIG. 6A: response of human α 9 α 10 nachrs to ACh. FIG. 6B: response of rat α 9 α 10nAChR to ACh.

Fig. 7A-7B: pharmacokinetic profile of pegylated conotoxin peptide analogs in rats. A profile of the mean concentration vs. time of pegylated conotoxin peptide analogue IIa and conotoxin peptide analogue Ia administered intravenously and subcutaneously at 1mg/kg to rats (n ═ 3). FIG. 7A: pharmacokinetic profile of pegylated conotoxin peptide analog IIa (ii) ((iii))

1mg/kg IV administration;

1mg/kg SC administration). FIG. 7B: pharmacokinetic profile of conotoxin peptide analogues Ia (II)

1mg/kg IV administration;

1mg/kg SC administration).

FIG. 8: pharmacokinetic profile of pegylated conotoxin peptide analogue IIa in monkeys. Profile of the mean concentration vs time of compound 21 administered intravenously and subcutaneously to cynomolgus macaques (cynomolgus macaque) at 1mg/kg (n ═ 3). (

1mg/kg IV administration;

1mg/kg SC administration).

FIG. 9: analgesic efficacy and duration of action of conotoxin peptide analog Ib 'in a rat model of chronic stress injury (CCI) (■: 0.1 mg/kg/day conotoxin peptide analog Ib'; ●: vehicle).

Fig. 10A to 10C: conotoxin peptide analog CSP-4-NH₂And CSP-4-NH₂The analgesic efficacy and duration of action of the pegylated derivatives of (a) in the rat CIPN model. FIG. 10A: mechanical hyperalgesia was measured by the Randall-Selitto test, which showed CSP-4-NH within 96h after dosing₂And analgesic efficacy (normalized response) of pegylated conotoxin peptides VIII and X in rat CIPN model. FIG. 10B: mechanical hyperalgesia was measured by the Randall-Selitto test, which showed CSP-4-NH within 96h after dosing₂And analgesic potency of pegylated conotoxin peptides VII, IX, XI and XII in rat CIPN model (normalized response). FIG. 10C: CSP-4-NH₂And CSP-4-NH₂Summary of the duration of potency of pegylated derivatives of (a) shows the duration of potency versus the pegylation conjugation chemistry (branched vs. linear) and the size of the conjugated PEG polymer of (b)Are correlated.

Fig. 11A to 11C: analgesic efficacy and duration of action 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 0.5mg/kg dose of conotoxin peptide analogue Ia' to rats with oxaliplatin-induced peripheral neuropathy at 14 days after induction of neuropathy resulted in a statistically significant reduction of mechanical hyperalgesia for up to 24h compared to vehicle-treated control animals. (■: 0.5mg/kg conotoxin peptide analogue Ia'; ●: vehicle). FIG. 11B: a single 0.5mg/kg dose of Ia' administered to rats with retained nerve injury on day 14 post-surgical challenge model resulted in a statistically significant reduction in mechanical hyperalgesia for up to 24h compared to vehicle-treated control animals. (■: 0.5mg/kg conotoxin peptide analogue Ia'; ●: vehicle). FIG. 11C: a single 0.5mg/kg dose of conotoxin peptide analog Ia or pegylated conotoxin peptide analog IIa administered to rats with oxaliplatin-induced peripheral neuropathy 14 days after induction of neuropathy resulted in a statistically significant reduction of mechanical hyperalgesia up to 4h (conotoxin peptide analog Ia) or 72h (conotoxin peptide analog Ia) after dosing compared to vehicle-treated control animals. (

A 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). Bidirectional analysis of variance,. about.p<0.05，**p<0.01，***p<0.001。

5. Detailed description of the invention

It is to be understood that this disclosure is not limited to the particular embodiments described herein, and that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

5.1. Abbreviations as used herein

It will be understood that throughout the present disclosure, amino acids are referred to in terms of single letter or 3-letter codes. The amino acids forming all or part of a peptide may be derived from the known 21 naturally occurring amino acids, which are indicated by their single letter abbreviations and their common three letter abbreviations. In the peptide sequences provided herein, conventional amino acid residues have their conventional meaning. Thus, "Leu" is leucine, "Ile" is isoleucine, "Nle" is norleucine, and the like. To assist the reader, conventional amino acids and their corresponding 3-letter and one-letter abbreviations are as follows:

Alanine	Ala	(A)
			Arginine	Arg	(R)
Asparagine	Asn	(N)
			Aspartic acid	Asp	(D)
Cysteine	Cys	(C)
			Glutamic acid	Glu	(E)
Glutamine	Gln	(Q)
			Glycine	Gly	(G)
Histidine	His	(H)
			Isoleucine	Ile	(I)
Leucine	Leu	(L)
			Lysine	Lys	(K)
Methionine	Met	(M)
			Phenylalanine	Phe	(F)
Proline	Pro	(P)
			Serine	Ser	(S)
Threonine	Thr	(T)
			Tryptophan	Trp	(W)
Tyrosine	Tyr	(Y)
			Valine	Val	(V)

In the peptide sequences provided herein, other amino acids and their corresponding 3-letter abbreviations are as follows:

(S) -5-azidovaline 5-azido NVa

(S) -propargylglycine Pra

(S) -3-azido-alanine 3-azido Ala

(S) -homopropargylglycine homo Pra

(S) -gamma-azido-homoalanine azido homoAla

(S) -bis-homopropargylglycine bishomo Pra

Citrulline Cit

5.2. Conotoxin peptide RgIA analogue

In one aspect, provided herein is a conotoxin peptide analog of formula (I) (SEQ ID NO: 93):

or a pharmaceutically acceptable salt thereof,

wherein

C in the formula (I)¹And C²The notation "(S)" near carbon indicates C ¹And C²The absolute configuration of the carbon.

In various embodiments, the triazole bridge is

Wherein the single wave line

C representing triazole bridge and conotoxin peptide analogues²A point of attachment for carbon; and wherein x is 1, 2, 3 or 4; y is 2, 3 or 4. In a specific embodiment, the triazole bridge is

In a specific embodiment, the triazole bridge is

In particular embodiments, x is 1, 2, 3 or 4. In a preferred embodiment, x is 1, 2 or 3. In particular embodiments, x is 1 or 2. In one embodiment, x is 1. In another embodiment, x is 2.

In particular embodiments, y is 2, 3 or 4. In particular embodiments, y is 2 or 3. In one embodiment, y is 3. In another embodiment, y is 2.

In a specific embodiment, x is 1, 2 or 3 and y is 2 or 3. In particular embodiments, x is 1, 2 or 3 and y is 2. In particular embodiments, 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 a preferred embodiment, x is 1 and y is 3. In a preferred embodiment, x is 2 and y is 3. In a preferred embodiment, x is 2 and y is 2.

In a specific embodiment, theThe triazole bridge is

Wherein the single wave line

In a specific embodiment, the triazole bridge is

Wherein the single wave line

In a specific embodiment, X is X_AA ¹. In a specific embodiment, X is X_AA ¹X_AA ²。

In a specific embodiment, X_AA ¹Is Tyr, Phe, Trp, or the D-isomer of Tyr, Phe or Trp. In a specific embodiment, X_AA ¹Is Tyr, Phe or Trp. In a specific embodiment, X_AA ¹Is Tyr, D-Tyr or Phe. In a specific embodiment, X_AA ¹Is Tyr or D-Tyr. In a specific embodiment, X_AA ¹Is Tyr or Phe. In a specific embodiment, X_AA ¹Is D-Tyr or Phe. In a specific embodiment, X_AA ¹Is Phe. In particular toIn an embodiment, X_AA ¹Is D-Phe. In a specific embodiment, X_AA ¹Is Trp. In a specific embodiment, X_AA ¹Is D-Trp. In a specific embodiment, X _AA ¹Is D-Tyr. In a preferred embodiment, X_AA ¹Is Tyr.

In a specific embodiment, X_AA ²Is N-Me-Gly, D-Tyr or N-Me-Tyr. In a specific embodiment, X_AA ²Is N-Me-Gly or D-Tyr. In a specific embodiment, X_AA ²Is D-Tyr or N-Me-Tyr. In a specific embodiment, X_AA ²Is N-Me-Gly or N-Me-Tyr. In a specific embodiment, X_AA ²Is N-Me-Gly. In a specific embodiment, X_AA ²Is N-Me-Tyr. In a specific embodiment, X_AA ²Is D-Tyr. In a specific embodiment, X_AA ²Is optionally present. In a specific embodiment, X_AA ²Is present. In a specific embodiment, X_AA ²Is absent.

In a specific embodiment, X is selected from Tyr, Phe, D-Tyr, (Tyr) - (D-Tyr), (Tyr) - (N-Me-Gly), (Tyr) - (N-Me-Tyr), N-Me-Tyr, D-Arg, N-Me-D-Tyr, β -Tyr, and N-Me-Arg. In a specific embodiment, X is selected from Tyr, Phe, D-Tyr, (Tyr) - (D-Tyr), (Tyr) - (N-Me-Gly), and (Tyr) - (N-Me-Tyr). In a specific embodiment, X is selected from 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, β -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. In a specific embodiment, 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 β -Tyr. In a specific embodiment, X is N-Me-Arg.

In particular embodiments, the C-terminus of the conotoxin peptide analog is a carboxylic acid or an amide group. In a 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。

In a specific embodiment, the conotoxin peptide analog of formula (I) or a pharmaceutically acceptable salt thereof has formula (Ia) (SEQ ID NO: 94):

wherein R is¹Is OH or NH₂. In a specific embodiment, R¹Is OH. In a specific embodiment, R¹Is NH₂。

In a specific embodiment, the conotoxin peptide analog of formula (I) or a pharmaceutically acceptable salt thereof has formula (Ig) (SEQ ID NO: 30):

in a specific embodiment, the conotoxin peptide analog of formula (I) or a pharmaceutically acceptable salt thereof has formula (Ih) (SEQ ID NO: 33):

in a specific embodiment, the conotoxin peptide analog of formula (I) or pharmaceutically acceptable salt thereof has formula (Ii) (SEQ ID NO: 36):

in a specific embodiment, the conotoxin peptide analog of formula (I) or a pharmaceutically acceptable salt thereof has formula (Ik) (SEQ ID NO: 42):

in a specific embodiment, the conotoxin peptide analog of formula (I) or pharmaceutically acceptable salt thereof has formula (Il) (SEQ ID NO: 45):

In a specific embodiment, the conotoxin peptide analog represented by formula (I) or a pharmaceutically acceptable salt thereof has formula (Im) (SEQ ID NO: 48):

in a specific embodiment, the conotoxin peptide analog of formula (I) or a pharmaceutically acceptable salt thereof has formula (In) (SEQ ID NO: 51):

in a specific embodiment, the conotoxin peptide analog of formula (I) or a pharmaceutically acceptable salt thereof has formula (Io) (SEQ ID NO: 54):

in a specific embodiment, the conotoxin peptide analog of formula (I) or a pharmaceutically acceptable salt thereof has formula (Ip) (SEQ ID NO: 57):

in another aspect, provided herein is a conotoxin peptide analog of formula (Ib) (SEQ ID NO: 104):

or a pharmaceutically acceptable salt thereof,

wherein R is²Is OH or NH₂。

In a specific embodiment, R²Is OH. In a specific embodiment, R²Is NH₂。

In another aspect, provided herein Is a conotoxin peptide analog selected from the group consisting of conotoxin peptide analogs Ia, Ia ', 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.

In a particular embodiment, the conotoxin peptide analog is selected from conotoxin peptide analogs Ia, Ia ', Ib and Ib'. In a preferred embodiment, the conotoxin peptide analog is conotoxin peptide analog Ia or Ia'. In a preferred embodiment, the conotoxin peptide analog is conotoxin peptide analog Ia.

PEGylated conotoxin peptide RgIA analogs

In one embodiment, the conotoxin peptide analogs of the present invention are pegylated, specifically covalently linked to one or more PEG polymers.

In particular embodiments, the conotoxin peptide analog is covalently attached to a PEG polymer. In a specific embodiment, the conotoxin peptide analog is covalently attached to more than one PEG polymer. In a specific embodiment, the conotoxin peptide analog is covalently attached to two PEG polymers. In a specific embodiment, the conotoxin peptide analog is covalently attached to 3 PEG polymers.

In a preferred embodiment, the PEG polymers are covalently attached to the N-terminus of the conotoxin peptide analogs, most preferably one PEG polymer is attached to the N-terminus only. In a specific embodiment, the PEG polymer is covalently attached to the C-terminus of the conotoxin peptide analog. In a specific embodiment, the PEG polymer is covalently attached to the non-conotoxin peptide analog at an amino acid residue position at the N-terminus or C-terminus.

In a specific embodiment, the PEG polymer is covalently attached to the conotoxin peptide analog via a linking group. In a specific embodiment, the PEG polymer is directly covalently attached to the conotoxin peptide analog.

In a preferred embodiment, the linking group is a valerate linker having the formula:

in a specific embodiment, the linking group is butylene. In a specific embodiment, the linking group is butylene. In a specific embodiment, the linking group is a carbonyl group.

In a specific embodiment, the PEG polymer is a linear or branched PEG polymer. In a specific embodiment, the PEG polymer is a branched PEG polymer. In a preferred embodiment, the PEG polymer is a linear PEG polymer.

In a specific embodiment, the PEG polymer has a molecular weight in the range of 10kDa to 40 kDa. In a preferred embodiment, the PEG polymer is a 30kDa PEG polymer. In a specific embodiment, the PEG polymer is a linear 30kDa PEG polymer. In a specific embodiment, the PEG polymer is a linear 30kDa mPEG polymer.

In preferred embodiments, one PEG polymer is attached to the amino terminus of the conotoxin peptide analogAnd attaching the PEG polymer to the conotoxin peptide analog via a linking group, and the linking group is a valerate linker having the formula:

and the PEG polymer is a linear 30kDa mPEG polymer.

In a specific embodiment, provided herein is a PEGylated conotoxin peptide analog or pharmaceutically acceptable salt, wherein the conotoxin peptide analog is of formula (I) (SEQ ID NO: 93):

wherein

In various embodiments, the triazole bridge is

Wherein the single wave line

C representing triazole bridge and conotoxin peptide analogues²A point of attachment for carbon; and wherein x is 1, 2, 3 or 4; y is 2, 3 or 4. In the concrete examples In one embodiment, the triazole bridge is

In a specific embodiment, the triazole bridge is

In particular embodiments, x is 1, 2, 3, or 4. In a preferred embodiment, x is 1, 2 or 3. In particular embodiments, x is 1 or 2. In one embodiment, x is 1. In another embodiment, x is 2.

In particular embodiments, x is 1, 2 or 3 and y is 2 or 3. In particular embodiments, x is 1, 2 or 3 and y is 2. In particular embodiments, 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 a preferred embodiment, x is 1 and y is 3. In a preferred embodiment, x is 2 and y is 3. In a preferred embodiment, x is 2 and y is 2.

In a specific embodiment, the triazole bridge is

Wherein the single wave line

In a specific embodiment, the triazole bridge is

Wherein the single wave line

In a specific embodiment, X_AA ¹Is Tyr, Phe, Trp or the D-isomer of Tyr, Phe or Trp. In a specific embodiment, X_AA ¹Is Tyr, Phe or Trp. In a specific embodiment, X_AA ¹Is Tyr, D-Tyr or Phe. In a specific embodiment, X_AA ¹Is Tyr or D-Tyr. In a specific embodiment, X_AA ¹Is Tyr or Phe. In a specific embodiment, X_AA ¹Is D-Tyr or Phe. In a specific embodiment, X_AA ¹Is Phe. In a specific embodiment, X_AA ¹Is D-Phe. In a specific embodiment, X_AA ¹Is Trp. In a specific embodiment, X_AA ¹Is D-Trp. In a specific embodiment, X _AA ¹Is D-Tyr. In a preferred embodiment, X_AA ¹Is Tyr.

in another aspect, provided herein is a pegylated conotoxin peptide analog, or a pharmaceutically acceptable salt thereof, wherein the conotoxin peptide analog is of formula (Ib) (SEQ ID NO: 104):

wherein R is²Is OH or NH₂(ii) a And

In a particular embodiment of formula (Ib), R²Is OH. In a particular embodiment of formula (Ib), R²Is NH₂。

5.4. Pharmaceutical composition

Also provided herein are "pharmaceutical compositions" comprising a conotoxin peptide analog provided herein or a pegylated conotoxin peptide analog provided herein, and one or more pharmaceutically acceptable carriers. In a specific embodiment, the conotoxin peptide analog is present in a therapeutically effective amount. In a specific embodiment, the conotoxin peptide analog is present in a prophylactically effective amount. The pharmaceutical compositions may be used according to the methods and uses provided herein. Thus, for example, the pharmaceutical compositions can be administered to a subject to practice the therapeutic or prophylactic methods and uses provided herein. The pharmaceutical compositions provided herein can be formulated to be suitable for the intended method or route of administration; exemplary routes of administration are illustrated herein.

The pharmaceutical compositions generally include a therapeutically effective amount of at least one of a conotoxin peptide analog or a pegylated conotoxin peptide analog 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 paraben, p-hydroxybenzoate), emulsifiers, suspending agents, dispersants, solvents, buffers, lubricants, fillers, and/or diluents. For example, a suitable vehicle may be a physiological saline solution. Typical buffering agents that may be used include, but are not limited to, pharmaceutically acceptable weak acids, weak bases, or mixtures thereof. The buffer component may also include water soluble agents such as phosphoric acid, tartaric acid, succinic acid, citric acid, acetic acid, and salts thereof.

The vehicle may contain other pharmaceutically acceptable excipients that alter or maintain the pH, osmolarity, viscosity, or stability of the pharmaceutical composition. In a specific embodiment, the vehicle is an aqueous buffer. In particular embodiments, the vehicle comprises, for example, sodium chloride.

The pharmaceutical compositions provided herein may contain other pharmaceutically useful agents for altering or maintaining the release rate of the conotoxin peptide analogs described herein. Such formulations include, for example, those skilled in the artThose known to the man to prepare sustained or controlled release formulations. For pharmaceutically useful formulations, see, e.g., Remington's Pharmaceutical Sciences, 18 th edition (1990, Mack Publishing Co., Easton, Pa.18042) page 1435-1712, andThe Merck Index12 th edition (1996, Merck Publishing Group, Whitehouse, NJ).

In particular embodiments, the pharmaceutical composition is provided in a sterile vial as a solution, suspension, gel, emulsion, or dehydrated or lyophilized powder. These compositions may be stored, for example, in ready-to-use form, in lyophilized form requiring reconstitution prior to use, or in liquid form requiring dilution prior to use. In particular embodiments, the pharmaceutical composition is provided in a disposable container (e.g., a disposable vial, ampoule, syringe, or auto injector). In particular embodiments, the pharmaceutical composition is provided in a multi-purpose container (e.g., a multi-purpose vial or cartridge). Any drug delivery device can be used to deliver the conotoxin peptide analogs or pegylated conotoxin peptide analogs or pharmaceutical compositions described herein, including implants (e.g., implantable pumps) and catheters. In particular embodiments, depot injections can be used to release a conotoxin peptide analog or pegylated conotoxin peptide analog or pharmaceutical composition described herein over a defined period of time. Depot injections are usually administered subcutaneously or intramuscularly.

The pharmaceutical compositions can be formulated to be compatible with the intended route of administration as described herein.

The pharmaceutical compositions may be in the form of a sterile injectable aqueous suspension. Such suspensions 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. In one embodiment, the sterile injectable solution may be, for example, a solution in 1, 3-butanediol. Useful diluents, solvents or dispersion vehicles which may be used include water, ringer's solution, saline, Cremophor EL^TMPhosphate Buffered Saline (PBS), ethanol, polyols (e.g., glycerol or liquid polyEthylene glycol) and mixtures thereof. Prolonged absorption of a particular injectable preparation can be brought about by the inclusion of an agent which delays absorption, for example, aluminum monostearate or gelatin.

The pharmaceutical composition may also include a carrier to protect the composition against degradation or elimination from the body. Various antibacterial and antifungal agents, for example, parabens, chlorobutanol, ascorbic acid, thimerosal, may be included in the pharmaceutical composition.

5.5. Methods of treatment or prevention

In one embodiment, provided herein is a method of treating or preventing a condition that contributes to treatment or prevention by inhibiting α 9-containing nicotinic acetylcholine receptors (nachrs), comprising administering to a subject a therapeutically effective amount of a conotoxin peptide analog, or a pharmaceutically acceptable salt thereof, as described herein, or a pegylated conotoxin peptide analog, or a pharmaceutically acceptable salt thereof, as described herein, or a pharmaceutical composition, as described herein.

In a preferred embodiment, a condition that contributes to treatment or prevention by inhibiting α 9-containing nicotinic acetylcholine receptors (nachrs) is one that contributes to treatment or prevention by inhibiting the α 9 α 10 subtype of nachrs.

In particular embodiments, the condition that contributes to treatment or prevention by inhibiting α 9-containing nachrs, e.g., the α 9 α 10 subtype of nachrs, is pain or inflammation.

In a preferred embodiment, the methods provided herein are methods of treating a condition conducive to treatment by inhibition of α 9-containing nicotinic acetylcholine receptors (nachrs), comprising administering to a subject a therapeutically effective amount of a conotoxin peptide analog, or a pharmaceutically acceptable salt thereof, as described herein, or a pegylated conotoxin peptide analog, or a pharmaceutically acceptable salt thereof, as described herein, or a pharmaceutical composition, as described herein.

In a preferred embodiment, the condition conducive to treatment by inhibition of α 9-containing nicotinic acetylcholine receptors (nachrs) is a condition conducive to treatment by inhibition of the α 9 α 10 subtype of nachrs.

In a particular embodiment, the condition that contributes to treatment by inhibiting α 9-containing nachrs, e.g., the α 9 α 10 subtype of nachrs, is pain or inflammation.

In a preferred embodiment, the methods provided herein are methods of preventing a condition conducive to prevention by inhibition of α 9-containing nicotinic acetylcholine receptors (nachrs), 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 a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described herein.

In a preferred embodiment, a condition that contributes to prophylaxis by inhibiting α 9-containing nicotinic acetylcholine receptors (nachrs) is one that contributes to treatment by inhibiting the α 9 α 10 subtype of nachrs.

In particular embodiments, the condition that contributes to prevention by inhibiting α 9-containing nachrs, e.g., the α 9 α 10 subtype of nachrs, is pain or inflammation.

In a particular embodiment, a condition that contributes to the treatment or prevention by inhibiting α 9-containing nicotinic acetylcholine receptors, e.g., the α 9 α 10 subtype of nAChR, is pain. In particular embodiments, the pain is selected from the group consisting of general pain, chronic pain, neuropathic pain, nociceptive pain, inflammatory pain, pain caused by peripheral nerve injury, pain caused by inflammatory conditions, pain caused by metabolic conditions, pain caused by cancer, pain caused by chemotherapy, pain caused by surgical procedures, and pain caused by burns. In a specific embodiment, the pain is cancer-related chronic pain. In particular embodiments, the pain is pain associated with a neuropathy, which may be, for example, a drug (e.g., a cancer chemotherapeutic agent) -induced neuropathy or an infection-induced neuropathy. In a particular embodiment, the pain is neuropathic pain, including (but not limited to) postherpetic neuralgia, e.g., postherpetic neuralgia due to herpes zoster.

In particular embodiments, a condition that contributes to the treatment or prevention by inhibiting α 9-containing nicotinic acetylcholine receptors, e.g., the α 9 α 10 subtype of nAChR, is an inflammatory condition. In particular embodiments, the inflammatory condition is selected from the group consisting of inflammation, chronic inflammation, rheumatic diseases, sepsis, fibromyalgia, inflammatory bowel disease, sarcoidosis, endometriosis, uterine fibroids, inflammatory skin diseases, inflammatory lung diseases, diseases associated with nervous system inflammation, periodontal disease, and cardiovascular disease. In particular embodiments, the inflammatory condition is mediated by immune cells. In particular embodiments, the inflammatory condition is long-term inflammation and/or peripheral neuropathy after injury.

In particular embodiments, the condition associated with α 9-containing nicotinic acetylcholine receptors, e.g., the α 9 α 10 subtype of nAChR, is pain and inflammation. In particular embodiments, the conditions associated with the α 9 α 10 subtype conditions of nachrs are inflammation and neuropathy.

In another aspect, provided herein is a method of treating or preventing a condition associated with activation of α 9-containing nachrs, e.g., the α 9 α 10 subtype of nachrs, in a subject, 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 pegylated conotoxin peptide analog as described herein or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described herein.

In another aspect, provided herein is 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 a pharmaceutically acceptable salt thereof, or a pegylated conotoxin peptide analog as described herein or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition as described herein. In particular embodiments of the method of treating or preventing pain, the pain is pain associated with a neuropathy, for example, it can be a drug (e.g., a cancer chemotherapeutic agent) -induced neuropathy or an infection-induced neuropathy. In a particular embodiment, the pain is neuropathic pain, including (but not limited to) postherpetic neuralgia, e.g., postherpetic neuralgia due to herpes zoster.

In particular embodiments, the methods provided herein are methods of treating pain or inflammation. In a specific embodiment, the methods provided herein are methods of treating pain. In a specific embodiment, the methods provided herein are methods of treating inflammation.

In particular embodiments, the methods provided herein are methods of preventing pain or inflammation. In a specific embodiment, the methods provided herein are methods of preventing pain. In a specific embodiment, the methods provided herein are methods of preventing inflammation.

In a specific embodiment, the method of preventing pain is preventing pain associated with neuropathy. In particular embodiments of these methods, the pain associated with the neuropathy is neuropathic pain. In a specific embodiment, the neuropathic pain is postherpetic neuralgia. In a particular embodiment, the method of preventing pain is preventing pain caused by cancer chemotherapy (and thus administering a conotoxin peptide analog or pharmaceutically acceptable salt thereof to a patient prior to administering a cancer chemotherapeutic agent to the patient). In particular embodiments, the method of preventing pain is preventing pain caused by a surgical procedure; in particular embodiments of these methods, the conotoxin peptide analog or pharmaceutically acceptable salt thereof is administered prior to and/or concurrently with a surgical procedure, particularly wherein it is reasonably expected that the surgical procedure will cause pain and/or inflammation.

In another aspect, provided herein is a method of inhibiting α 9-containing nicotinic acetylcholine receptors (nachrs), e.g., the α 9 α 10 subtype of nachrs, in a subject, 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.

In particular embodiments, "treating" or the like refers to an action (e.g., administration of a conotoxin peptide analog or pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising a conotoxin peptide analog or pharmaceutically acceptable salt thereof) that, after a disease, disorder or condition or symptom thereof or the like has been diagnosed, observed, initiates to temporarily or permanently eliminate, reduce, inhibit, alleviate or ameliorate at least one of the underlying causes of the disease, disorder or condition experienced by the subject or at least one of the symptoms associated with the disease, disorder or condition experienced by the subject. Thus, treatment may include inhibiting (i.e., terminating the development or further development of the disease, disorder or condition or the clinical symptoms associated therewith) active disease.

In particular embodiments of the invention in which the method of prevention is practiced, the prevention need not be complete; in particular embodiments, the prevention is partial inhibition or partial prevention or reduction in a disease, condition, or disorder that would otherwise be expected to occur, relative to administration in the absence of a conotoxin peptide analog or pharmaceutically acceptable salt.

In particular embodiments, the administration may be by injection or otherwise physical delivery of the conotoxin peptide analog to the patient, such as by mucosal, intradermal, intravenous, intramuscular delivery, and/or any other physical delivery method described herein or known in the art. Administration of a conotoxin peptide analog or a pharmaceutically acceptable salt thereof typically occurs after onset of the disease, disorder or condition or symptom thereof, when the disease, disorder or condition or symptom thereof is being treated. Administration of a conotoxin peptide analog or a pharmaceutically acceptable salt thereof typically occurs prior to the onset of a disease, disorder or condition, or symptom thereof, when the disease, disorder or condition, or symptom thereof, is being prevented. In particular embodiments, the conotoxin peptide analog or pharmaceutically acceptable salt thereof is administered before and after onset of pain and/or inflammation to prevent and treat pain and/or inflammation.

In various embodiments, a conotoxin peptide analog as described herein, including (but not limited to) a pegylated conotoxin peptide analog or a pharmaceutically acceptable salt thereof or a pharmaceutical composition thereof, is used in any of the methods provided herein. For example, conotoxin peptide analogs provided herein, including (but not limited to) pegylated conotoxin peptide analogs or pharmaceutically acceptable salts or pharmaceutical compositions thereof, can be used in methods of treatment or prevention of a disease or disorder or condition described herein. Thus, conotoxin peptide analogs provided herein, including (but not limited to) pegylated conotoxin peptide analogs or pharmaceutically acceptable salts or pharmaceutical compositions thereof, can be used as medicaments. Conotoxin peptide analogs provided herein, including (but not limited to) pegylated conotoxin peptide analogs or pharmaceutically acceptable salts or pharmaceutical compositions thereof, can be used in methods of treating or preventing conditions that are conducive to treatment or prevention by inhibiting α 9-containing nicotinic acetylcholine receptors (nachrs), e.g., the α 9 α 10 subtype of nachrs. Conotoxin peptide analogs provided herein, including (but not limited to) pegylated conotoxin peptide analogs or pharmaceutically acceptable salts or pharmaceutical compositions thereof, can be used in methods of treating or preventing conditions that are conducive to treatment or prevention by inhibiting α 9-containing nicotinic acetylcholine receptors (nachrs), e.g., the α 9 α 10 subtype of nachrs. For example, conotoxin peptide analogs provided herein, including (but not limited to) pegylated conotoxin peptide analogs or pharmaceutically acceptable salts or pharmaceutical compositions thereof, can be used in methods of treatment or prevention of pain or inflammation.

5.6. Route of administration and dosage

Conotoxin peptide analogs as described herein, including (but not limited to) pegylated conotoxin peptide analogs or pharmaceutically acceptable salts or pharmaceutical compositions thereof, can 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. In particular embodiments, conotoxin peptide analogs described herein, including (but not limited to) pegylated conotoxin peptide analogs or pharmaceutically acceptable salts or pharmaceutical compositions thereof, are administered by subcutaneous administration. In particular embodiments, the conotoxin peptide analogs described herein, including but not limited to pegylated conotoxin peptide analogs or pharmaceutically acceptable salts or pharmaceutical compositions thereof, are administered by intravenous administration. In particular embodiments, the conotoxin peptide analogs described herein, including but not limited to pegylated conotoxin peptide analogs or pharmaceutically acceptable salts or pharmaceutical compositions thereof, are administered by intra-articular administration.

The amount of conotoxin peptide analog, including but not limited to pegylated conotoxin peptide analogs or pharmaceutically acceptable salts thereof, administered to a 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 a physician.

The exact dosage and regimen employed in the composition will also depend on the route of administration and the severity of the disease and should be determined at the discretion of the physician and the condition of each patient. The appropriate dosage can be determined by one skilled in the medical arts. The total daily dose may be divided and administered in several portions throughout the day or by providing continuous delivery.

In a specific embodiment, the conotoxin peptide analog or pharmaceutically acceptable salt thereof is administered to the human subject at a dose of 0.01 to about 50mg/kg body weight. In a specific embodiment, 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 dose of about 0.5mg/kg body weight. In a specific embodiment, the human dose is 1 to 1000 mg/day. In specific embodiments, the human daily dose is from 1 to 750 mg/day; or 10 to 500 mg/day.

The conotoxin peptide analogs disclosed herein, e.g., pegylated conotoxin peptide analogs or pharmaceutically acceptable salts or pharmaceutical compositions thereof, can be administered once or multiple times at intervals. It will be appreciated that the exact dosage and duration of treatment may vary with the age, weight and condition of the patient to be treated and may be determined empirically using known testing protocols or by extrapolation of in vivo or in vitro testing or diagnostic data. It is further understood that for any particular individual, the particular dosage regimen may be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the formulation.

5.7. Patient's health

The patient referred to in the present disclosure may be, but is not limited to, a human or non-human vertebrate, such as a wild, domestic or farm animal. In particular embodiments, the patient is a mammal, e.g., a human, cow, dog, cat, goat, horse, sheep, pig, rabbit, rat, or mouse. In a preferred embodiment, the patient is a human patient.

In a specific embodiment, the human patient is an adult (at least 16 years of age). In another specific embodiment, the human patient is young (12-15 years old). In another specific embodiment, the patient is a child (under 12 years of age).

5.8. Method for preparing conotoxin peptide analogue

The present disclosure provides a method for preparing a conotoxin peptide analog, wherein an intermediate conotoxin peptide analog (prior to triazole bridge formation) is subjected to triazole formation conditions to form a conotoxin peptide analog of the present invention.

5.8.1. Intermediate conotoxin peptide analogs

The invention discloses synthesis of an intermediate conotoxin peptide analogue used for synthesizing the conotoxin peptide analogue through a triazole bridge. Intermediate conotoxin peptide analogs can be synthesized, for example, using solid phase peptide synthesis. When solid phase peptide synthesis is performed, an essential amino acid having a reactive group suitable for forming a triazole bridge (e.g., an azide group or an acetylene group) may be introduced into a peptide (e.g., a conotoxin peptide of RgIA). The essential amino acids may be introduced at the 3-and 12-positions of the conotoxin peptide analogues of RgIA.

An amino acid residue having an ethynyl group (e.g., (S) -propargylglycine, (S) -homopropargylglycine, or (S) -bishomopropargylglycine) is brought into one of the 3-and 12-positions, and an amino acid having an azido group (e.g., (S) -azidoalanine, (S) -azidohomoalanine, (S) -azidovaline) is introduced into the other position, whereby the ethynyl and azido groups within the same intermediate conotoxin peptide can form a triazole ring when the intermediate conotoxin peptide is subjected to triazole forming conditions, thereby forming a triazole bridge in the resulting conotoxin peptide analog.

In a specific embodiment, the essential amino acids for forming the triazole bridge are selected, for example, from (S) -propargylglycine, (S) -azidoalanine, (S) -homopropargylglycine, (S) -azidohomoalanine, (S) -azidovaline and (S) -bishomopropargylglycine.

In another aspect, provided herein is an intermediate conotoxin peptide analog or salt thereof, wherein the amino acid sequence of the conotoxin peptide analog is

Gly-Cys-X_AA ³-Thr-Asp-Pro-Arg-Cys-X_AA ⁹-Trp-Gln-X_AA ¹²-X(SEQ ID NO：106)，

Wherein

X_AA ⁹Is citrulline;

wherein the C-terminal of the intermediate conotoxin peptide analog is carboxylic acid or amido.

In a specific embodiment, X_AA ³Is (S) -propargyl glycine or (S) -azido alanine. In a specific embodiment, wherein X_AA ¹²Is (S) -azidovaline or (S) -bishomopropargyl glycine. In a specific embodiment, X_AA ³Is (S) -propargylglycine and X_AA ¹²Is (S) -azidovaline. In a specific embodiment, X_AA ³Is (S) -homopropargylglycine and X_AA ¹²Is (S) -azidovaline. In a specific embodiment, X _AA ³Is (S) -homopropargylglycine and X_AA ¹²Is (S) -azidohomoalanine. In a specific embodiment, X_AA ³Is (S) -azidohomoalanine and X_AA ¹²Is (S) -homopropargyl glycine. In a specific embodiment, X_AA ³Is (S) -azidohomoalanine and X_AA ¹²Is (S) -homopropargyl glycine.

In a specific embodiment, X_AA ¹Is Tyr, Phe, Trp or the D-isomer of Tyr, Phe or Trp. In a specific embodiment, X_AA ¹Is Tyr, Phe or Trp. In a specific embodiment, X_AA ¹Is Tyr, D-Tyr or Phe. In a specific embodiment, X_AA ¹Is Tyr or D-Tyr. In a specific embodiment, X_AA ¹Is Tyr or Phe. In a specific embodiment, X_AA ¹Is D-Tyr or Phe. In a specific embodiment, X_AA ¹Is Phe. In a specific embodiment, X_AA ¹Is D-Phe. In a specific embodiment, X_AA ¹Is Trp. In a specific embodiment, X_AA ¹Is D-Trp. In a specific embodiment, X_AA ¹Is D-Tyr. In a preferred embodiment, X_AA ¹Is Tyr.

In a specific embodiment, X_AA ²Is N-Me-Gly, D-Tyr or N-Me-Tyr. In a specific embodiment, X_AA ²Is N-Me-Gly or D-Tyr. In a specific embodiment, X _AA ²Is D-Tyr or N-Me-Tyr. In a specific embodiment, X_AA ²Is N-Me-Gly or N-Me-Tyr. In a specific embodiment, X_AA ²Is N-Me-Gly. In a specific embodiment, X_AA ²Is N-Me-Tyr. In a specific embodiment, X_AA ²Is D-Tyr. In a specific embodiment, X_AA ²Is optionally present. In a specific embodiment, X_AA ²Is present. In a specific embodiment, X_AA ²Is absent.

In a specific embodiment, X_AA ³Is (S) -propargylglycine, X_AA ¹²Is (S) -azidovaline, and X is Tyr. In thatIn one embodiment, the C-terminus of the conotoxin peptide analog is a carboxyl group. In another embodiment, the C-terminal end of the conotoxin peptide analog is an amide group.

In a specific embodiment, X _AA ³Is (S) -azidoalanine, X_AA ¹²Is (S) -di-homopropargyl glycine, and X is Tyr; and wherein the C-terminus of the conotoxin peptide analog is a carboxyl group.

In a specific embodiment, X_AA ³Is (S) -propargylglycine, X_AA ¹²Is (S) -azidovaline, and X is Tyr-N-Me-Tyr; and wherein the C-terminus of the conotoxin peptide analog is a carboxyl group.

Gly-Cys-X_AA ³-Thr-Asp-Pro-Arg-Cys-X_AA ⁹-X_AA ¹⁰-Gln-X_AA ¹²Tyr (SEQ ID NO: 107), wherein

X_AA ³Is (S) -propargylglycine;

X_AA ⁹is citrulline;

X_AA ¹⁰is 3-iodo-Tyr;

X_AA ¹²is (S) -azidovaline;

In a specific embodiment, the C-terminus of the intermediate conotoxin peptide analog is a carboxyl group.

In a specific embodiment, the intermediate conotoxin peptide analog has an amide group at the C-terminus.

5.8.2. Triazole bridge formation

Following synthesis of an intermediate conotoxin peptide having the groups necessary to form a triazole bridge at the 3-and 12-positions (having one ethynyl group and one azido group respectively), the ethynyl and azido groups react under triazole-forming conditions to provide a triazole, thereby forming a triazole bridge in the resulting conotoxin peptide analogue.

In a specific embodiment, provided herein is a method for preparing a conotoxin peptide analog of formula (I) (SEQ ID NO: 93) or a pharmaceutically acceptable salt thereof,

wherein

Which comprises subjecting an intermediate conotoxin peptide analog or a salt thereof to triazole formation conditions, wherein the amino acid sequence of the intermediate conotoxin peptide analog is Gly-Cys-X_AA ³-Thr-Asp-Pro-Arg-Cys-X_AA ⁹-Trp-Gln-X_AA ¹²-X(SEQ ID NO：106)，

Wherein

X_AA ⁹is citrulline;

x is as defined above for the conotoxin peptide analogue of formula (I); and

wherein under the triazole forming conditions, X _AA ³And X_AA ¹²Reacting to form a triazole bridge in the conotoxin peptide analogue of formula (I).

In another aspect, provided herein is a method of preparing a conotoxin peptide analog of formula (Ib) (SEQ ID NO: 104) or a pharmaceutically acceptable salt thereof,

Wherein

X_AA ³Is (S) -propargylglycine;

X_AA ⁹is citrulline;

X_AA ¹⁰is 3-iodo-Tyr;

X_AA ¹²is (S) -azidovaline; and

In a specific embodiment, the triazole formation conditions are in Hein et alHuman, pharm. Res.2008,25(10):2216-2230 (i.e., 1, 3-dipolar cycloaddition of azide and terminal alkyne). In a specific embodiment, the triazole forming conditions comprise the presence of a copper catalyst. In a specific embodiment, the triazole forming conditions include the presence of a copper catalyst and a reducing agent. In one embodiment, the copper catalyst is a cu (ii) salt. In one embodiment, the copper catalyst is CuSO ₄. In one embodiment, the reducing agent is L-ascorbic acid. In another embodiment, the reducing agent is sodium ascorbate. In one embodiment, the triazole forming conditions comprise the presence of a ruthenium catalyst. In one embodiment, the ruthenium catalyst is Cp RuCl (PPh)₃). In certain embodiments, the triazole forming conditions are catalyst-free conditions.

In a particular embodiment, the salt of a conotoxin peptide analog of formula (I) or (Ib) may be subjected to a salt exchange step to provide a pharmaceutically acceptable salt. In one embodiment, for example, the TFA salt of a conotoxin peptide analog of formula (I) is reacted with a base in aqueous solution (pH 7.0-8.0) and then reacted with an appropriate acid to provide a pharmaceutically acceptable salt of the conotoxin peptide analog of formula (I). In one embodiment, the base is NH₄HCO₃(aqueous solution). In one embodiment, after reaction with a base in an aqueous solution, the conotoxin peptide analog is reacted with acetic acid to provide an acetate salt of the conotoxin peptide analog.

PEGylation of

The synthetic conotoxin peptide analogs can be further covalently conjugated to one or more polyethylene glycol (PEG) polymers. The conotoxin peptide analogs can be attached to one or more PEG polymers, e.g., directly or through a linking group.

In a specific embodiment, provided herein is a method of preparing a pegylated conotoxin peptide analog or pharmaceutically acceptable salt thereof, comprising contacting a conotoxin peptide analog or salt thereof with one or more reactive polyethylene glycol (PEG) polymers under reaction conditions to form a pegylated conotoxin peptide analog, wherein the reactive PEG polymers each comprise a reactive group that is covalently attached, optionally through a linking group, to the 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 through a linking group, to one or more PEG polymers. In one embodiment, the conotoxin peptide analog is a conotoxin peptide analog of formula (I) (SEQ ID NO: 93):

wherein

In a specific embodiment, provided herein is a method of preparing a pegylated conotoxin peptide analog or pharmaceutically acceptable salt thereof, comprising contacting a conotoxin peptide analog or salt thereof with one or more reactive polyethylene glycol (PEG) polymers under reaction conditions to form a pegylated conotoxin peptide analog, wherein the reactive PEG polymers each comprise a reactive group that is covalently attached, 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 one or more PEG polymers,

wherein the conotoxin peptide analog is a conotoxin peptide analog represented by the chemical formula (Ib) (SEQ ID NO: 104):

wherein R is²Is OH or NH₂。

Wherein

X_AA ⁹is citrulline;

In another aspect, provided herein is a method of preparing a pegylated conotoxin peptide analog or pharmaceutically acceptable salt thereof, comprising contacting a conotoxin peptide analog or salt thereof with one or more reactive polyethylene glycol (PEG) polymers under reaction conditions to form a pegylated conotoxin peptide analog, wherein the reactive PEG polymers each comprise a reactive group that is covalently attached, 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 one or more PEG polymers,

Wherein

X_AA ³Is (S) -propargylglycine;

X_AA ⁹is citrulline;

X_AA ¹⁰is 3-iodo-Tyr;

X_AA ¹²is (S) -azidovaline;

In a specific embodiment, the reaction conditions are standard pegylation conditions. In one embodiment, the reaction conditions are standard amide formation conditions. Certain standard amide formation conditions are described in Valeur and Bradley, Chemical Society Reviews,2009,38: 606-. In one embodiment, the amide forming conditions include the presence of a coupling agent 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 ] 3-pyridinenium Hexafluorophosphate (HATU). In another embodiment, the reaction conditions are standard amine formation conditions. In one embodiment, the amine forming conditions are reductive amination conditions.

6. Examples of the embodiments

The following non-limiting examples disclose the preparation and testing of conotoxin peptide analogs of conotoxin RgIA and pegylated conotoxin peptide analogs of conotoxin RgIA.

It is to be understood that the following examples are illustrative and not restrictive.

6.1. Example 1: disulfide bond shuffling of RgIA conotoxin peptide analogs CSP-4-OH and CSP-4-NH2

RgIA conotoxin peptide analogs CSP-4-OH and CSP-4-NH₂Having the amino acid sequence Gly-Cys-Cys-Thr-Asp-Pro-Arg-Cys- (Cit) - (3-iodo-Tyr) -Gln-Cys-Tyr; wherein CSP-4-OH (SEQ ID NO: 4) has a carboxylic acid group at the C-terminus and CSP-4-NH₂Having an amide group at the C-terminal. CSP-4-desTyr-OH has the amino acid sequence 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.

The conotoxin peptide analogs CSP-4-OH and CSP-4-NH were found when incubated in human and rat plasma and serum₂Susceptible to disulfide bond shuffling. This disulfide rearrangement results in the formation of isomers of RgIA derivatives, which lose activity on α 9 α 10 nachrs.

CSP-4-NH₂Consists of two disulfide bonds, one between Cys2 and Cys8 and the second between Cys3 and Cys 12. CSP-4-NH₂Cys2-8, Cys3-12 "native" forms of (A) are active on both human and rat α 9 α 10 nAChRs. CSP-4-NH upon exposure to human and rat serum or plasma₂In its natural form Most underwent isomerization to a "ribbon" form containing the replacement disulfide bonds Cys2-Cys12 and Cys3-Cys8 and a "bead" form containing the replacement disulfide bonds Cys2-Cys3 and Cys8-Cys 12.

CSP-4-NH in plasma and serum samples₂Isomerization was predominant between native and banded forms with minimal bead formation (fig. 1A, B). Neither the band nor bead forms were active on human or rat α 9 α 10 nachrs, suggesting that disulfide shuffling helps reduce the efficacy of the molecule. Furthermore, CSP-4-NH is relative to the C-terminal carboxylic acid in CSP-4-OH₂The presence of the medium C-terminal amide does not reduce isomerization. Finally, disulfide isomerization also occurred in vivo following intravenous and subcutaneous injection of compound 6 at a dose of 1mg/kg into Sprague Dawley rats. 1h after injection, only 40-50% of the peptides recovered from the blood remained in native form by HPLC-MS/MS analysis, indicating that disulfide isomerization occurred in vivo similar to in vitro observations.

Conotoxin peptide analogs CSP-4-OH and CSP-4-NH were confirmed by in vitro incubation of the peptides at 37 ℃ for up to 24h in serum or plasma from Sprague Dawley rats and humans at final concentrations of 0.1-0.5mg/mL₂Shuffling of disulfide bonds. With anticoagulants, including citrate, K ₂EDTA and heparin treated plasma samples. Incubated samples were removed at several time points and for each time point, the precipitated samples were centrifuged at 10,000 Xg by protein precipitation (3 volumes of methanol) followed by Millipore Ultrafree-MC GV centrifugal filters to extract the resulting peptide isomers from the matrix. The resulting clarified sample was recovered and 5. mu.L of sample was injected on an Agilent 1260Infinity HPLC equipped with a Phenomenex Aeris C-18 column (2.1X 150mm, 3.6 μm). Peptides were separated by elution with a 0.3mL/min gradient over 45min, 0% to 50% B; mobile phase A (19: 1: 0.01H)₂O/CH₃CN/TFA) and mobile phase B (CH with 0.05% TFA)₃CN). Under these conditions, CSP-4-NH₂The relative retention times of the various isomers of (a) are shown below: band < beads < native (fig. 1B).

1: 1 mixture (10 mM each) treatment of CSP-4-NH₂(40. mu.g/mL) for 1h, followed by treatment with 8% formic acid (quenching), resulting in the formation of a ribbon isomer with the same mass and chromatographic retention time as the product observed in serum or plasma, indicating that disulfide bond isomerization mediates instability of the peptide in biomatrix (FIG. 1D). Glutathione reduction results in a mixture of native and ribbon-like forms.

After separation, collection and freeze-drying of each HPLC peak as described above, LC-MS analysis on the Orbitrap Elite was used to determine each CSP-4-OH and CSP-4-NH₂Identity of the isomers (figure 1C). Aliquots of non-reducing samples were taken in water: acetonitrile: formic acid (98: 2: 0.1%) 1: 100 was diluted to an estimated concentration of 1.2-1.5 pmol/. mu.L. mu.L was injected into a sample of 100. mu.m i.d.IntegraFrit 2cm long by Easy-nLC II HPLC system (Thermo Scientific)^TMWell (New Objective) coupled 75 μm i.d. PicoTip^TM25cm long fused silica nanopillars (New Objective). The columns and wells were configured in an open configuration (wickreader et al, Analytical Chemistry,2002,74,3076-

Pore size, Michrom biolesources, Auburn, CA) and the wells were packed with the same material (

Pore size, Michrom). Use was made of mobile phase A [ ultrapure water/formic acid (0.1% by volume)]And mobile phase B [ acetonitrile/formic acid (0.1% by volume)]The gradient of composition separates the sample on the column at a flow rate of 400nL/min according to the following conditions: starting with 95% "a" and 5% "B", for 0 to 2 minutes, at which time the mobile phase "B" rose from 5% to 7%, then 7% "B" rose to 35% "B" over 60 minutes, then 35% "B" rose to 50% "B" over 2 minutes, at which time "B" was held for 1 minute, then 50% "B" rose to 90% "B" over 5 minutes and held for 8 minutes, and finally a period of time to allow "B" to drop back to 5% over 1 minute to re-equilibrate to the starting conditions. The mass spectrometer is in a data dependent mode at 400-1800m/z Operating within the range. For each cycle of the instrument, the first 3 most abundant ions were selected from the precursor scan (precorsor scan) (where the orbitrap resolution was set to 120k in profile mode). MS/MS data was acquired in centroid mode using the same settings as listed above, except that the separation width was set to 2.0 m/z. The dynamic exclusion settings used were as follows: the repeat count is 1, the count duration is 15 seconds, the exclusion list size is 500 and the exclusion duration is 30 seconds. Native and banding isomers have the same mass-to-charge ratio as determined using mass spectrometry, indicating that the two HPLC peaks have the same properties (fig. 1C).

Isolated CSP-4-NH was found₂Does not have blocking activity on human α 9 α 10 nachrs. CSP-4-NH as described above₂Incubation in human serum for 1 hour was followed by preparative HPLC and collection of peaks corresponding to the ribbon isomers. The disulfide linkage of Cys2-Cys12 and Cys3-Cys8 was also used to chemically synthesize ribbon isomers. Two-electrode voltage-clamp electrophysiology was used to test the activity of the chemically synthesized ribbon isomers and the isomers isolated after incubation in human serum on human α 9 α 10 nachrs.

The ability of each analogue to block acetylcholine-induced α 9 α 10nAChR currents in Xenopus oocytes was measured. As shown in Table 1, native CSP-4-NH was present at a concentration of 100nM₂Blocks > 99% of ACh-induced current, while CSP-4-NH₂Both the chemically synthesized and serum-derived ribbon isomers of (a) are inactive at concentrations of 0.1-10. mu.M. As shown in FIGS. 1A and 1B, in plasma and serum samples, the conotoxin peptide analog CSP-4-NH₂Isomerization between native and banded forms with minimal bead formation.

Decreased activity of the banded form on human or rat α 9 α 10 nachrs indicates that disulfide shuffling helps to reduce the potency of the molecule. Furthermore, CSP-4-NH is relative to the C-terminal carboxylic acid in CSP-4-OH₂The presence of the medium C-terminal amide does not reduce isomerization. Finally, disulfide isomerization also occurred in vivo after intravenous and subcutaneous injection of CSP-4-OH at a dose of 1mg/kg into Sprague Dawley rats. 1h after injection, analysis by HPLC-MS/MS, Only40-50% of the peptides recovered from the blood remained in native form, indicating that disulfide isomerization occurred in vivo similar to in vitro observations.

Table 1: CSP-4-NH₂Activity of the natural and zonal isomers of Xenopus laevis (Xenopus) on human α 9 α 10nAChR expressed in Xenopus oocytes (percentage of 100 μ M acetylcholine-induced current retained in the presence of compound compared to control response in the absence of compound)

6.2. Example 2: preparation of conotoxin peptide analogs

Example 1.1 conotoxin peptide analog Ia (L-tyrosine, glycyl-L-cysteinyl-L-alanyl-L-threonyl-L-alpha-aspartyl-L-prolyl-L-arginyl-L-cysteinyl-N5- (carbamoyl) -L-ornyl-L-tryptophanyl-L-glutaminyl-L-N-valyl-ring (2 → 8) -disulfide-ring 3³,12⁵- (1H-1,2, 3-triazole-4, 1-diyl)) (SEQ ID NO: 6):

FMOC-Tyr (OtBu) -OH-supporting 2-chlorotrityl resin (1): 2-Chlorotriphenylmethyl chloride resin (MFCD00040399, 15.0g, 18mmol) was added to the reaction vessel and CH was used₂Cl₂(100mL) followed by washing in CH₂Cl₂(100mL) for 10 min. After draining the reaction vessel, FMOC-Tyr (OtBu) -OH (2.5g, 5.4mmol) in CH was added₂Cl₂(150mL), followed by dropwise addition of DIPEA (1.8mL, 10.8 mmol). The reaction vessel was then shaken at room temperature for 1.5 h. The solvent was drained from the resin, followed by washing with DMF (6X 100mL), then MeOH (2X 100mL) and Final Et₂O (2X 100mL) rinse. The resin was dried in vacuo for 2h and the FMOC loading test indicated a peptide loading of 0.29 mmol/g.

Solid phase peptide synthesis on polystyrene support (SPPS) of peptide sequence FMOC-Gly-cys (trt) -Pra-thr (tbu) -asp (otbu) -Pro-arg (pbf) -cys (trt) -Cit-trp (boc) -gln (trt) -5-azido-NVa-tyr (tbu) -O- (2-chlorotrityl) (2): 2-Chlorotribenzyl resin (1, 17.3g, 5mmol) loaded with FMOC-Tyr (OtBu) was washed with DMF (100mL) and then drained completely. By using 1: 4 (2X 100mL, 15min each), remove the FMOC group from the attached Tyr group, and then wash the resin with DMF (6X 100 mL). Then, HATU (3.80g, 10mmol), HOAt (1.35g, 10mmol) and DIPEA (2.5mL, 15mmol) in DMF (100mL) and FMOC-5-azido-Nva-OH (CAS #1097192-04-5, 3.80g, 10mmol) were added. The reaction vessel was shaken for at least 2.5h, then washed with DMF (6X 100 mL). The reaction completeness was determined by ninhydrin test. If negative (colorless), the coupling is considered complete and the synthesis is continued. If positive (blue), a second equivalent of the same FMOC-amino acid (10mmol) in DMF (100mL) was coupled using the resin described above and HATU/HOAt (10mmol/10 mmol). Once the ninhydrin test indicates completion of the reaction, FMOC is removed from each sequentially added amino acid, followed by sequential coupling of FMOC-amino acids-OH in the peptide sequence as described above. The following were used, in the following order: FMOC-Gln (Trt) -OH (CAS #132327-80-1, 6.1g, 10mmol), FMOC-Trp (Boc) -OH (CAS #143824-78-6, 5.2g, 10mmol), FMOC-Cit-OH (CAS #133174-15-9, 3.9g, 10mmol), FMOC-Cys (Trt) -OH (CAS #103213-32-7, 5.8g, 10mmol), FMOC-Arg (Pbf) -OH (CAS #154445-77-9, 6.4g, 10mmol), FMOC-Pro-OH (CAS #71989-31-6, 3.3g, 10mmol), FMOC-Asp (OtBu) -OH (CAS #71989-14-5, 4.1g, 10mmol), FMOC-Thr (tBu) -OH (CAS #71989-35-0, 3.9g, 10mmol), CAS # PrOC-398-07 (CAS #198561 mmol), 3.3g, 10mmol), FMOC-Cys (Trt) -OH (5.8g, 10mmol) and FMOC-Gly-OH (CAS #29022-11-5, 2.9g, 10 mmol). After the linear peptide coupling was complete, the resin was washed with DMF (100mL × 6), followed by MeOH (100mL × 2) and finally Et ₂O (100 mL. times.2) and then dried in vacuo for 2 h.

Resin cleavage of the amino acid sequence H-Gly-Cys-Pra-Thr-Asp-Pro-Arg-Cys-Cit-Trp-Gln-5-azido NVa-Tyr-OH (3) (SEQ ID NO: 7): to the dry resin (2, 28.0g, 5.0mmol) in the flask was added 230mL of cleavage solution (TFA/EDT/phenylthiomethane/anisole: 90: 3: 5: 2), and the mixture was shaken at room temperature for 2 h. After this time, the resin was filtered and washed with TFA (2X 50 mL). The filtrates were combined and 10-fold volume of cold (0 ℃ C.) Et was added₂O, which results in precipitation of the peptide. The precipitated peptide was centrifuged at 5,000rpm for 10min and Et at cold (0 ℃ C.)₂O (3X 50mL) rinse. The crude product was dried in vacuo for 2h to afford 3(6.8g, 4.2mmol, 84%) which was used further without further purification.

Cysteine disulfide bond formation to generate H-Gly-Cys-Pra-Thr-Asp-Pro-Arg-Cys-Cit-Trp-Gln-5-azido Nva-Tyr-OH, (Cys2 → Cys8) disulfide bond (4) (SEQ ID NO: 8): peptide 3(2.0g, 1.2mmol) was dissolved in CH₃CN (100mL), DMSO (100mL) and H₂O (1800 mL). Then, H was added dropwise₂O₂(30% in H)₂O, 0.25mL) and the mixture was stirred at room temperature for 1.5 h. After this, the mixture was filtered, and the filtrate was purified by HPLC method J to provide 800mg of the title compound 4 as a white solid (yield: 38%, TFA salt). LC-MS (ESI) m/z: 540.7[ M +3H ]/3⁺，810.3[M+2H]/2⁺Purity 95% (214 nm). The remaining 4.8g of 3 were divided into two batches and oxidized and purified as described to obtain a total of 2.5g of 4 (total yield: 35%).

1, 3-dipolar cyclization/click reaction (triazole bridge formation) to produce conotoxin peptide analogue Ia: peptide 4(400mg, 0.25mmol) was dissolved in iPrOH (100mL) and H₂O (300 mL). Then, CuSO is added₄(157mg, 1mmol) in H₂O (2mL), followed by dropwise addition of L-ascorbic acid (176mg, 1mmol) in H₂Solution in O (2 mL). The mixture was stirred at room temperature for 2 h. The solution is then loaded directly into a flash chromatography column (e.g.,

SNAP Ultra C18, 120g) with water (0.1% TFA) and CH₃And (4) eluting CN. Combinations comprising conotoxin peptide analogues IaThe eluate was partially and lyophilized to obtain 305mg of TFA salt of conotoxin peptide analog Ia as a white solid (305mg, TFA salt, yield: 69%). LC-MS (ESI) m/z: 540.7[ M +3H]/3⁺，810.3[M+2H]/2⁺Purity 90% (214 nm). The remaining 2.1g of 4 were divided into 3 lots and treated with the conditions as described above, respectively, to thereby provide 1.7g of the title conotoxin peptide analog Ia (overall combination yield: 61%, TFA salt).

The obtained TFA salt (1.0g) of conotoxin peptide analog Ia was dissolved in H₂O (20mL) and CH₃CN (2 mL). Then, 20mM NH was added dropwise ₄HCO₃To adjust the pH to 7.0-8.0. The mixture was then purified using HPLC method H. The product-containing fractions were lyophilized to provide acetate salt of conotoxin peptide analog Ia as a white solid (650mg, acetate salt, total yield: 62%). In the same manner, the remaining 700mg of conotoxin peptide analog Ia-TFA salt was purified and converted to acetate to provide 350mg (48%, acetate). The total combined amount obtained was 1.0g of the acetate salt of conotoxin peptide analog Ia (56%). LC-MS (ESI-TOF) m/z: 540.2[ M +3H]/3⁺，810.0[M+2H]/2⁺，1617.66[M+H]/⁺(ii) a HPLC method a; retention time: 10.1 min; the purity is 95.7%.

The synthesis of conotoxin peptide analog Ia is shown in scheme 1.

Step: a step of; required for reach amino acid: repeating for each amino acid

Example 1.2 conotoxin peptide analog Ia' (L-tyrosinamide, glycyl-L-cysteinyl-L-alanyl-L-threonyl-L-alpha-aspartyl-L-prolyl-L-arginyl-L-cysteinyl-N5- (carbamoyl) -L-ornyl-L-tryptophanyl-L-glutaminyl-L-N-valyl-ring (2 → 8) -disulfide-ring 3³,12⁵- (1H-1,2, 3-triazole-4, 1-diyl)) (SEQ ID NO: 9):

intermediate conotoxin peptide analogue Ya' (H-Gly-Cys-Pra-Thr-Asp-Pro-Arg-Cys-Cit-Trp-Gln-5-azido NVa-Tyr-NH) ₂) (SEQ ID NO: 10): the intermediate conotoxin peptide analog Ya' was synthesized using the same procedure described for compound 3, by using Rink Amide resin MFCD00677976 instead of 2-chlorotrityl chloride resin MFCD 00040399.

Intermediate conotoxin peptide analog Za' (H-Gly-Cys-Pra-Thr-Asp-Pro-Arg-Cys-Cit-Trp-Gln-5-azido NVa-Tyr-NH)₂(Cys2 → Cys8) disulfide bond) (SEQ ID NO: 11): intermediate conotoxin peptide analogue Za 'was synthesized by replacing compound 3 with intermediate conotoxin peptide analogue Ya' using the same procedure described for compound 4.

Conotoxin peptide analogue Ia ' was synthesized using the same procedure described for conotoxin peptide analogue Ia ' by using conotoxin peptide analogue Za ' instead of compound 4. LC-MS (Single quadrupole ESI) m/z: 1617.5(M +1H) </>⁺(MW calculated: 1616.5); HPLC method E; retention time: 14.0 min; the purity is 96.9%.

Example 1.3 conotoxin peptide analog Ib (L-tyrosine, glycyl-L-cysteinyl-L-alanyl-L-threonyl-L-alpha-aspartyl-L-prolyl-L-arginyl-L-cysteinyl-N5- (carbamoyl) -L-ornithiyl-L-3-iodotyrosyl-L-glutaminyl-L-N-valyl-ring (2 → 8) -disulfide-ring 3 ³,12⁵- (1H-1,2, 3-triazole-4, 1-diyl)) (SEQ ID NO: 12):

the intermediate product of the conotoxin peptide analogue Yb (H-Gly-Cys-Pra-Thr-Asp-Pro-Arg-Cys-Cit-3-I-Tyr-Gln-5-azido NVa-Tyr-OH) (SEQ ID NO: 13): the intermediate conotoxin peptide analog Yb was synthesized by using FMOC-3-I-Tyr-OH in place of FMOC-trp (boc) -OH in the peptide synthesis of residue 10 using the same procedure described for compound 3.

Intermediate conotoxin peptide analog Zb (H-Gly-Cys-Pra-Thr-Asp-Pro-Arg-Cys-Cit-3-I-Tyr-Gln-5-azido NVa-Tyr-OH, (Cys2 → Cys8) disulfide bond) (SEQ ID NO: 14): intermediate conotoxin peptide analog Zb was synthesized by replacing compound 3 with intermediate conotoxin peptide analog Yb using the same procedure described for compound 4.

Intermediate conotoxin peptide analog Ib was synthesized using the same procedure described for conotoxin peptide analog Ia by replacing compound 4 with intermediate conotoxin peptide analog Zb. LC-MS (ESI-TOF) m/z: 574.7[ M +3H]/3⁺，861.5[M+2H]/2⁺(MW calculated: 1720.54); HPLC method C; retention time: 11.7 min; the purity is 94.8%.

Example 1.4 conotoxin peptide analog Ib' (L-tyrosinamide, glycyl-L-cysteinyl-L-alanyl-L-threonyl-L-alpha-aspartyl-L-prolyl-L-arginyl-L-cysteinyl-N5- (carbamoyl) -L-ornityl-L-3-iodotyrosyl-L-glutaminyl-L-N-valyl-ring (2 → 8) -disulfide-ring 3 ³,12⁵- (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-azido NVa-Tyr-NH₂) (SEQ ID NO: 16): the intermediate conotoxin peptide analogue Yb' was synthesized using the same procedure described for the intermediate conotoxin peptide analogue Yb by using Rink Amide resin MFCD00677976 instead of 2-chlorotrityl chloride resin MFCD00040399 in solid phase peptide synthesis.

Intermediate conotoxin peptide analogue Zb' (H-Gly-Cys-Pra-Thr-Asp-Pro-Arg-Cys-Cit-3-I-Tyr-Gln-5-azido NVa-Tyr-NH₂(Cys2 → Cys8) disulfide bond) (SEQ ID NO: 17): by replacing compound 3 with the intermediate conotoxin peptide analog Yb', the same procedure as described for compound 4 was usedSynthesizing intermediate conotoxin peptide analog Zb'.

Intermediate conotoxin peptide analogue Ib 'was synthesized using the same procedure described for conotoxin peptide analogue Ia by replacing compound 4 with intermediate conotoxin peptide analogue Zb'. LC-MS (ESI-TOF) m/z: 574.2[ M +3H]/3⁺，860.8[M+2H]/2⁺(MW calculated: 1719.55); HPLC method B; retention time: 10.5 min; the purity was 93.8%.

Example 1.4 conotoxin peptide analog Ic (L-tyrosinamide, glycyl-L-alanyl-L-cysteinyl-L-threonyl-L-alpha-aspartyl-L-prolyl-L-arginyl-L-N-valinyl-N5- (carbamoyl) -L-ornityl-L-3-iodotyrosyl-L-glutaminyl-L-cysteinyl-Ring 2 ³,8⁵- (1H-1,2, 3-triazole-4, 1-diyl) -ring- (3 → 12) -disulfide) (SEQ ID NO: 18):

intermediate conotoxin peptide analog Yc (H-Gly-Pra-Cys-Thr-Asp-Pro-Arg-5-azido NVa-Cit-3-I-Tyr-Gln-Cys-Tyr-NH₂) (SEQ ID NO: 19): intermediate conotoxin peptide analog Yc was synthesized by using FMOC-cys (trt) -OH in place of FMOC-5-azido-Nva-OH (residue 12), FMOC-5-azido-Nva-OH in place of FMOC-cys (trt) -OH (residue 8), FMOC-cys (trt) -OH in place of FMOC-Pra-OH (residue 3) and FMOC-Pra-OH in place of FMOC-cys (trt) -OH (residue 2) in solid phase peptide synthesis using the same procedure described for intermediate conotoxin peptide analog Yb'.

Intermediate conotoxin peptide analog Zc (H-Gly-Pra-Cys-Thr-Asp-Pro-Arg-5-azido NVa-Cit-3-I-Tyr-Gln-Cys-Tyr-NH₂(Cys3 → Cys12) disulfide bond) (SEQ ID NO: 20): intermediate conotoxin peptide analog Zc was synthesized using the same procedure described for compound 4 by replacing compound 3 with intermediate conotoxin peptide analog Yc.

By replacing intermediate conotoxin peptide analogue Zb' with intermediate conotoxin peptide analogue ZcConotoxin peptide analog Ic was synthesized by the same procedure described for conotoxin peptide analog Ib'. LC-MS (ESI-TOF) m/z: 574.2[ M +3H ]/3⁺，860.7[M+2H]/2⁺(MW calculated: 1719.55); HPLC method B; retention time: 10.2 min; the purity is 98.3%.

EXAMPLE 1.5 conotoxin peptide analog Id (L-tyrosinamide, glycyl-L-N-valyl-L-cysteinyl-L-threonyl-L-alpha-aspartyl-L-prolyl-L-arginyl-L-alanyl-N5- (carbamoyl) -L-ornityl-L-3-iodotyrosyl-L-glutaminyl-L-cysteinyl-Ring 2⁵,8³- (1H-1,2, 3-triazole-1, 4-diyl) -ring- (3 → 12) -disulfide) (SEQ ID NO: 21):

intermediate conotoxin peptide analog Yd (H-Gly-5-azido NVa-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 using the same procedure described for intermediate conotoxin peptide analog Yb' in solid phase peptide synthesis using FMOC-cys (trt) -OH in place of FMOC-5-azido-Nva-OH (residue 12), FMOC-Pra-OH in place of FMOC-cys (trt) -OH (residue 8), FMOC-cys (trt) -OH in place of FMOC-Pra-OH (residue 3) and FMOC-5-azido-Nva-OH in place of FMOC-cys (trt) -OH (residue 2).

Intermediate conotoxin peptide analogue ZD (H-Gly-5-azido NVa-Cys-Thr-Asp-Pro-Arg-Pra-Cit-3-I-Tyr-Gln-Cys-Tyr-NH ₂(Cys3 → Cys12) disulfide bond) (SEQ ID NO: 23): by replacing the intermediate conotoxin peptide analog Yb ' with the intermediate conotoxin peptide analog Yd ', the intermediate conotoxin peptide analog Zd was synthesized using the same procedure described for intermediate conotoxin peptide analog Zb '.

Conotoxin peptide analogue Id was synthesized using the same procedure described for conotoxin peptide analogue Ib 'by replacing intermediate conotoxin peptide analogue Zb' with intermediate conotoxin peptide analogue Zd. LC-MS (ESI-TOF) m/z: 860.7[M+2H]/2⁺(MW calculated: 1719.55); HPLC method B; retention time: 10.2 min; the purity is 99.2%.

Example 1.6 conotoxin peptide analog Ie (L-tyrosinamide, glycyl-L-cysteinyl-L-N-valyl-L-threonyl-L-alpha-aspartyl-L-prolyl-L-arginyl-L-cysteinyl-N5- (carbamoyl) -L-ornithiyl-L-3-iodotyrosyl-L-glutaminyl-L-alaninyl-cyclo (2 → 8) -disulfide-cyclo 3⁵,12³- (1H-1,2, 3-triazole-1, 4-diyl)) (SEQ ID NO: 24):

intermediate conotoxin peptide analog Ye (H-Gly-Cys-5-azido NVa-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 using FMOC-Pra-OH in place of FMOC-5-azido-Nva-OH (residue 12) and FMOC-5-azido-Nva-OH in place of FMOC-Pra-OH (residue 3) in solid phase peptide synthesis using the same procedure described for intermediate conotoxin peptide analog Yb'.

Intermediate conotoxin peptide analog Ze (H-Gly-Cys-5-azido NVa-Thr-Asp-Pro-Arg-Cys-Cit-3-I-Tyr-Gln-Pra-Tyr-NH₂(Cys2 → Cys8) disulfide bond) (SEQ ID NO: 26): by replacing the intermediate conotoxin peptide analog Yb 'with the intermediate conotoxin peptide analog Ye, the intermediate conotoxin peptide analog Ze was synthesized using the same procedure described for intermediate conotoxin peptide analog Zb'.

By replacing the intermediate conotoxin peptide analog Zb 'with the intermediate conotoxin peptide analog Ze, conotoxin peptide analog Ie was synthesized using the same procedure described for conotoxin peptide analog Ib'. LC-MS (ESI-TOF) m/z: 574.4[ M +3H]/3⁺，861.1[M+2H]/2⁺(MW calculated: 1719.55); HPLC method C; retention time: 12.6 min; the purity is 92.1%.

Example 1.7 conotoxin peptide analog If (L-tyrosinamide, glycyl-L-cysteine)acyl-L-alanyl-L-threonyl-L-alpha-aspartyl-L-prolyl-L-arginyl-L-cysteinyl-N5- (carbamoyl) -L-ornityl-L-3-iodotyrosyl-L-glutaminyl-L-alanyl-ring (2 → 8) -disulfide-ring 3³,12³- (1H-1,2, 3-triazole-4, 1-diyl)) (SEQ ID NO: 27):

intermediate conotoxin peptide analog Yf (H-Gly-Cys-Pra-Thr-Asp-Pro-Arg-Cys-Cit-3-I-Tyr-Gln-3-azido Ala-Tyr-NH) ₂) (SEQ ID NO: 28): intermediate conotoxin peptide analog Yf was synthesized by using FMOC-3-azido-Ala-OH in place of FMOC-Pra-OH (residue 12) and FMOC-Pra-OH in place of FMOC-5-azido-Nva-OH (residue 3) in solid phase peptide synthesis using the same procedure described for intermediate conotoxin peptide analog Yb'.

Intermediate conotoxin peptide analog Zf (H-Gly-Cys-Pra-Thr-Asp-Pro-Arg-Cys-Cit-3-I-Tyr-Gln-3-azido Ala-Tyr-NH)₂(Cys2 → Cys8) disulfide bond) (SEQ ID NO: 29): by replacing the intermediate conotoxin peptide analog Yb 'with the intermediate conotoxin peptide analog Yf, the intermediate conotoxin peptide analog Zf was synthesized using the same procedure described for intermediate conotoxin peptide analog Zb'.

Conotoxin peptide analogue If was synthesized using the same procedure described for conotoxin peptide analogue Ib 'by replacing intermediate conotoxin peptide analogue Zb' with intermediate conotoxin peptide analogue Zf. LC-MS (ESI-TOF) m/z: 847.1[ M +2H]/2⁺；1693.1[M+1H]/⁺(MW calculated: 1691.52); HPLC method E; retention time: 12.3 min; the purity is 94.6%.

Example 1.8 conotoxin peptide analog Ig (L-tyrosine, glycyl-L-cysteinyl- (2S) -2-aminobutyryl-L-threonyl-L-alpha-aspartyl-L-prolyl-L-arginyl-L-cysteinyl-N5- (carbamoyl) -L-ornityl-L-tryptophanyl-L-glutaminyl-L-N-valyl-cyclo (2 → 8) -di Sulfide-ring 3⁴,12⁵- (1H-1,2, 3-triazole-4, 1-diyl)) (SEQ ID NO: 30):

intermediate conotoxin peptide analog Yg (H-Gly-Cys-homo Pra-Thr-Asp-Pro-Arg-Cys-Cit-Trp-Gln-5-azido Nva-Tyr-OH) (SEQ ID NO: 31): the intermediate conotoxin peptide analog Yg was synthesized using the same procedure described for compound 3 by using FMOC-homopropargylglycine instead of FMOC-Pra-OH (residue 3-position) in solid phase peptide synthesis.

Intermediate conotoxin peptide analog Zg (H-Gly-Cys-homo Pra-Thr-Asp-Pro-Arg-Cys-Cit-Trp-Gln-5-azido Nva-Tyr-OH, (Cys2 → Cys8) disulfide bond) (SEQ ID NO: 32): intermediate conotoxin peptide analogue Zg was synthesized using the same procedure described for compound 4 by replacing compound 3 with intermediate conotoxin peptide analogue Yg.

Conotoxin peptide analog Ig was synthesized using the same procedure described for conotoxin peptide analog Ia by replacing compound 4 with intermediate conotoxin peptide analog Zg. LC-MS (Single quadrupole ESI) m/z: 545.0[ M +3H]/3⁺，817.3[M+2H]/2⁺(MW calculated: 1631.67); HPLC method a; retention time: 12.5 min; the purity is 98.2%.

Example 1.9 conotoxin peptide analog Ih (L-tyrosine, glycyl-L-cysteinyl- (2S) -2-aminobutyryl-L-threonyl-L- α -aspartyl-L-prolyl-L-arginyl-L-cysteinyl-N5- (carbamoyl) -L-ornityl-L-tryptophanyl-L-glutaminyl-L- (2S) -2-aminobutyryl-ring (2 → 8) -disulfide-ring 3 ⁴,12⁴- (1H-1,2, 3-triazole-4, 1-diyl)) (SEQ ID NO: 33):

intermediate conotoxin peptide analog Yh (H-Gly-Cys-homo Pra-Thr-Asp-Pro-Arg-Cys-Cit-Trp-Gln-azido homo Ala-Tyr-OH) (SEQ ID NO: 34): intermediate conotoxin peptide analogue Yh was synthesized by using FMOC-homopropargyl glycine instead of FMOC-Pra-OH (residue 3-position) and FMOC- γ -azido-homoalanine instead of FMOC-5-azido-Nva-OH (residue 12-position) in solid phase peptide synthesis using the same procedure as described for compound 3.

Intermediate conotoxin peptide analog Zh (H-Gly-Cys-homo Pra-Thr-Asp-Pro-Arg-Cys-Cit-Trp-Gln-azido-homo Ala-Tyr-OH, (Cys2 → Cys8) disulfide bond) (SEQ ID NO: 35): intermediate conotoxin peptide analog Zh was synthesized using the same procedure described for compound 4 by substituting intermediate conotoxin peptide analog Yh for compound 3.

Conotoxin peptide analog Ih was synthesized using the same procedure described for conotoxin peptide analog Ia by replacing compound 4 with intermediate conotoxin peptide analog Zh. LC-MS (Single quadrupole ESI) m/z: 540.6[ M +3H]/3⁺，809.9[M+2H]/2⁺(MW calculated: 1617.66); HPLC method a; retention time: 12.4 min; the purity was 83.9%.

Example 1.10 conotoxin peptide analogs Ii (L-tyrosine, glycyl-L-cysteinyl- (2S) -2-aminobutyryl-L-threonyl-L- α -aspartyl-L-prolyl-L-arginyl-L-cysteinyl-N5- (carbamoyl) -L-ornityl-L-tryptophanyl-L-glutaminyl-L- (2S) -2-aminobutyryl-cyclo (2 → 8) -disulfide-cyclo 3⁴,12⁴- (1H-1,2, 3-triazole-1, 4-diyl)) (SEQ ID NO: 36):

intermediate conotoxin peptide analog Yi (H-Gly-Cys-azido homo Ala-Thr-Asp-Pro-Arg-Cys-Cit-Trp-Gln-homo Pra-Tyr-OH) (SEQ ID NO: 37): intermediate conotoxin peptide analogue Yi was synthesized by using FMOC- γ -azido-homoalanine instead of FMOC-Pra-OH (residue 3) and FMOC-homopropargyl glycine instead of FMOC-5-azido-Nva-OH (residue 12) in solid phase peptide synthesis using the same procedure as described for compound 3.

Intermediate conotoxin peptide analog Zi (H-Gly-Cys-azidohomoala-Thr-Asp-Pro-Arg-Cys-Cit-Trp-Gln-homopra-Tyr-OH, (Cys2 → Cys8) disulfide bond) (SEQ ID NO: 38): intermediate conotoxin peptide analogue Zi was synthesized using the same procedure described for compound 4 by substituting intermediate conotoxin peptide analogue Yi for compound 3.

Conotoxin peptide analog Ii was synthesized using the same procedure described for conotoxin peptide analog Ia by replacing compound 4 with intermediate conotoxin peptide analog Zi. LC-MS (Single quadrupole ESI) m/z: 540.7[ M +3H]/3⁺，810.5[M+2H]/2⁺(MW calculated: 1617.66); HPLC method a; retention time: 12.3 min; the purity is 85.8%.

EXAMPLE 1.11 conotoxin peptide analog Ij (L-tyrosine, glycyl-L-cysteinyl- (2S) -2-aminopentanoyl-L-alanyl-L-threonyl-L-alpha-aspartyl-L-prolyl-L-arginyl-L-cysteinyl-N5- (carbamoyl) -L-ornithio-L-3-iodotyrosyl-L-glutaminyl-L-alanyl-cyclo (2 → 8) -disulfide-cyclo 3⁵,12³- (1H-1,2, 3-triazole-4, 1-diyl)) (SEQ ID NO: 39):

intermediate conotoxin peptide analog Yj (H-Gly-Cys-double high Pra-Thr-Asp-Pro-Arg-Cys-Cit-Trp-Gln-3-azido Ala-Tyr-OH) (SEQ ID NO: 40): intermediate conotoxin peptide analogue Yj was synthesized by using FMOC-bis-homopropargyl glycine instead of FMOC-Pra-OH (residue 3-position) and FMOC-3-azido-Ala-OH instead of FMOC-5-azido-Nva-OH (residue 12-position) in solid phase peptide synthesis using the same procedure as described for compound 3.

Intermediate conotoxin peptide analog Zj (H-Gly-Cys-bishomopro Pra-Thr-Asp-Pro-Arg-Cys-Cit-Trp-Gln-3-azido Ala-Tyr-OH, (Cys2 → Cys8) disulfide bond) (SEQ ID NO: 41): intermediate conotoxin peptide analog Zj was synthesized using the same procedure described for compound 4 by replacing compound 3 with intermediate conotoxin peptide analog Yj.

Conotoxin peptide analog Ij was synthesized using the same procedure described for conotoxin peptide analog Ia by replacing compound 4 with intermediate conotoxin peptide analog Zj. LC-MS (Single quadrupole ESI) m/z: 540.5[ M +3H]/3⁺，810.1[M+2H]/2⁺(MW calculated: 1617.66); HPLC method a; retention time: 12.04 min; the purity is 97.6%.

EXAMPLE 1.12 conotoxin peptide analog Ik (L-tyrosine, glycyl-L-cysteinyl-L-alanyl-L-threonyl-L-alpha-aspartyl-L-prolyl-L-arginyl-L-cysteinyl-N5- (carbamoyl) -L-ornyl-L-tryptophanyl-L-glutaminyl-L- (2S) -2-aminopentanoyl-cyclo (2 → 8) -disulfide-cyclo 3³,12⁵- (1H-1,2, 3-triazole-1, 4-diyl)) (SEQ ID NO: 42):

intermediate conotoxin peptide analog Yk (H-Gly-Cys-3-azido Ala-Thr-Asp-Pro-Arg-Cys-Cit-Trp-Gln-double-high Pra-Tyr-OH) (SEQ ID NO: 43): the intermediate conotoxin peptide analogue Yk was synthesized by using FMOC-3-azido-Ala-OH instead of FMOC-Pra-OH (residue 3) and FMOC-bis-homopropargyl glycine instead of FMOC-5-azido-Nva-OH (residue 12) in solid phase peptide synthesis using the same procedure as described for compound 3.

Intermediate conotoxin peptide analog Zk (H-Gly-Cys-3-azidoala-Thr-Asp-Pro-Arg-Cys-Cit-Trp-Gln-bishomo Pra-Tyr-OH, (Cys2 → Cys8) disulfide bond) (SEQ ID NO: 44): intermediate conotoxin peptide analog Zk was synthesized using the same procedure described for compound 4 by replacing compound 3 with intermediate conotoxin peptide analog Yk.

Conotoxin peptide analog Ik was synthesized using the same procedure described for conotoxin peptide analog Ia by replacing compound 4 with intermediate conotoxin peptide analog Zk. LC-MS (Single quadrupole ESI) m/z: 540.6[ M +3H]/3⁺，810.0[M+2H]/2⁺(MW calculated: 1617.66); HPLC method a; retention time: 12.24 min; the purity is 97.6%.

EXAMPLE 1.13 conotoxin peptide analog Il (L-phenylalanine, glycyl-L-cysteinyl-L-alanyl-L-threonyl-L-alpha-aspartyl-L-prolyl-L-arginyl-L-cysteinyl-N5- (carbamoyl) -L-ornithiyl-L-tryptophanyl-L-glutaminyl-L-norvalyl-cyclo (2 → 8) -disulfide-cyclo 3³,12⁵- (1H-1,2, 3-triazole-4, 1-diyl)) (SEQ ID NO: 45):

2-chlorotrityl resin-supported FMOC-Phe (OtBu) -OH (5): 2-Chlorotriphenylmethyl resin-supported FMOC-Phe (OtBu) -OH was synthesized by using FMOC-Phe (OtBu) -OH in place of FMOC-Tyr (OtBu) -OH using the same procedure as described for 2-chlorotrityl resin 1.

Intermediate conotoxin peptide analog Yl (H-Gly-Cys-Pra-Thr-Asp-Pro-Arg-Cys-Cit-Trp-Gln-5-azido NVa-Phe-OH) (SEQ ID NO: 46): the intermediate conotoxin peptide analog Yl was synthesized by using FMOC-phe (otbu) -OH-supported 2-chlorotrityl resin-supported FMOC-phe (otbu) -OH (5) instead of FMOC-tyr (otbu) -supported 2-chlorotrityl resin (1) as the C-terminal starting point in solid phase peptide synthesis using the same procedure described for compound 3.

Intermediate conotoxin peptide analog Zl (H-Gly-Cys-Pra-Thr-Asp-Pro-Arg-Cys-Cit-Trp-Gln-5-azido NVa-Phe-OH, (Cys2 → Cys8) disulfide bond) (SEQ ID NO: 47): intermediate conotoxin peptide analog Zl was synthesized using the same procedure described for compound 4 by replacing compound 3 with intermediate conotoxin peptide analog Yl.

Conotoxin peptide analog Il was synthesized using the same procedure described for conotoxin peptide analog Ia by replacing compound 4 with intermediate conotoxin peptide analog Zl. LC-MS (Single quadrupole ESI) m/z: 535.2[ M +3H]/3⁺，802.2[M+2H]/2⁺(MW calculated: 1601.66); HPLC methodA; retention time: 13.47 min; the purity is 95.6%.

EXAMPLE 1.14 conotoxin peptide analog Im (D-tyrosine, glycyl-L-cysteinyl-L-alanyl-L-threonyl-L-alpha-aspartyl-L-prolyl-L-arginyl-L-cysteinyl-N5- (carbamoyl) -L-ornyl-L-tryptophanyl-L-glutaminyl-L-N-valyl-ring (2 → 8) -disulfide-ring 3 ³,12⁵- (1H-1,2, 3-triazole-4, 1-diyl)) (SEQ ID NO: 48):

2-chlorotrityl resin-supported FMOC-D-Tyr (OtBu) -OH (6): 2-Chlorotriphenylmethyl resin-supported FMOC-D-Tyr (OtBu) -OH was synthesized by using FMOC-D-Tyr (OtBu) -OH in place of FMOC-Tyr (OtBu) -OH using the same procedure as described for 2-chlorotrityl resin 1.

Intermediate conotoxin peptide analog Ym (H-Gly-Cys-Pra-Thr-Asp-Pro-Arg-Cys-Cit-Trp-Gln-5-azido NVa-D-Tyr-OH) (SEQ ID NO: 49): the intermediate conotoxin peptide analogue Ym was synthesized using the same procedure as described for compound 3 by using FMOC-D-tyr (otbu) -OH-supported 2-chlorotrityl resin (6) instead of FMOC-tyr (otbu) -supported 2-chlorotrityl resin (1) as C-terminal starting point in solid phase peptide synthesis.

Intermediate conotoxin peptide analog Zm (H-Gly-Cys-Pra-Thr-Asp-Pro-Arg-Cys-Cit-Trp-Gln-5-azido NVa-D-Tyr-OH, (Cys2 → Cys8) disulfide bond) (SEQ ID NO: 50): intermediate conotoxin peptide analog Zm was synthesized using the same procedure described for compound 4 by substituting intermediate conotoxin peptide analog Ym for compound 3.

Conotoxin peptide analogue Im was synthesized using the same procedure described for conotoxin peptide analogue Ia by replacing compound 4 with intermediate conotoxin peptide analogue Zm. LC-MS (Single quadrupole ESI) m/z: 540.6[ M +3H ]/3⁺，810.0[M+2H]/2⁺(MW calculated: 1617.66); HPLC method a; retention time: 12.07 min; the purity is 86.6%.

Example 1.15 conotoxin peptide analog In (Glycine, glycyl-L-cysteinyl-L-alanyl-L-threonyl-L-alpha-aspartyl-L-prolyl-L-arginyl-L-cysteinyl-N5- (carbamoyl) -L-ornithiyl-L-tryptophanyl-L-glutaminyl-L-norvalyl-L-tyrosyl-N-methyl-cyclo (2 → 8) -disulfide-cyclo 3³,12⁵- (1H-1,2, 3-triazole-4, 1-diyl)) (SEQ ID NO: 51):

2-Chlorotriphenylmethyl resin loaded FMOC-N-Me-Gly-OH (7): 2-Chlorotriphenylmethyl resin loaded FMOC-N-Me-Gly-OH was synthesized by using FMOC-N-Me-Gly-OH in place of FMOC-Tyr (OtBu) -OH using the same procedure described for 2-chlorotrityl resin 1.

Intermediate conotoxin peptide analog Yn (H-Gly-Cys-Pra-Thr-Asp-Pro-Arg-Cys-Cit-Trp-Gln-5-azido NVa-Tyr-N-Me-Gly-OH) (SEQ ID NO: 52): the intermediate conotoxin peptide analogue Yn was synthesized by sequential addition of amino acids using FMOC-N-Me-Gly-OH-supported 2-chlorotrityl resin (7) using the same procedure described for compound 3, in the following order: FMOC-Tyr (OtBu) -OH, FMOC-5-azido-Nva-OH, FMOC-Gln (Trt) -OH, FMOC-Trp (Boc) -OH, FMOC-Cit-OH, FMOC-Cys (Trt) -OH, FMOC-Arg (Pbf) -OH, FMOC-Pro-OH, FMOC-Asp (OtBu) -OH, FMOC-Thr (tBu) -OH, FMOC-Pra-OH, FMOC-Cys (Trt) -OH and FMOC-Gly-OH.

Intermediate conotoxin peptide analog Zn (H-Gly-Cys-Pra-Thr-Asp-Pro-Arg-Cys-Cit-Trp-Gln-5-azido NVa-Tyr-N-Me-Gly-OH, (Cys2 → Cys8) disulfide bond) (SEQ ID NO: 53): the intermediate conotoxin peptide analogue Zn was synthesized using the same procedure described for compound 4 by replacing compound 3 with the intermediate conotoxin peptide analogue Yn.

Conotoxin peptide analog In was synthesized using the same procedure described for conotoxin peptide analog Ia by replacing compound 4 with intermediate conotoxin peptide analog Zn. LC-MS (Single quadrupole ESI) m/z：564.2[M+3H]/3⁺，846.0[M+2H]/2⁺(MW calculated: 1688.69); HPLC method a; retention time: 11.63 min; the purity is 95.8%.

EXAMPLE 1.16 conotoxin peptide analog Io (D-tyrosine, glycyl-L-cysteinyl-L-alanyl-L-threonyl-L-alpha-aspartyl-L-prolyl-L-arginyl-L-cysteinyl-N5- (carbamoyl) -L-ornyl-L-tryptophanyl-L-glutaminyl-L-norvalyl-L-tyrosyl-ring (2 → 8) -disulfide-ring 3³,12⁵- (1H-1,2, 3-triazole-4, 1-diyl)) (SEQ ID NO: 54):

intermediate conotoxin peptide analog Yo (H-Gly-Cys-Pra-Thr-Asp-Pro-Arg-Cys-Cit-Trp-Gln-5-azido NVa-Tyr-D-Tyr-OH) (SEQ ID NO: 55): the intermediate conotoxin peptide analogue Yo was synthesized by sequential addition of amino acids using FMOC-D-tyr (otbu) -OH-supported 2-chlorotrityl resin using the same procedure described for compound 3, in the following order: FMOC-Tyr (OtBu) -OH, FMOC-5-azido-Nva-OH, FMOC-Gln (Trt) -OH, FMOC-Trp (Boc) -OH, FMOC-Cit-OH, FMOC-Cys (Trt) -OH, FMOC-Arg (Pbf) -OH, FMOC-Pro-OH, FMOC-Asp (OtBu) -OH, FMOC-Thr (tBu) -OH, FMOC-Pra-OH, FMOC-Cys (Trt) -OH and FMOC-Gly-OH.

Intermediate conotoxin peptide analog Zo (H-Gly-Cys-Pra-Thr-Asp-Pro-Arg-Cys-Cit-Trp-Gln-5-azido NVa-Tyr-D-Tyr-OH, (Cys2 → Cys8) disulfide bond) (SEQ ID NO: 56): intermediate conotoxin peptide analog Zo was synthesized using the same procedure described for compound 4 by replacing compound 3 with intermediate conotoxin peptide analog Yo.

Conotoxin peptide analog Io was synthesized using the same procedure described for conotoxin peptide analog Ia by replacing compound 4 with intermediate conotoxin peptide analog Zo. LC-MS (Single quadrupole ESI) m/z: 595.0[ M +3H]/3⁺，891.7[M+2H]/2⁺(MW calculated: 1780.72); HPLC method a; retention time: 12.43 min;the purity is 97.4%.

Example 1.17 conotoxin peptide analog Ip (L-tyrosine, glycyl-L-cysteinyl-L-alanyl-L-threonyl-L-alpha-aspartyl-L-prolyl-L-arginyl-L-cysteinyl-N5- (carbamoyl) -L-ornyl-L-tryptophanyl-L-glutaminyl-L-norvalyl-L-tyrosyl-N-methyl-ring (2 → 8) -disulfide-ring 3³,12⁵- (1H-1,2, 3-triazole-4, 1-diyl)) (SEQ ID NO: 57):

2-Chlorotriphenylmethyl resin-loaded FMOC-N-Me-Tyr-OH (8): 2-Chlorotriphenylmethyl resin loaded FMOC-N-Me-Tyr-OH was synthesized by using FMOC-N-Me-Tyr-OH in place of FMOC-Tyr (OtBu) -OH using the same procedure described for 2-chlorotrityl resin 1.

Intermediate conotoxin peptide analog Yp (H-Gly-Cys-Pra-Thr-Asp-Pro-Arg-Cys-Cit-Trp-Gln-5-azido NVa-Tyr-N-Me-Tyr-OH) (SEQ ID NO: 58): the intermediate conotoxin peptide analogue Yp was synthesized by sequential addition of amino acids using FMOC-N-Me-Tyr-OH-supported 2-chlorotrityl resin (8) using the same procedure described for compound 3, in the following order: FMOC-Tyr (OtBu) -OH, FMOC-5-azido-Nva-OH, FMOC-Gln (Trt) -OH, FMOC-Trp (Boc) -OH, FMOC-Cit-OH, FMOC-Cys (Trt) -OH, FMOC-Arg (Pbf) -OH, FMOC-Pro-OH, FMOC-Asp (OtBu) -OH, FMOC-Thr (tBu) -OH, FMOC-Pra-OH, FMOC-Cys (Trt) -OH and FMOC-Gly-OH.

Intermediate conotoxin peptide analog Zp (H-Gly-Cys-Pra-Thr-Asp-Pro-Arg-Cys-Cit-Trp-Gln-5-azido NVa-Tyr-N-Me-Tyr-OH, (Cys2 → Cys8) disulfide bond) (SEQ ID NO: 59): intermediate conotoxin peptide analogue Zp was synthesized using the same procedure described for compound 4 by replacing compound 3 with intermediate conotoxin peptide analogue Yp.

Conotoxin peptide was synthesized using the same procedure described for conotoxin peptide analog Ia, by replacing compound 4 with intermediate conotoxin peptide analog Zp The analog Ip. LC-MS (Single quadrupole ESI) m/z: 599.7[ M +3H]/3⁺，898.7[M+2H]/2⁺(MW calculated: 1794.74); HPLC method a; retention time: 12.56 min; the purity is 95.5%.

EXAMPLE 1.18 conotoxin peptide analog Iq (L-valine, glycyl-L-cysteinyl-L-alanyl-L-threonyl-L-alpha-aspartyl-L-prolyl-L-arginyl-L-cysteinyl-N5- (carbamoyl) -L-ornithiyl-L-tryptophanyl-L-glutaminyl-cyclo (2 → 8) -disulfide-cyclo 3³,12⁵- (1H-1,2, 3-triazole-4, 1-diyl)) (SEQ ID NO: 60):

2-chlorotrityl resin-supported FMOC-5-azido-Nva-OH (9): 2-Chlorotriphenylmethyl resin-loaded FMOC-5-azido-Nva-OH was synthesized by using FMOC-5-azido-Nva-OH in place of FMOC-Tyr (OtBu) -OH using the same procedure described for 2-chlorotrityl resin 1.

Intermediate conotoxin peptide analog Yq (H-Gly-Cys-Pra-Thr-Asp-Pro-Arg-Cys-Cit-Trp-Gln-5-azido NVa-OH) (SEQ ID NO: 61): the intermediate conotoxin peptide analogue Yq was synthesized by sequential addition of amino acids using FMOC-5-azido-Nva-OH-supported 2-chlorotrityl resin (9) using the same procedure described for compound 3, in the following order: FMOC-Gln (Trt) -OH, FMOC-Trp (Boc) -OH, FMOC-Cit-OH, FMOC-Cys (Trt) -OH, FMOC-Arg (Pbf) -OH, FMOC-Pro-OH, FMOC-Asp (OtBu) -OH, FMOC-Thr (tBu) -OH, FMOC-Pra-OH, FMOC-Cys (Trt) -OH and FMOC-Gly-OH.

Intermediate conotoxin peptide analog Zq (H-Gly-Cys-Pra-Thr-Asp-Pro-Arg-Cys-Cit-Trp-Gln-5-azido NVa-OH, (Cys2 → Cys8) disulfide bond) (SEQ ID NO: 62): intermediate conotoxin peptide analog Zq was synthesized using the same procedure described for compound 4 by replacing compound 3 with intermediate conotoxin peptide analog Yq.

By using intermediate conotoxin peptide analogue Zq instead of compound 4Conotoxin peptide analog Iq was synthesized by the same procedure described for conotoxin peptide analog Ia. LC-MS (ESI-TOF) m/z: 1455.5[ M +1H]/⁺(MW calculated: 1454.59); HPLC method a; retention time: 11.4 min; the purity is 95.0%.

EXAMPLE 1.19 conotoxin peptide analog Ir (L-tyrosine, glycyl-L-cysteinyl-L-alanyl-L-threonyl-L-alpha-aspartyl-L-prolyl-L-arginyl-L-cysteinyl-N5- (carbamoyl) -L-ornyl-L-tryptophanyl-L-glutaminyl-L-N-valyl-N-methyl-cyclo (2 → 8) -disulfide-cyclo 3³,12⁵- (1H-1,2, 3-triazole-4, 1-diyl)) (SEQ ID NO: 63):

intermediate conotoxin peptide analog Yr (H-Gly-Cys-Pra-Thr-Asp-Pro-Arg-Cys-Cit-Trp-Gln-5-azido NVa-N-Me-Tyr-OH) (SEQ ID NO: 64): the intermediate conotoxin peptide analogue Yr was synthesized by the sequential addition of amino acids using FMOC-N-Me-Tyr-OH-supported 2-chlorotrityl resin using the same procedure described for compound 3, in the following order: FMOC-5-azido-Nva-OH, FMOC-Gln (Trt) -OH, FMOC-Trp (Boc) -OH, FMOC-Cit-OH, FMOC-Cys (Trt) -OH, FMOC-Arg (Pbf) -OH, FMOC-Pro-OH, FMOC-Asp (OtBu) -OH, FMOC-Thr (tBu) -OH, FMOC-Pra-OH, FMOC-Cys (Trt) -OH and FMOC-Gly-OH.

Intermediate conotoxin peptide analog Zr (H-Gly-Cys-Pra-Thr-Asp-Pro-Arg-Cys-Cit-Trp-Gln-5-azido NVa-N-Me-Tyr-OH, (Cys2 → Cys8) disulfide bond) (SEQ ID NO: 65): the intermediate conotoxin peptide analogue Zr was synthesized by replacing compound 3 with the intermediate conotoxin peptide analogue Yr using the same procedure as described for compound 4.

Conotoxin peptide analog Ir was synthesized using the same procedure described for conotoxin peptide analog Ia by replacing compound 4 with intermediate conotoxin peptide analog Zr. LC-MS (Single quadrupole ESI) m/z: 545.3[ M +3H]/3⁺，817.0[M+2H]/2⁺(calculation MW: 1631.67); HPLC method a; retention time: 12.01 min; the purity is 96.4%.

Example 1.20 conotoxin peptide analogs Is (D-arginine, glycyl-L-cysteinyl-L-alanyl-L-threonyl-L-alpha-aspartyl-L-prolyl-L-arginyl-L-cysteinyl-N5- (carbamoyl) -L-ornyl-L-tryptophanyl-L-glutaminyl-L-N-valyl-cyclo (2 → 8) -disulfide-cyclo 3³,12⁵- (1H-1,2, 3-triazole-4, 1-diyl)) (SEQ ID NO: 66):

2-Chlorotribenzyl resin-Supported FMOC-D-Arg-OH (10): 2-Chlorotriphenylmethyl resin-loaded FMOC-D-Arg-OH was synthesized by using FMOC-D-Arg-OH in place of FMOC-Tyr (OtBu) -OH using the same procedure as described for 2-chlorotrityl resin 1.

Intermediate conotoxin peptide analog Ys (H-Gly-Cys-Pra-Thr-Asp-Pro-Arg-Cys-Cit-Trp-Gln-5-azido NVa-D-Arg-OH) (SEQ ID NO: 67): the intermediate conotoxin peptide analogue Ys was synthesized by sequential addition of amino acids using FMOC-D-Arg-OH-supported 2-chlorotrityl resin (10) using the same procedure described for compound 3, in the following order: FMOC-5-azido-Nva-OH, FMOC-Gln (Trt) -OH, FMOC-Trp (Boc) -OH, FMOC-Cit-OH, FMOC-Cys (Trt) -OH, FMOC-Arg (Pbf) -OH, FMOC-Pro-OH, FMOC-Asp (OtBu) -OH, FMOC-Thr (tBu) -OH, FMOC-Pra-OH, FMOC-Cys (Trt) -OH and FMOC-Gly-OH.

Intermediate conotoxin peptide analog Zs (H-Gly-Cys-Pra-Thr-Asp-Pro-Arg-Cys-Cit-Trp-Gln-5-azido NVa-D-Arg-OH, (Cys2 → Cys8) disulfide bond) (SEQ ID NO: 68): intermediate conotoxin peptide analogue Zs was synthesized using the same procedure described for compound 4 by replacing compound 3 with intermediate conotoxin peptide analogue Ys.

Conotoxin peptide analogue Is was synthesized using the same procedure described for conotoxin peptide analogue Ia by replacing compound 4 with intermediate conotoxin peptide analogue Zs. LC (liquid Crystal)MS (Single quadrupole ESI) m/z: 539.2[ M +3H ]/3⁺，807.0[M+2H]/2⁺(MW calculated: 1610.69); HPLC method a; retention time: 10.52 min; the purity is 97.1%.

Example 1.21 conotoxin peptide analog It (D-tyrosine, glycyl-L-cysteinyl-L-alanyl-L-threonyl-L-alpha-aspartyl-L-prolyl-L-arginyl-L-cysteinyl-N5- (carbamoyl) -L-ornyl-L-tryptophanyl-L-glutaminyl-L-N-valyl-N-methyl-cyclo (2 → 8) -disulfide-cyclo 3³,12⁵- (1H-1,2, 3-triazole-4, 1-diyl)) (SEQ ID NO: 69):

2-Chlorotriphenylmethyl resin-loaded FMOC-N-Me-D-Tyr-OH (11): 2-Chlorotriphenylmethyl resin loaded FMOC-N-Me-D-Tyr-OH was synthesized by using FMOC-N-Me-D-Tyr-OH in place of FMOC-Tyr (OtBu) -OH using the same procedure described for 2-chlorotrityl resin 1.

Intermediate conotoxin peptide analog Yt (H-Gly-Cys-Pra-Thr-Asp-Pro-Arg-Cys-Cit-Trp-Gln-5-azido NVa-N-Me-D-Tyr-OH) (SEQ ID NO: 70): the intermediate conotoxin peptide analogue Yt was synthesized by sequential addition of amino acids using FMOC-N-Me-D-Tyr-OH-supported 2-chlorotrityl resin (11) using the same procedure described for compound 3, in the following order: FMOC-5-azido-Nva-OH, FMOC-Gln (Trt) -OH, FMOC-Trp (Boc) -OH, FMOC-Cit-OH, FMOC-Cys (Trt) -OH, FMOC-Arg (Pbf) -OH, FMOC-Pro-OH, FMOC-Asp (OtBu) -OH, FMOC-Thr (tBu) -OH, FMOC-Pra-OH, FMOC-Cys (Trt) -OH and FMOC-Gly-OH.

Intermediate conotoxin peptide analog Zt (H-Gly-Cys-Pra-Thr-Asp-Pro-Arg-Cys-Cit-Trp-Gln-5-azido NVa-N-Me-D-Tyr-OH, (Cys2 → Cys8) disulfide bond) (SEQ ID NO: 71): intermediate conotoxin peptide analogue Zt was synthesized using the same procedure described for compound 4 by replacing compound 3 with intermediate conotoxin peptide analogue Yt.

By using intermediate conotoxinConotoxin peptide analog It was synthesized using the same procedure described for conotoxin peptide analog Ia, substituting compound 4 with the peptide analog Zt. LC-MS (Single quadrupole ESI) m/z: 545.3[ M +3H]/3⁺，817.0[M+2H]/2⁺(MW calculated: 1631.67); HPLC method a; retention time: 10.72 min; the purity is 96.6%.

EXAMPLE 1.22 conotoxin peptide analogs Iu ((R) -3- (amino) -3- (4-hydroxyphenyl) propionic acid, glycyl-L-cysteinyl-L-alanyl-L-threonyl-L-alpha-aspartyl-L-prolyl-L-arginyl-L-cysteinyl-N5- (carbamoyl) -L-ornithiyl-L-tryptophanyl-L-glutaminyl-L-N-valinyl-cyclo (2 → 8) -disulfide-cyclo 3³,12⁵- (1H-1,2, 3-triazole-4, 1-diyl)) (SEQ ID NO: 72):

2-chlorotrityl resin-supported FMOC-beta-Tyr-OH (12): 2-Chlorotriphenylmethyl resin-loaded FMOC-Tyr-OH was synthesized by using FMOC-Tyr-OH in place of FMOC-Tyr (OtBu) -OH using the same procedure described for 2-chlorotrityl resin 1.

Intermediate conotoxin peptide analog Yu (H-Gly-Cys-Pra-Thr-Asp-Pro-Arg-Cys-Cit-Trp-Gln-5-azido NVa-beta-Tyr-OH) (SEQ ID NO: 73): the intermediate conotoxin peptide analogue Yu was synthesized by sequential addition of amino acids using FMOC- β -Tyr-OH-supported 2-chlorotrityl resin (12) using the same procedure described for compound 3, in the following order: FMOC-5-azido-Nva-OH, FMOC-Gln (Trt) -OH, FMOC-Trp (Boc) -OH, FMOC-Cit-OH, FMOC-Cys (Trt) -OH, FMOC-Arg (Pbf) -OH, FMOC-Pro-OH, FMOC-Asp (OtBu) -OH, FMOC-Thr (tBu) -OH, FMOC-Pra-OH, FMOC-Cys (Trt) -OH and FMOC-Gly-OH.

Intermediate conotoxin peptide analog Zu (H-Gly-Cys-Pra-Thr-Asp-Pro-Arg-Cys-Cit-Trp-Gln-5-azido NVa- β -Tyr-OH, (Cys2 → Cys8) disulfide bond) (SEQ ID NO: 74): intermediate conotoxin peptide analog Zu was synthesized using the same procedure described for compound 4 by replacing compound 3 with intermediate conotoxin peptide analog Yu.

Conotoxin peptide analog Iu was synthesized using the same procedure described for conotoxin peptide analog Ia by replacing compound 4 with intermediate conotoxin peptide analog Zu. LC-MS (Single quadrupole ESI) m/z: 544.5[ M +3H ]/3⁺，816.3[M+2H]/2⁺(MW calculated: 1631.67); HPLC method a; retention time: 10.28 min; the purity is 98.8%.

EXAMPLE 1.23 conotoxin peptide analog Iv (L-arginine, glycyl-L-cysteinyl-L-alanyl-L-threonyl-L-alpha-aspartyl-L-prolyl-L-arginyl-L-cysteinyl-N5- (carbamoyl) -L-ornyl-L-tryptophanyl-L-glutaminyl-L-N-valyl-N-methyl-cyclo (2 → 8) -disulfide-cyclo 3³,12⁵- (1H-1,2, 3-triazole-4, 1-diyl)) (SEQ ID NO: 75):

2-Chlorotriphenylmethyl resin-loaded FMOC-N-Me-Arg-OH (13): 2-Chlorotriphenylmethyl resin-loaded FMOC-N-Me-Arg-OH was synthesized by using FMOC-N-Me-Arg-OH in place of FMOC-Tyr (OtBu) -OH using the same procedure described for 2-chlorotrityl resin 1.

Intermediate conotoxin peptide analog Yv (H-Gly-Cys-Pra-Thr-Asp-Pro-Arg-Cys-Cit-Trp-Gln-5-azido NVa-N-Me-Arg-OH) (SEQ ID NO: 76): the intermediate conotoxin peptide analogue Yv was synthesized by sequential addition of amino acids using FMOC-N-Me-Arg-OH-supported 2-chlorotrityl resin (13) using the same procedure described for compound 3, in the following order: FMOC-5-azido-Nva-OH, FMOC-Gln (Trt) -OH, FMOC-Trp (Boc) -OH, FMOC-Cit-OH, FMOC-Cys (Trt) -OH, FMOC-Arg (Pbf) -OH, FMOC-Pro-OH, FMOC-Asp (OtBu) -OH, FMOC-Thr (tBu) -OH, FMOC-Pra-OH, FMOC-Cys (Trt) -OH and FMOC-Gly-OH.

Intermediate conotoxin peptide analog Zv (H-Gly-Cys-Pra-Thr-Asp-Pro-Arg-Cys-Cit-Trp-Gln-5-azido NVa-N-Me-Arg-OH, (Cys2 → Cys8) disulfide bond) (SEQ ID NO: 77): intermediate conotoxin peptide analog Zv was synthesized using the same procedure described for compound 4 by replacing compound 3 with intermediate conotoxin peptide analog Yv.

Conotoxin peptide analog Iv was synthesized using the same procedure described for conotoxin peptide analog Ia by replacing compound 4 with intermediate conotoxin peptide analog Zv. LC-MS (Single quadrupole ESI) m/z: 542.9[ M +3H]/3⁺，813.5[M+2H]/2⁺(MW calculated: 1624.71); HPLC method a; retention time: 9.29 min; the purity is 95.9%.

Example 1.24 conotoxin peptide analog Iw (L-tyrosinamide, glycyl-L-cysteinyl-L-glutamyl-L-threonyl-L- α -aspartyl-L-prolyl-L-arginyl-L-cysteinyl-N5- (carbamoyl) -L-ornityl-L-3-iodotyrosyl-L-glutaminyl-L-lysyl-ring (2 → 8) -disulfide-ring- (3 → 12) -lactam) (SEQ ID NO: 78):

synthesis of the Linear peptide intermediate (X1) (SEQ ID NO: 79): FMOC-Tyr (OtBu) -loaded Rink amide MBHA resin (1.7g, 0.5mmol) was washed with DMF (10ml) and then completely drained. By using 1: 4 (2X 10mL, 15min each), remove the FMOC group from the attached Tyr group, and then wash the resin with DMF (6X 10 mL). HATU (CAS #148893-10-1, 0.38g, 1mmol), HOAt (CAS #39968-33-7, 0.14g, 1mmol) and DIPEA (0.25mL, 1.5mmol) were then added to DMF (10mL) along with FMOC-Lys (ivDde) -OH (CAS #204777-78-6, 0.57g, 1 mmol). The reaction vessel was shaken for at least 2.5h, then washed with DMF (6X 10 mL). The reaction completeness was determined by ninhydrin test. If negative (colorless), the coupling is considered complete and the synthesis is continued. If positive (blue), a second equivalent of the same FMOC-amino acid (1mmol) in DMF (10mL) was coupled using the resin described above and HATU/HOAt (1mmol/1 mmol). Once the ninhydrin test indicates completion of the reaction, the FMOC is removed from each of the sequentially added amino acids, then the reaction is run as described above FMOC-amino acid-OH is coupled in sequence in the peptide sequence. The following were used, in the following order: FMOC-Gln (Trt) -OH (CAS #132327-80-1, 0.61g, 1mmol), FMOC- (3-I-Tyr) -OH (CAS #134486-00-3, 0.53g, 1mmol), FMOC-Cit-OH (CAS #133174-15-9, 0.39g, 1mmol), FMOC-Cys (Trt) -OH (CAS #103213-32-7, 0.58g, 1mmol), FMOC-Arg (Pbf) -OH (CAS #154445-77-9, 0.64g, 1mmol), FMOC-Pro-OH (CAS #71989-31-6, 0.33g, 1mmol), FMOC-Asp (OtBu) -OH (CAS #71989-14-5, 0.41g, 1mmol), FMOC-Thr (tBu) -OH (CAS #71989-35-0, 0.84-39 g, 1mmol), FMOC-Glu (ODmab) (CAS #268730-86-5, 0.68g, 1mmol), FMOC-Cys (Trt) -OH (0.58g, 1mmol), and FMOC-Gly-OH (CAS #29022-11-5, 0.29g, 1 mmol). After the linear peptide coupling was complete, the resin was washed with DMF (10 mL. times.6), followed by MeOH (10 mL. times.2) and finally Et₂O (10 mL. times.2) and then dried in vacuo for 2 h.

Lactam formation to yield intermediate X2(SEQ ID NO: 80) on the resin: to remove the ivDde and ODmab protecting groups from the Lys12 and Glu3 residues, respectively, the peptide resin was washed with DMF (10mL) and then drained completely, and then the resin was incubated with 2% hydrazine hydrate in DMF (10mL) at room temperature for 1 h. The resin was then washed with DMF (6X 10mL) and resuspended in DMF (10mL) and then treated with HATU (0.38g, 1mmol), HOAt (0.14g, 1mmol) and DIPEA (0.25mL, 1.5 mmol). The reaction vessel was shaken for at least 2.5h, then washed with DMF (6X 10mL), then MeOH (10mL X2) and finally Et ₂O (10 mL. times.2) and then dried in vacuo for 2 h.

Cleavage from the resin to provide intermediate X3(SEQ ID NO: 81): the lactam was cleaved from the resin by incubating intermediate X2 in 23mL of cleavage solution (TFA/EDT/phenylthiomethane/anisole: 30: 3: 5: 2) and the mixture was shaken for 2h at room temperature. In thatAfter this time, the resin was filtered and washed with TFA (2X 5 mL). The filtrates were combined and 10-fold volume of cold (0 ℃ C.) Et was added₂O, which results in precipitation of the peptide. The precipitated peptide was centrifuged at 5,000rpm for 10min and Et at cold (0 ℃ C.)₂O (3X 5mL) rinse. The crude product X3 was dried in vacuo for 2h to give the crude monocyclic 3 → 12 lactam peptide intermediate (X3, MW 1727.7g/mol, 0.4g, 0.23mmol, 46%) which was used without further purification.

Disulfide bond formation to provide conotoxin peptide analog Iw: intermediate X3(0.4g, 0.23mmol) was dissolved in CH₃CN (10mL), DMSO (10mL) and H₂O (180 mL). Then, H was added dropwise₂O₂(30% in H)₂O, 25 μ L) and the mixture was stirred at room temperature for 1.5 h. After this, the mixture was filtered and the filtrate was purified by HPLC method J to provide 50mg of conotoxin peptide analog Iw (MW 1725.6g/mol, 29 μmol, yield: 12.6%, TFA salt) as a white solid. Purity 82% (230 nm).

The peptide was further purified by preparative HPLC simultaneously with TFA anion exchange to acetate according to the method described for conotoxin peptide analogue Ia. The isolation yield of conotoxin peptide analogue Iw (acetate) was 20mg (12. mu. mol, total yield 2.4%). LC-MS (Single quadrupole ESI) m/z: 576.1[ M +3H]/3⁺，863.5[M+2H]/2⁺(MW calculated: 1723.56); HPLC method K; retention time: 7.0 min; the purity is 97%.

Example 1.25 conotoxin peptide analog Ix (L-tyrosinamide, glycyl-L-cysteinyl-L-lysyl-L-threonyl-L- α -aspartyl-L-prolyl-L-arginyl-L-cysteinyl-N5- (carbamoyl) -L-ornityl-L-3-iodotyrosyl-L-glutaminyl-ring (2 → 8) -disulfide-ring- (3 → 12) -lactam) (SEQ ID NO: 82):

conotoxin peptide analog Ix was synthesized using the same procedure described for conotoxin peptide analog Iw by using FMOC-glu (odmab) in place of FMOC-lys (ivdde) -OH and FMOC-lys (ivdde) -OH in place of FMOC-glu (odmab). LC-MS (ESI-ion trap) m/z: 862.9[ M +2H]/2⁺，1724.3[M+H]/⁺(MW calculated: 1723.56); HPLC method K; retention time: 7.1 min; the purity is 95%.

6.3. Example 2: preparation of PEGylated conotoxin peptide analogs

Example 2.1 PEGylated conotoxin peptide analog IIa (SEQ ID NO: 83) (30 kDa-mPEG-Valerate (VA) -conotoxin peptide analog Ia):

the acetate salt of conotoxin peptide analog Ia (25mg, 0.015mmol) was dissolved in PBS buffer (pH 8.0, 12.5mL) and then added to H₂mPEG-30kDa-VA-NHS ester (560mg, 0.018 mmol; NOF America, SUNBRIGHT ME-300 HS; final linker ═ pentanoic acid amide ("VA");

average n 675). After shaking the reaction vessel at room temperature for 2h, the progress of the reaction was monitored using analytical HPLC. When > 90% of the conotoxin peptide analogue Ia had been consumed, the resulting pegylated peptide (pegylated conotoxin peptide analogue IIa) was purified by HPLC method I to remove unreacted conotoxin peptide analogue Ia (RT ═ 5.0min), as determined by HPLC analysis. The HPLC peak of pegylated conotoxin peptide analog IIa was divided into three portions and each portion was analyzed by HPLC method I using ELSD detection (on-line, after UV detector) to determine peptide purity (UV RT 16.65min, ELSD retention time 16.75min), amount of residual free PEG (ELSD RT 17.00 min). Fractions containing < 5% free PEG with > 95% peptide purity based on the ELSD signal (free PEG versus ELSD signal for peptide) were combined and lyophilized. Overall isolation yield was 0.21g (44%); HPL C, method I: purity: 95% (UV 214nm), 97% (ELSD). Characterization data for the pegylated conotoxin peptide analog IIa are listed in table 2 below.

TABLE 2 characterization data of PEGylated conotoxin peptide analog IIa

Example 2.2 PEGylated conotoxin peptide analog IVa (SEQ ID NO: 84) (30 kDa-mPEG-bAmine-conotoxin peptide analog Ia):

the acetate salt of conotoxin peptide analog Ia (25mg, 0.013mmol) was dissolved in MeOH (10mL) and a solution of mPEG-30 kDa-butyraldehyde (CAS No.9004-74-4, 414mg, 0.013mmol) in MeOH (10mL) was added. The mixture was shaken at room temperature for 3 h. After this time, a solution of PPTS (3.3mg, 0.013mmol) in MeOH (0.1mL) was added, followed by the addition of NaCNBH₃(1.6mg, 0.026mmol) in MeOH (0.1 mL). Shaking was continued for 12h, after which the reaction mixture was directly purified by preparative HPLC method G to provide the TFA salt of pegylated conotoxin peptide analog IVa (92mg, total yield 21%, TFA salt). HPLC purity: 95.7% (UV 214 nm).

Subsequent salt exchange was achieved by dissolving the TFA salt (92mg, 0.0027mmol) from the pegylated conotoxin peptide analog IVa above in 3mL of water. Adding NH dropwise to the solution₄HCO₃(aqueous solution) to adjust the pH to 7.0-8.0. The mixture was then directly purified by preparative RP-HPLC (mobile phase: A: 0.1% AcOH in water, B: 0.1% AcOH in ACN) and lyophilized to provide the acetate salt of PEGylated conotoxin peptide analog IVa as a white solid (31mg, total yield 7%, acetate salt). LC/MS purity: 95.5% (UV 214nm), 98.7% (ELSD). Characterization data for pegylated conotoxin peptide analog IVa are listed in table 3 below.

TABLE 3 characterization data of PEGylated conotoxin peptide analog IVa

Example 2.3C-terminally PEGylated conotoxin peptide analog Va (SEQ ID NO: 85) (conotoxin peptide analog Ia-30kDa-PEG)

N-terminal N-acetyl conotoxin peptide analog Ia intermediate (14): conotoxin peptide analog Ia (75mg, 46. mu. mol) was dissolved in anhydrous DMF (35mL) and Ac was added₂O (7.5. mu.L, 0.3mmol) and anhydrous pyridine (32. mu.L, 0.4 mmol). The solution was stirred at room temperature for 2 h. The product was then purified by preparative HPLC method G (table 7). After lyophilization of the product-containing fractions, compound 14(44.5mg, 27 μmol, 98% purity, 59% yield) was obtained as an off-white solid. LC-MS (ESI) m/z: 554.6[ M +3H]/3⁺，831.0[M+2H]/2⁺Purity 98% (UV 214 nm).

C-terminal pegylated conotoxin peptide analog Va: compound 14(15mg, 9.0. mu. mol) was dissolved in anhydrous DMF (2.5mL) and a solution of 30 kDa-mPEG-propylamine (300mg, 10. mu. mol, 1.1 eq) in anhydrous DMF (2.5mL) was added. Then, a solution of HBTU (3.7mg, 9.7. mu. mol) in DMF (0.3mL) and a solution of DIPEA (458. mu.L, 26. mu. mol) in anhydrous DMF (45mL) were added, respectively. The mixture was stirred at room temperature for 2h, then purified by HPLC method G (table 7). The purified product was lyophilized to give the TFA salt of C-terminally pegylated conotoxin peptide analog Va (100mg, 3.1 μmol, 35% yield) as a TFA salt (91% purity at 214 nm). The TFA salt was then dissolved in H ₂O：CH₃CN (3 mL: 1mL), and then NH was added dropwise₄HCO₃Dilute solution (aqueous solution) to obtain pH 7.0-8.0. The mixture was then purified by HPLC method H (table 7) to provide the acetate salt of C-terminal polypegylated conotoxin peptide analog Va. After lyophilization, a white solid was obtained (50mg, yield: 15.8. mu. mol, 17.5%, acetate). LC-purity:90.4% (UV 214nm), 99.3% (ELSD). Characterization data for pegylated conotoxin peptide analog Va are listed in table 4 below.

TABLE 4 characterization data for PEGylated conotoxin peptide analogs Va

Example 2.47-Arg-PEGylated conotoxin peptide analog VIa (SEQ ID NO: 86) (Ac-conotoxin peptide analog Ia (Arg7-VA-30kDa-PEG)

N-terminal N-acetyl conotoxin peptide analog Ia intermediate 14(15mg, 9.0. mu. mol) was dissolved in anhydrous DMF (2.5mL) and a solution of mPEG-30 kDa-pentanoic acid-NHS ester reagent (0.54g, 18. mu. mol) in anhydrous DMF (2.5mL) was added. Then, a solution of DBU (4. mu.L, 27. mu. mol) in DMF (0.3mL) was added. The mixture was heated to 40 ℃ for 4h, then the product was purified by HPLC method G (table 7). Subsequent lyophilization afforded 35mg of the acetate salt of 7-Arg-PEGylated conotoxin peptide analog VIa (Table 6; 35mg, 1.1. mu. mol, 12.2%, TFA salt). Purity 80% (UV 214 nm). Salt exchange was performed as described to provide the acetate salt of 7-Arg-pegylated conotoxin peptide analog VIa as a white solid (28mg, 9.8% total yield, acetate salt). Purity: 99.1% (UV 214nm), 81.4% (ELSD). Characterization data for the pegylated conotoxin peptide analog VIa are presented in table 5 below.

TABLE 5 characterization data of PEGylated conotoxin peptide analogs VIa

TABLE 6 PEGylation reagents

Table 7 below summarizes the HPLC methods described herein.

Table 7: summary of the HPLC procedure

6.4. Stability analysis

To address the instability of the disulfide bond in RgIA derivatives, conotoxin peptide analogs were synthesized with a triazole bridge replacing the disulfide bridge and evaluated in human and Sprague Dawley rat plasma and serum.

Reverse phase HPLC analysis of samples from each triazole-stable conotoxin peptide analogue showed a single isolated peak consistent with the native conformation of the peptide. For example, fig. 2A shows that conotoxin peptide analog Ia shows a single isolated peak on reverse phase HPLC at 0h, 8h and 24h in rat plasma. There was no evidence of disulfide rearrangement, suggesting that the replacement of one native disulfide bond by a triazole mimetic was sufficient to prevent disulfide shuffling (disulfide shuffling) of RgIA derivatives in plasma.

In contrast, conotoxin peptide analog CSP-4-NH as in FIG. 2B₂Shown by the reverse phase HPLC trace, the conotoxin peptide analog CSP-4-NH₂("native" form, consisting of two disulfide bonds, one between Cys2 and Cys8 and the second between Cys3 and Cys 12) undergoes a significant portion that becomes "ribbon-like" (ri) containing the replacement disulfide bonds Cys2-Cys12 and Cys3-Cys8 bbon) "and" bead "forms containing a replacement Cys2-Cys3 and Cys8-Cys12 disulfide bond. Conotoxin peptide analog CSP-4-NH in plasma and serum samples₂Isomerization was predominant between the native form and the banded form with minimal bead formation (fig. 2B).

6.5. Biological assay

6.5.1. Two-electrode voltage clamp method for rat and human nAChR

Example 4.1

And (3) determination:

xenopus laevis oocytes (Xenopus laevis, Miss 1, MI) were used for heterologous expression of cloned rat or human nAChR subtypes. Recordings were made 1-5 days after injection. Briefly, oocytes were placed in 100. mu.L chambers (4mM diameter, 62mM depth) made by Sylgard and gravity-perfused with ND96(96mM NaCl, 1.8mM CaCl) at a constant flow rate (2mL/min)₂，2.0mM KCl，1.0mM MgCl₂5mM HEPES, pH 7.1-7.5). The membrane potential of the oocytes was maintained at-70 mV using a GeneClamp500 two-electrode voltage clamp amplifier. nAChRs were stimulated once per minute with 100 μ M acetylcholine (ACh)1-sec pulses and ACh-gated currents were collected. After achieving a stable baseline for ACh pulse using ND96, the solution was switched to ND96 containing various compound concentrations and observed for blocking response of ACh pulse. After peptide application, the nAChR response to ACh was calculated as the "% response" of the value observed at baseline. The data for each oocyte were compared to the Hill equation by non-linear regression analysis and using constraints with bottom equal to 0 and top equal to 100: reaction% + 100/{1+ ([ toxin) ]/IC₅₀}^nHFitted (GraphPad Prism), resulting in a concentration-response curve for ACh-gated current inhibition. Fitting independent concentration response curves to each oocyte and integrating IC₅₀The values are averaged. Will IC₅₀The mean of (d) is reported as mean ± SEM. The two-tailed unpaired t-test was used for significant differences (GraphPad Prism).

As a result:

6.5.1.1. replacement of disulfide bridges with triazole bridges improved stability and maintained sufficient activity for both rat and human nachrs

TABLE 8 inhibition of rat and human α 9 α 10 nicotinic acetylcholine receptors (nAChR) by conotoxin peptides RgIA and analogs thereof

Note: NT was not tested.

The activity of conotoxin peptide analogs prepared in sections 6.2 and 6.3 and pegylated conotoxin peptide analogs on both rat and human nachrs were determined and the results are summarized in tables 8 and 9, respectively.

CSP-4-NH as shown in Table 8₂Shows nanomolar activity (IC) on both rat and human nAChRs₅₀: 0.4 ± 0.2nM for rat α 9 α 10nAChR and 3.5 ± 1.5nM for human α 9 α 10 nAChR). Nevertheless, due to CSP-4-NH₂Having two disulfide bridges (one disulfide bridge between the cysteine residues in the 2-and 8-positions; the other disulfide bridge between the cysteine residues in the 3-and 8-positions), thus CSP-4-NH ₂Suffer from the problem of disulfide shuffling and have poor stability (see fig. 1A-1D and 2B).

As shown in table 8, conotoxin peptide analogs Iw and Ix, in which the disulfide bridges at the 3-and 12-positions were replaced with lactam bridges, did not maintain sufficient activity on human α 9 α 10 nachrs. Conotoxin peptide analogues Iw and Ix, respectively, have been shown to be>IC of 300nM and 123. + -.22 nM₅₀. Certain conotoxin peptide analogs having a triazole bridge formed between Pra at the 3-position and azido Nva at the 12-position maintain sufficient biological activity (e.g., IC) for human α 9 α 10nAChR₅₀<100 nM). For example, the IC of conotoxin peptide analogs Ia and Ia' on human α 9 α 10nAChR₅₀The results of measurement of (A) were 0.7. + -. 0.03nM and 26. + -. 4nM, respectively. IC of intermediate conotoxin peptide analogs Ib and Ib' to human alpha 9 alpha 10nAChR₅₀The results of the measurement of (A) were 37. + -. 1.3nM and 48. + -. 9nM, respectively. Conotoxin peptide analogs Ia and Ib having a carboxylic acid at the C-terminal end and conotoxin peptide analogs Ia 'and Ib' having an amide group at the C-terminal end maintained sufficient activity. Conotoxin peptide analogs Ia and Ib, both having an amide group at the C-terminus, respectively, exhibit lower IC than the corresponding conotoxin peptide analogs having a carboxylic acid at the C-terminus₅₀(Ia vs. Ia 'and Ib vs. Ib').

In contrast, conotoxin peptide analogs Ic and Id having a triazole bridge formed between the 2-and 8-positions did not maintain sufficient activity on human α 9 α 10 nachrs. IC of conotoxin peptide analogs Ic and Id₅₀The measurement results of (b) are 152. + -. 71nM and 1000nM, respectively. Still in contrast, conotoxin peptide analogs If and Ij having a triazole bridge formed between the 3-and 12-positions, but a triazole bridge formed at the 12-position by Pra or azido Ala, respectively, did not maintain sufficient activity on human α 9 α 10 nachrs. IC of conotoxin peptide analogs If and Ij₅₀The results of the measurement of (A) were 1521. + -.116 nM and 152. + -.71 nM, respectively.

Deletion of the terminal amino acid of RgIA and replacement by an amide (Cys 12-amide) has previously been shown to have no significant effect on peptide binding to rat α 9 α 10nAChR (Ellison et al, 2008, J.mol.biol.377: 1216-1227; Armishaw,2010, Toxins 2: 1471-1499; US 20120220539A 1). Furthermore, other alpha-conotoxins, such as vc1.1 and ImI, do not have an amino acid at position 13 (see fig. 4), indicating that this residue would not be necessary for channel binding.

However, it was unexpectedly found that in the context of conotoxin peptide analog Ia, C-terminal amino acid deletions or substitutions affected human α 9 α 10nAChR potency. Specifically, as shown in table 8, when Tyr at position 13 is replaced by Phe (conotoxin peptide analog Il) or D-Tyr (conotoxin peptide analog Im), the efficacy is decreased. IC of conotoxin peptide analogs Il and Im ₅₀The results of measurement of (A) were 11. + -. 1.4nM and 50. + -. 1.2nM, respectively. In contrast, when Tyr at position 13 Is deleted (conotoxin peptide analog Iq), replaced by N-Me-Tyr (conotoxin peptide analog Ir), D-Arg (conotoxin peptide analog Is), N-Me-D-Tyr (conotoxin peptide analog It), β -Tyr (conotoxin peptide analog Iu) or N-Me-Arg (conotoxin peptide analog Iv), It provides a conotoxin peptide analog with low potency for human α 9 α 10 nAChR.

The effect of deletion of the amino acid residue at position 13 of the conotoxin peptide analogs with the triazole bridge formed between positions 3-and 12-on their efficacy on human α 9 α 10nAChR was evaluated relative to the effect of deletion of the residue at position 13 of CSP-4-OH (the corresponding conotoxin peptide analogs with 3-and 12-disulfide bridges). Surprisingly, it was found that deletion of Tyr at position 13-results in a greater than 20-fold decrease in binding affinity to human α 9 α 10nAChR, relative to CSP-4-OH (Cys3,12 disulfide bridge) (fig. 5A). However, the deletion of amino acid 13 resulted in an approximately 10-fold loss of activity to the rat channel and a greater than 100-fold loss of activity to the human channel (conotoxin peptide analog 1a vs 1q) relative to conotoxin peptide analog 1a (with the replacement of the triazole bridge between the 3-and 12-positions) (table 8, fig. 5B). These data show that deletion of the amino acid at the C-terminus (position 13) reduces the activity of conotoxin peptide analogs having a triazole bridge between the 3-and 12-positions, but does not reduce those having a disulfide bridge between the 3-and 12-positions. These data indicate that in the context of stable disulfide bond mimetics, the interaction in loop 2 and the carboxy terminus of the entire peptide is important for determining human channel potency, and that changes in the secondary structure of loop 2 and the carboxy terminus produce unpredictable interactions between conotoxin peptide RgIA analogues and α 9 α 10 nachrs.

Furthermore, the addition of one other amino acid (e.g., N-Me-Gly, D-Tyr, or N-Me-Tyr) to the C-terminus of conotoxin peptide analog Ia provided conotoxin peptide analogs In, Io, and Ip having 14 residues that maintained potency at human α 9 α 10 nAChR. As shown In Table 8, for example, the IC of conotoxin peptide analogs In (having an additional N-Me-Gly at position 14 as compared to Ia), Io (having an additional D-Tyr at position 14 as compared to Ia) and Ip (having an additional N-Me-Tyr at position 14 as compared to Ia)₅₀The results of measurement of (A) were 37. + -.15 nM, 59nM and 26. + -. 1.4nM, respectively.

Selectivity of channel binding: in an evaluation of the potential off-target effects involving 88 receptors, transporters, enzymes and kinases, conotoxin peptide analogs Ia 'and Ib' containing triazole showed no off-target effects at 10 μ M, except for partial inhibition of α 7nAChR (52% and 56%, respectively). In this regard, the triazole bridge replacement of the disulfide bridge in conotoxin peptide RgIA did not significantly alter the selectivity of the conotoxin peptide analogs.

6.5.1.2. PEGylation of conotoxin peptide analog Ia at the N-terminus unexpectedly retains sufficient activity for both rat and human nAChRs

The addition of 30kDa valerate-linear PEG (N-terminally acylated) to the N-terminus of conotoxin peptide analog Ia resulted in the production of pegylated conotoxin peptide analog IIa. When its activity was evaluated on human and rat nachrs, the pegylated conotoxin peptide analog IIa produced similar blockade with low nanomolar potency on rat and human α 9 α 10 nachrs (fig. 6A and 6B). PEGylated conotoxin peptide analog IIa results in the production of an IC nearly equal to that of unconjugated conotoxin peptide analog Ia (0.7 + -0.03 nM) for human α 9 α 10nAChR ₅₀(Table 9; 0.7. + -. 0.1 nM). In contrast to the literature teachings that pegylation would potentially result in structural constraints, leading to reduced binding potency of the peptide to rodent and human channels (fisherburn, 2008, j. pharm. sci.97: 4167-4183; parrot and DeSimone,2011, nat. chem.4:13-14), pegylated conotoxin peptide analogs with an approximately 18.5-fold increase in molecular weight by attachment to PEG polymers unexpectedly retain the potency of conotoxin peptide Ia (0.7 ± 0.1nM for human α 9 α 10 nAChR).

TABLE 9 inhibition of rat and human α 9 α 10 nicotinic acetylcholine receptors (nAChR) by conotoxin peptide analog Ia and its PEGylated conotoxin peptide analogs IIa, IIIa, IVa, Va and VIa

Note: NT was not tested.

The effect of pegylation at different attachment positions was evaluated by testing the activity of pegylated conotoxin peptide analog Va (where the linear mPEG polymer was covalently attached to the C-terminus of conotoxin peptide analog Ia) and VIa (where the linear mPEG polymer was covalently attached to the arginine at the 7-position of conotoxin peptide analog Ia) on human α 9 α 10 nAChR. PEGylated conotoxin peptide analog IIa (IC) with a linear mPEG polymer covalently attached to the N-terminus of conotoxin peptide analog Ia ₅₀0.7 ± 0.1nM) versus pegylated conotoxin peptide analog Va (IC)₅₀300nM) and VIa (IC)₅₀300nM) was observed to be significant in human nAChRSignificantly lower IC₅₀. Conjugation to PEG at the C-terminus (Va) or position 7(VIa) of conotoxin peptide analog Ia does not provide a pegylated conotoxin peptide analog with sufficient activity.

The efficacy of pegylated conotoxin peptide analogs using different linkers was also investigated. The activity of pegylated conotoxin peptide analog IVa with a linear mPEG polymer attached to the N-terminus of conotoxin peptide analog Ia via a butylene linker was tested for human α 9 α 10 nAChR. IC of PEGylated conotoxin peptide analog IVa as shown in Table 9₅₀PEGylated conotoxin peptide analog IIa (IC) in which a PEG polymer is attached to the N-terminus of the peptide (conotoxin peptide analog Ia) via a valerate linker₅₀0.7 ± 0.1nM) IC₅₀100-fold higher (IC)₅₀100 nM). These results indicate that the choice of linker type and position on the peptide to which the PEG polymer is attached is critical for the pegylation derivatization of conotoxin peptide analog Ia, which retains its potency on human α 9 α 10 nAChR. Surprisingly, it was found that pegylated conotoxin peptide analog IIa with a 30kDa linear mPEG covalently linked to the N-terminus of conotoxin peptide analog Ia through a valerate ester linker is the most optimal pegylated conotoxin peptide analog.

Similar unexpected results were also demonstrated by PEGylated CSP-4-OH. PEGylated conotoxin peptide analog XI (straight chain 30kDa mPEG-VA-CSP-4-NH) generated by adding 30kDa PEG to the N-terminus of CSP-4-OH through a valerate linker₂) Low nanomolar potency (IC) was maintained for rat α 9 α 10nAChR₅₀6.0 ± 0.1nM), indicating that the N-terminus of these peptides allows the addition of large molecules without significantly altering their in vitro biological potency.

6.5.2. Pharmacokinetic study of conotoxin peptide analogs and pegylated conotoxin peptide analogs in rats

Pharmacokinetic studies of conotoxin peptide analogue Ia in rats: conotoxin peptide analogue Ia was injected intravenously or subcutaneously into 10-week-old male Sprague Dawley rats (n ═ 3 per group) at 1mg/kg in vehicle (10mM sodium phosphate, 0.8% sodium chloride, 0.05% tween 20, pH 6.0). At a different placeTime points (pre-dose and 0.083 to 24 hours post-dose) blood was collected via tail vein to K₂EDTA collection tubes, centrifugation and storage of plasma at-60 to-90 ℃ until analysis. The concentration of conotoxin peptide analog 1a was determined using a peptide-specific LC-MS/MS assay as described below. Raw data were generated and plotted by GraphPad Prism using non-linear regression with sigmoidal dose-response (variable slope). Pharmacokinetic parameters were calculated using PKSolver 2.0 software.

LC-MS/MS analysis of plasma PK samples of conotoxin peptide analogue 1 a: sample analysis of conotoxin peptide analog 1a was performed using a Shimadzu liquid chromatography system (Shimadzu UFLC-XR) and AB Sciex API 5000 triple quadrupole tandem mass spectrometer. The HPLC system consisted of a Shimadzu liquid chromatography system equipped with two LC-20AC XR pumps, a CBM-20A communication module, a SIL-20AC XR autosampler, a CTO-20A column oven, and an online SPD-20A UV detector. The reaction was carried out on a Thermo Scientific fluorhase PFP column (2.1 x 50mm, 5 μm,

) The chromatographic separation was carried out at 40 ℃. Mobile phase a was water with 0.05% acetic acid and mobile phase B was acetonitrile with 0.05% acetic acid. A linear gradient of B (5% -90%) was applied at a flow rate of 0.3mL/min for 0-6min, then 90% was maintained for 6.0-7.8min, then column equilibration was returned to 5% B for 2 min. The samples were kept in an autosampler at 4 ℃ and for each analysis a volume of 5 μ Ι _ was injected into the HPLC system.

Mass spectrometry detection was performed on a triple quadrupole tandem mass spectrometer API 5000 equipped with a vortex ion spray source operating in negative ionization mode. The operating conditions of the MS were optimized as follows: the voltage of the ion spray is set to be-4500 KV, and the temperature of the ion source is kept at 500 ℃; the collision energy was set to-35V. Nitrogen was used as the collision gas. The flow rates of the gas curtain gas, the ion source gas 1 and the gas 2 were set to 10, 40 and 40L/min, respectively. The manipulation and data analysis of LC-MS/MS was performed using Analyst 2.1 software (AB Sciex). Quantitative data were obtained by Multiple Reaction Monitoring (MRM) mode of shift using conotoxin peptide analog Ia at m/z 809.0/792.5.

K from a 1.0mg/mL stock of the correct purity peptide in DMSO accurately weighed using an HP D300 digital dispenser (Tecan)₂A standard curve (10-5000ng/mL) of conotoxin peptide analog Ia was prepared in EDTA rat plasma (Biorecamation). 50 μ L of PK sample plasma, conotoxin peptide analog Ia standard and conotoxin peptide analog Ia QC sample were aliquoted onto a Phere Phospholipid Removal Plate (Phenomenex), treated with 450 μ L of methanol (1: 10 dilution) and mixed thoroughly. The sample was then filtered through a phre plate into a 96-deep well sample plate via a vacuum manifold and analyzed using the LC-MS/MS method. The concentration of conotoxin peptide analog Ia was obtained from the standard curve by AB-Sciex MultiQuant software using single parameter non-linear regression, and PK parameters were calculated using PKSolver 2.0 software.

Pharmacokinetic (PK) study of pegylated conotoxin peptide analogue IIa in rats: pegylated conotoxin peptide analog IIa was injected intravenously ("IV") or subcutaneously ("SC") in vehicle (10mM sodium phosphate, 0.8% sodium chloride, 0.05% tween 20, pH 6.0) at 1mg/kg (core peptide content,. about.19 mg/kg pegylated peptide content) into 10-week old male Sprague Dawley rats (n ═ 3 per group). Blood was collected via tail vein at different time points (pre-dose and 0.083-120 hours post-dose) to K ₂EDTA collection tubes, centrifugation and storage of plasma at-60 to-90 ℃ until analysis. The amount of conotoxin peptide analogue IIa was quantified by a PEG-conjugate specific ELISA assay as described below. The plasma concentrations obtained (net peptide) were generated by GraphPad Prism using non-linear regression with sigmoidal dose-response (variable slope). PK parameters were calculated using PKSolver 2.0 software.

ELISA antibodies, reagents and methods for the analysis of PEGylated conotoxin peptide analogue IIa PK samples: rabbit polyclonal anti-conotoxin capture antibodies were generated by immunizing untreated NZW rabbits with conotoxin peptide analog Ia conjugated to KLH carrier protein, boosted with conotoxin peptide analog Ia. Then, an affinity protein G Sepharose (GE Healthcare Life Sciences) gravity column was used, followed by a size-exclusion chromatography (SEC) column (GE Hea) of 16/600Superdex 75pG using PBS as mobile phaselthcare Life Sciences) and buffer exchange, polyclonal IgG antibodies were purified from immune sera. The SEC fractions containing purified antibody were mixed and the concentration calculated by bicinchoninic acid assay (BCA assay). The purified polyclonal antibodies have demonstrated cross-reactivity recognizing conotoxin peptide analog Ia and conotoxin peptide analog IIa. Microtiter plate wells were coated with 50 μ L per well of capture antibody (purified anti-conotoxin peptide analog Ia 7626R pAb, batch #26R-1-D54-0618, 5 μ g/ml) in coating buffer (0.1M NaHCO3 pH 9.6) at 4 ℃ for 12h, or shaken at 37 ℃ for 1h, then blocked with 200 μ L per well of 5% BSA in PBS for 2h at room temperature. Plasma samples, QC samples, and standards (50 μ L) were loaded twice and incubated at room temperature for 1.5-2h with shaking, then 50 μ L of anti-PEG antibody (1ug/mL) per well in diluent buffer (0.5% BSA in PBS) was added and shaken at room temperature for an additional 1 h. 50 μ L of each well was diluted 1: 200 diluted streptavidin HRP was added to the plate and incubated for 1h at room temperature with shaking, then 50. mu.L chromogenic substrate (TMB) was added and incubated for 5-6min at room temperature. With 100. mu. L H ₂SO₄The reaction was stopped and absorbance at 450nm was measured using an ELISA plate reader. After each step, plates were washed 3 times with wash buffer (PBST, pH 7.4, containing 0.1% (v/v) Tween 20) and then 3-6 times with PBS. As a quantitative reference, the peptide analog IIa of conotoxin by PEGylation was at 10% K₂Standard curves were established for serial dilutions (400ng/mL-0.06ng/mL based on weight of core peptide content) in EDTA plasma (rat or monkey). The amount of pegylated conotoxin peptide analog IIa in each sample was quantified by extrapolating the sample signal to the linear range of the standard curve (signal vs concentration).

As a result: as discussed below, pegylation of conotoxin peptide analog Ia was shown to extend its circulation time. The pharmacokinetic profile of pegylated conotoxin peptide IIa was evaluated in rats and compared to the PK profile and clearance of conotoxin peptide analogue Ia. Fig. 7A and 7B show graphs of the mean concentration of conotoxin peptide analogue Ia and pegylated conotoxin peptide IIa in plasma versus time after a single 1mg/kg IV or SC dose. When PEG-conjugate PK profiles were usedTwo improvements due to pegylation are evident when the figure is compared to the spectrum of unconjugated conotoxin peptide analogue Ia alone: (i) by adding a PEG group, the half-life of conotoxin peptide analogue Ia is significantly increased, and (ii) the total exposure of pegylated conotoxin peptide IIa is much greater than that of the unconjugated conotoxin peptide analogue Ia. For SC administered compounds, the elimination half-life of pegylated conotoxin peptide IIa was approximately 42-fold longer (21 h versus 0.5h, respectively) than for the unconjugated conotoxin peptide analog Ia. Also improved are Cmax and AUC of PEGylated conotoxin peptide IIa, wherein Cmax of PEGylated conotoxin peptide IIa is nearly four-fold greater than unconjugated conotoxin peptide analogue Ia, and AUC of PEGylated conotoxin peptide IIa is nearly 140-fold greater than AUC of conotoxin peptide analogue Ia (Cmax: PEGylated conotoxin peptide IIa: 2518 ng/mL; conotoxin peptide analogue Ia: 648ng/mL) (AUC) _0-t: pegylated conotoxin peptide IIa: 137729ng/mL × hr; conotoxin peptide analog Ia: 981ng/mL hr). The Pharmacokinetic (PK) and Pharmacodynamic (PD) parameters of conotoxin peptide analogue Ia and pegylated conotoxin peptide IIa in rats are shown in table 10 below.

TABLE 10 Pharmacokinetic (PK) and Pharmacodynamic (PD) parameters of conotoxin peptide analogs Ia and PEGylated conotoxin peptides IIa in rats

An enhancement of the PK profile by pegylation of conotoxin peptide analogue Ia was also observed in monkeys. Male cynomolgus macaque (cynomolgus macaque) IV and SC were administered 1mg/kg (net peptide concentration) of pegylated conotoxin peptide IIa for PK studies. The highest exposure of pegylated conotoxin peptide IIa after IV administration was 0.083h post dose with a Cmax of 42,200ng/mL and decreased to 109ng/mL at 168h post dose. AUC was 603,594ng/ml × h. The highest exposure of pegylated conotoxin peptide IIa after SC administration was 24h post dose with a Cmax of 7,867ng/mL and decreased to 216ng/mL at 168h post dose. AUC was 453,077ng/ml × h and bioavailability of pegylated conotoxin peptide IIa after SC dose administration was 75%. The PK profile and parameters of pegylated conotoxin peptide IIa in monkeys are shown in fig. 8 and table 11.

TABLE 11 pharmacokinetic parameters of PEGylated conotoxin peptide IIa after single 1mg/kg dose administration (IV or SC) in monkeys

Note: NT was not tested.

6.5.3. Analgesic activity in animal pain models

The Chronic Compressive Injury (CCI) model is a model of mononeuropathy caused by attachment to the sciatic nerve (Bennett and Xie,1988, Pain,33(1): 87-107). Involves damage to peripheral nerves, and infiltration of mast cells, granulocytes, macrophages and T lymphocytes. Secretion of these cells by inflammatory mediators (e.g., pro-inflammatory cytokines and chemokines) can contribute to the production and maintenance of neuropathic pain. CCI is one of the common models of peripheral nerve injury.

And (3) determination: on day 0, male Sprague Dawley rats were anesthetized with 5% isoflurane (O2, at 1L/min) in an inhalation chamber. Once the appropriate plane of anesthesia has been reached, a constant flow of isoflurane at a maintenance level of 3% is administered through the nose cone. A 2cm skin incision was made in the left hind limb parallel to the femur. The sciatic nerve was exposed and 4 loose 4-0 chromium gut ligatures were tied at approximately 1mm intervals near the three bifurcations of the sciatic nerve. Bringing the muscles and skin into close proximity and allowing the animal to recover. Starting on the day of surgery and each of the 14 days thereafter, 0.1mg/kg conotoxin peptide analog 1b' in a vehicle consisting of 10mM sodium phosphate, 0.8% NaCl, 0.05% tween 20 was administered by subcutaneous injection.

As a result: data from animals treated with conotoxin peptide analog 1b' were compared to animals treated with vehicle control. Mechanical hyperalgesia was measured in the left hindpaw using a Ugo Basile hyperalgesia meter (n ═ 3 times) 7 and 14 days post surgery. Statistical analysis was performed by two-way analysis of variance. As shown in figure 9, daily treatment with 0.1mg/kg conotoxin peptide analog 1b' resulted in a statistically significant improvement in paw withdrawal threshold on both measurement days relative to vehicle-treated control animals. These data indicate that the triazole-substituted RgIA analogs effectively treated neuropathic pain in the rat CCI model.

6.5.4. Chemotherapy-induced peripheral neuropathy model

And (3) determination: chemotherapy-induced peripheral neuropathy (CIPN) is common in patients undergoing platinum salt chemotherapy. Chemotherapy-induced neuropathy was induced in male Sprague Dawley rats by treatment with 2.4mg/kg intravenous (i.v.) oxaliplatin twice a week for 3 weeks (6 i.v. injections). Pain thresholds were determined in rats using an algometer (Ugo Basile, Varese, Italy). Briefly, using a blunt tapered probe, a continuously increasing pressure is applied in a small area on the dorsal side of the hind paw. The mechanical pressure is increased until either a sound is produced or a back-off reflex occurs. The voicing or back-off reflectance threshold is expressed in grams. For the analgesia measurements, the application of mechanical pressure was terminated at 120 g. During the study, the experimenter was blinded to the nature of the injected compound. Data were analyzed by one-way analysis of variance using a Dunnett multiple comparison test (GraphPad Prism). Neuropathic pain was measured 14 days after a single subcutaneous dose (e.g., 0.5mg/kg) of conotoxin peptide analog administration.

6.5.4.1. Evaluation of PEGylated conotoxin peptide analog derivatives Using different PEG polymers

Preparing CSP-4-NH₂Including linear 20kDa mPEG-VA-CSP-4-NH₂(PEGylated conotoxin peptide analog VII, formula (VII), SEQ ID NO: 87), straight-chain 20kDa mPEG-bAmine-CSP-4-NH₂(PEGylated conotoxin peptide analog VIII, formula (VIII), SEQ ID NO: 88), branched 2X 10kDa mPEG-amine-CSP-4-NH₂(PEGylated conotoxin peptide analog IX, formula (IX), SEQ ID NO: 89), branched 2X 10kDa mPEG-carbonate-CSP-4-NH₂(PEGylated conotoxin peptide analog X, formula (X), SEQ ID NO: 90), straight-chain 30kDa mPEG-VA-CSP-4-NH₂(PEGylated conotoxin peptide analog XI, formula (XI), SEQ ID NO: 91) and straight chain 40kDa mPEG-VA-CSP-4-NH₂(PEGylated conotoxin peptidesAnalog XII, formula (XII), SEQ ID NO: 92).

CSP-4-NH was evaluated₂Duration of analgesic effect in rat CIPN model of various pegylated derivatives of (a). The pegylated derivatives tested varied in size (20kDa to 40kDa) and configuration (covalently linked to a linear or branched PEG polymer). CSP-4-NH at a single subcutaneous dose of 500. mu.g/kg ₂After pegylation of conotoxins VII, VIII, IX, X, XI and XII, the analgesic potency and duration of the pharmacodynamic action were determined in the rat CIPN model. Analgesic efficacy was tested 30min, 5h, 24h, 48h, 72h and 96h after dose administration. Mechanical hyperalgesia was measured by the Randall-Selitto test. Pain threshold decreased with time after IV administration of oxaliplatin twice weekly, with marked mechanical hyperalgesia induced at day 7 after oxaliplatin dose administration and continued to increase to day 14. On day 14, CSP-4-NH was tested₂And CSP-4-NH₂The PEG-derivatives of (A) have efficacy in reducing mechanical hyperalgesia. Mechanical hyperalgesia is measured to reach pre-chemotherapy levels 0.5-5h after drug administration. The duration of analgesic efficacy is prolonged as the molecular weight of the added PEG increases up to 30kDa linear PEG. PEGylated CSP-4-NH Using n-butyl linker₂(VIII) duration of potency compared to CSP-4-NH PEGylated by pentanoic acid linker₂The duration of potency of (VII), (XI), (XII) was significantly shorter (fig. 10A-10C). No additional benefit was obtained by conjugation with PEG molecules (XII) of linear PEG greater than 30kDa or with branched PEG (IX and X) (fig. 10C).

6.5.4.2. Evaluation of the efficacy of conotoxin peptide analogs Ia and Ia' and of PEGylated conotoxin peptide analog IIa

As shown in fig. 11A, a single subcutaneous dose (0.5mg/kg) of conotoxin peptide analog Ia' resulted in a statistically significant improvement in mechanical hyperalgesia at 0.5h, 4h and 24h post-dose. As shown in fig. 11B, a single subcutaneous injection of conotoxin peptide analog Ia' resulted in a statistically significant improvement in mechanical hyperalgesia at 0.5h, 4h, and 24h post dose relative to vehicle treated animals. Fig. 11C shows that a single 0.5mg/kg dose of conotoxin peptide analog Ia or pegylated conotoxin peptide analog IIa resulted in a significant reduction of mechanical hyperalgesia (up to 4h for Ia, or up to 72h for IIa) 14 days after the induction of oxaliplatin-induced peripheral neuropathy. These data further demonstrate the therapeutic potential of using these triazole-stabilized RgIA derivatives for the treatment or prevention of neuropathic pain.

7. Is incorporated by reference

All references cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual patent publication or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

Various modifications and alterations of this invention may be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The specific embodiments described herein are offered by way of example only, and the invention is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled.

Sequence listing

<110> Chenita Limited liability company for chronic pain

<120> conotoxin peptide analogues and uses for the treatment of pain and inflammatory conditions

<130> 14520-002-228

<140> TBA

<141>

<160> 107

<170> PatentIn version 3.5

<210> 1

<211> 13

<212> PRT

<213> genus Conus (Conus region)

<220>

<223> Natural RgIA (native RgIA)

<400> 1

Gly Cys Cys Ser Asp Pro Arg Cys Arg Tyr Arg Cys Arg

1 5 10

<210> 2

<211> 12

<212> PRT

<213> genus Conus (Conus region)

<220>

<223> Natural ImI (native ImI)

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Gly Cys Cys Ser Asp Pro Arg Cys Ala Trp Arg Cys

1 5 10

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<211> 15

<212> PRT

<213> genus Conus (Conus region)

<220>

<223> Vc1.1

<400> 3

Gly Cys Cys Ser Asp Pro Arg Cys Asn Tyr Asp His Pro Glu Ile

1 5 10 15

<210> 4

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> CSP-4-OH

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (10)..(10)

<223> Xaa = 3-I-Tyr

<400> 4

Gly Cys Cys Thr Asp Pro Arg Cys Xaa Xaa Gln Cys Tyr

1 5 10

<210> 5

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> CSP-4-desTyr-OH

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (10)..(10)

<223> Xaa = 3-I-Tyr

<400> 5

Gly Cys Cys Thr Asp Pro Arg Cys Xaa Xaa Gln Cys

1 5 10

<210> 6

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> conotoxin peptide analog Ia (Conotoxin peptide analog Ia)

<220>

<221> DISULFID

<222> (2)..(8)

<220>

<221> MISC_FEATURE

<222> (3)..(12)

<223> (S) -propargylglycine at position 3 and (S) -5-azidovaline at position 12 form a triazole bridge ((S) -propargylglycine at position 3 and (S) -5-azidinorvaline at position 12 form a triazole bridge)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -propargyl glycine ((S) -propargyl glycine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -5-azidovaline ((S) -5-azidonorrvaline)

<400> 6

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Trp Gln Xaa Tyr

1 5 10

<210> 7

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Compound 3 (Compound 3)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -propargyl glycine ((S) -propargyl glycine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -5-azidovaline ((S) -5-azidonorrvaline)

<400> 7

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Trp Gln Xaa Tyr

1 5 10

<210> 8

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Compound 4 (Compound 4)

<220>

<221> DISULFID

<222> (2)..(8)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -propargyl glycine ((S) -propargyl glycine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -5-azidovaline ((S) -5-azidonorrvaline)

<400> 8

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Trp Gln Xaa Tyr

1 5 10

<210> 9

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Conotoxin peptide analog Ia '(Conotoxin peptide analog Ia')

<220>

<221> DISULFID

<222> (2)..(8)

<220>

<221> MISC_FEATURE

<222> (3)..(12)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -propargyl glycine ((S) -propargyl glycine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -5-azidovaline ((S) -5-azidonorrvaline)

<400> 9

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Trp Gln Xaa Tyr

1 5 10

<210> 10

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Intermediate conotoxin peptide analog Ya '(Intermediate conotoxin peptide analog Ya')

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -propargyl glycine ((S) -propargyl glycine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -5-azidovaline ((S) -5-azidonorrvaline)

<400> 10

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Trp Gln Xaa Tyr

1 5 10

<210> 11

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Intermediate conotoxin peptide analog Za '(Intermediate conotoxin peptide analog Za')

<220>

<221> DISULFID

<222> (2)..(8)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -propargyl glycine ((S) -propargyl glycine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -5-azidovaline ((S) -5-azidonorrvaline)

<400> 11

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Trp Gln Xaa Tyr

1 5 10

<210> 12

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> conotoxin peptide analog Ib (Conotoxin peptide analog Ib)

<220>

<221> DISULFID

<222> (2)..(8)

<220>

<221> MISC_FEATURE

<222> (3)..(12)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -propargyl glycine ((S) -propargyl glycine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (10)..(10)

<223> Xaa = 3-I-Tyr

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -5-azidovaline ((S) -5-azidonorrvaline)

<400> 12

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Xaa Gln Xaa Tyr

1 5 10

<210> 13

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> intermediate conotoxin peptide analog Yb (Intermediate conotoxin peptide analog Yb)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -propargyl glycine ((S) -propargyl glycine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (10)..(10)

<223> Xaa = 3-I-Tyr

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -5-azidovaline ((S) -5-azidonorrvaline)

<400> 13

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Xaa Gln Xaa Tyr

1 5 10

<210> 14

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> intermediate conotoxin peptide analog Zb (intermediate conotoxin peptide analog Zb)

<220>

<221> DISULFID

<222> (2)..(8)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -propargyl glycine ((S) -propargyl glycine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (10)..(10)

<223> Xaa = 3-I-Tyr

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -5-azidovaline ((S) -5-azidonorrvaline)

<400> 14

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Xaa Gln Xaa Tyr

1 5 10

<210> 15

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Conotoxin peptide analog Ib '(Conotoxin peptide analog Ib')

<220>

<221> DISULFID

<222> (2)..(8)

<220>

<221> MISC_FEATURE

<222> (3)..(12)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -propargyl glycine ((S) -propargyl glycine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (10)..(10)

<223> Xaa = 3-I-Tyr

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -5-azidovaline ((S) -5-azidonorrvaline)

<400> 15

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Xaa Gln Xaa Tyr

1 5 10

<210> 16

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Intermediate conotoxin peptide analog Yb '(Intermediate conotoxin peptide analog Yb')

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -propargyl glycine ((S) -propargyl glycine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (10)..(10)

<223> Xaa = 3-I-Tyr

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -5-azidovaline ((S) -5-azidonorrvaline)

<400> 16

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Xaa Gln Xaa Tyr

1 5 10

<210> 17

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Intermediate conotoxin peptide analog Zb '(Intermediate conotoxin peptide analog Zb')

<220>

<221> DISULFID

<222> (2)..(8)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -propargyl glycine ((S) -propargyl glycine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (10)..(10)

<223> Xaa = 3-I-Tyr

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -5-azidovaline ((S) -5-azidonorrvaline)

<400> 17

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Xaa Gln Xaa Tyr

1 5 10

<210> 18

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> conotoxin peptide analog ic (Conotoxin peptide analog ic)

<220>

<221> DISULFID

<222> (3)..(12)

<220>

<221> MISC_FEATURE

<222> (2)..(8)

<223> (S) -propargylglycine at position 2 and (S) -5-azidovaline at position 8 form a triazole bridge ((S) -propargylglycine at position 2 and (S) -5-azidinorvaline at position 8 form a triazole bridge)

<220>

<221> MOD_RES

<222> (2)..(2)

<223> Xaa = (S) -propargyl glycine ((S) -propargyl glycine)

<220>

<221> MOD_RES

<222> (8)..(8)

<223> Xaa = (S) -5-azidovaline ((S) -5-azidonorrvaline)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (10)..(10)

<223> Xaa = 3-I-Tyr

<400> 18

Gly Xaa Cys Thr Asp Pro Arg Xaa Xaa Xaa Gln Cys Tyr

1 5 10

<210> 19

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> intermediate conotoxin peptide analog Yc (intermediate conotoxin peptide analog Yc)

<220>

<221> MOD_RES

<222> (2)..(2)

<223> Xaa = (S) -propargyl glycine ((S) -propargyl glycine)

<220>

<221> MOD_RES

<222> (8)..(8)

<223> Xaa = (S) -5-azidovaline ((S) -5-azidonorrvaline)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (10)..(10)

<223> Xaa = 3-I-Tyr

<400> 19

Gly Xaa Cys Thr Asp Pro Arg Xaa Xaa Xaa Gln Cys Tyr

1 5 10

<210> 20

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> intermediate conotoxin peptide analog Zc (intermediate conotoxin peptide analog Zc)

<220>

<221> DISULFID

<222> (3)..(12)

<220>

<221> MOD_RES

<222> (2)..(2)

<223> Xaa = (S) -propargyl glycine ((S) -propargyl glycine)

<220>

<221> MOD_RES

<222> (8)..(8)

<223> Xaa = (S) -5-azidovaline ((S) -5-azidonorrvaline)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (10)..(10)

<223> Xaa = 3-I-Tyr

<400> 20

Gly Xaa Cys Thr Asp Pro Arg Xaa Xaa Xaa Gln Cys Tyr

1 5 10

<210> 21

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> conotoxin peptide analog Id (Conotoxin peptide analog Id)

<220>

<221> DISULFID

<222> (3)..(12)

<220>

<221> MISC_FEATURE

<222> (2)..(8)

<220>

<221> MOD_RES

<222> (2)..(2)

<223> Xaa = (S) -5-azidovaline ((S) -5-azidonorrvaline)

<220>

<221> MOD_RES

<222> (8)..(8)

<223> Xaa = (S) -propargyl glycine ((S) -propargyl glycine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (10)..(10)

<223> Xaa = 3-I-Tyr

<400> 21

Gly Xaa Cys Thr Asp Pro Arg Xaa Xaa Xaa Gln Cys Tyr

1 5 10

<210> 22

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> intermediate conotoxin peptide analog Yd (intermediate conotoxin peptide analog Yd)

<220>

<221> MOD_RES

<222> (2)..(2)

<223> Xaa = (S) -5-azidovaline ((S) -5-azidonorrvaline)

<220>

<221> MOD_RES

<222> (8)..(8)

<223> Xaa = (S) -propargyl glycine ((S) -propargyl glycine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (10)..(10)

<223> Xaa = 3-I-Tyr

<400> 22

Gly Xaa Cys Thr Asp Pro Arg Xaa Xaa Xaa Gln Cys Tyr

1 5 10

<210> 23

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> intermediate conotoxin peptide analog Zd (intermediate conotoxin peptide analog Zd)

<220>

<221> DISULFID

<222> (3)..(12)

<220>

<221> MOD_RES

<222> (2)..(2)

<223> Xaa = (S) -5-azidovaline ((S) -5-azidonorrvaline)

<220>

<221> MOD_RES

<222> (8)..(8)

<223> Xaa = (S) -propargyl glycine ((S) -propargyl glycine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (10)..(10)

<223> Xaa = 3-I-Tyr

<400> 23

Gly Xaa Cys Thr Asp Pro Arg Xaa Xaa Xaa Gln Cys Tyr

1 5 10

<210> 24

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> conotoxin peptide analog ie (Conotoxin peptide analog ie)

<220>

<221> DISULFID

<222> (2)..(8)

<220>

<221> MISC_FEATURE

<222> (3)..(12)

<223> (S) -5-azidovaline to form a triazole bridge between the 3-position and the 12-position of (S) -propargylglycine ((S) -5-Azidonorvaline at position 3 and (S) -propargylglycine at position 12 for a triazole bridge)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -5-azidovaline ((S) -5-azidonorrvaline)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (10)..(10)

<223> Xaa = 3-I-Tyr

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -propargyl glycine ((S) -propargyl glycine)

<400> 24

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Xaa Gln Xaa Tyr

1 5 10

<210> 25

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> intermediate conotoxin peptide analog Ye (intermediate conotoxin peptide analog Ye)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -5-azidovaline ((S) -5-azidonorrvaline)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (10)..(10)

<223> Xaa = 3-I-Tyr

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -propargyl glycine ((S) -propargyl glycine)

<400> 25

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Xaa Gln Xaa Tyr

1 5 10

<210> 26

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> intermediate conotoxin peptide analog ze (intermediate conotoxin peptide analog ze)

<220>

<221> DISULFID

<222> (2)..(8)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -5-azidovaline ((S) -5-azidonorrvaline)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (10)..(10)

<223> Xaa = 3-I-Tyr

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -propargyl glycine ((S) -propargyl glycine)

<400> 26

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Xaa Gln Xaa Tyr

1 5 10

<210> 27

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> conotoxin peptide analog if (Conotoxin peptide analog if)

<220>

<221> DISULFID

<222> (2)..(8)

<220>

<221> MISC_FEATURE

<222> (3)..(12)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -propargyl glycine ((S) -propargyl glycine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (10)..(10)

<223> Xaa = 3-I-Tyr

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S)-3-azido-alanine

<400> 27

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Xaa Gln Xaa Tyr

1 5 10

<210> 28

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> intermediate conotoxin peptide analog Yf (intermediate conotoxin peptide analog Yf)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -propargyl glycine ((S) -propargyl glycine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (10)..(10)

<223> Xaa = 3-I-Tyr

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -3-azidoalanine ((S) -3-azido-alanine)

<400> 28

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Xaa Gln Xaa Tyr

1 5 10

<210> 29

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> intermediate conotoxin peptide analog Zf (intermediate conotoxin peptide analog Zf)

<220>

<221> DISULFID

<222> (2)..(8)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -propargyl glycine ((S) -propargyl glycine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (10)..(10)

<223> Xaa = 3-I-Tyr

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -3-azidoalanine ((S) -3-azido-alanine)

<400> 29

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Xaa Gln Xaa Tyr

1 5 10

<210> 30

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> conotoxin peptide analog Ig (Conotoxin peptide analog Ig)

<220>

<221> DISULFID

<222> (2)..(8)

<220>

<221> MISC_FEATURE

<222> (3)..(12)

<223> (S) -Homopropargyl glycine at position 3 and (S) -5-azidovaline at position 12 forming a triazole bridge ((S) -Homopropargyl glycine at position 3 and (S) -5-azidinorvaline at position 12 form a triazole bridge)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -homopropargyl glycine ((S) -homopropagyl glycine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -5-azidovaline ((S) -5-azidonorrvaline)

<400> 30

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Trp Gln Xaa Tyr

1 5 10

<210> 31

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> intermediate conotoxin peptide analog Yg (intermediate conotoxin peptide analog Yg)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -homopropargyl glycine ((S) -homopropagyl glycine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -5-azidovaline ((S) -5-azidonorrvaline)

<400> 31

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Trp Gln Xaa Tyr

1 5 10

<210> 32

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> intermediate conotoxin peptide analog Zg (intermediate conotoxin peptide analog Zg)

<220>

<221> DISULFID

<222> (2)..(8)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -homopropargyl glycine ((S) -homopropagyl glycine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -5-azidovaline ((S) -5-azidonorrvaline)

<400> 32

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Trp Gln Xaa Tyr

1 5 10

<210> 33

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> conotoxin peptide analog ih (Conotoxin peptide analog ih)

<220>

<221> DISULFID

<222> (2)..(8)

<220>

<221> MISC_FEATURE

<222> (3)..(12)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -homopropargyl glycine ((S) -homopropagyl glycine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -gamma-azido-homoalanine ((S) -gamma-azido-homoalanine)

<400> 33

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Trp Gln Xaa Tyr

1 5 10

<210> 34

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> intermediate conotoxin peptide analog Yh (intermediate conotoxin peptide analog Yh)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -homopropargyl glycine ((S) -homopropagyl glycine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -homopropargylglycine ((S) -gamma-azido-homoalanine)

<400> 34

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Trp Gln Xaa Tyr

1 5 10

<210> 35

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> intermediate conotoxin peptide analog Zh (intermediate conotoxin peptide analog Zh)

<220>

<221> DISULFID

<222> (2)..(8)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -homopropargyl glycine ((S) -homopropagyl glycine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -homopropargylglycine ((S) -gamma-azido-homoalanine)

<400> 35

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Trp Gln Xaa Tyr

1 5 10

<210> 36

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> conotoxin peptide analog Ii (Conotoxin peptide analog Ii)

<220>

<221> DISULFID

<222> (2)..(8)

<220>

<221> MISC_FEATURE

<222> (3)..(12)

<223> (S) -Gamma-azidohomoalanine at position 3 and (S) -homopropargylglycine at position 12 form a triazole bridge ((S) -Gamma-azido-homoalanine at position 3 and (S) -homopropargylglycine at position 12 for a triazole bridge)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -gamma-azido-homoalanine ((S) -gamma-azido-homoalanine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -homopropargyl glycine ((S) -homopropagyl glycine)

<400> 36

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Trp Gln Xaa Tyr

1 5 10

<210> 37

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> intermediate conotoxin peptide analog Yi (intermediate conotoxin peptide analog Yi)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -gamma-azido-homoalanine ((S) -gamma-azido-homoalanine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -homopropargyl glycine ((S) -homopropagyl glycine)

<400> 37

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Trp Gln Xaa Tyr

1 5 10

<210> 38

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> intermediate conotoxin peptide analog Zi (intermediate conotoxin peptide analog Zi)

<220>

<221> DISULFID

<222> (2)..(8)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -gamma-azido-homoalanine ((S) -gamma-azido-homoalanine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -homopropargyl glycine ((S) -homopropagyl glycine)

<400> 38

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Trp Gln Xaa Tyr

1 5 10

<210> 39

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> conotoxin peptide analog Ij (Conotoxin peptide analog Ij)

<220>

<221> DISULFID

<222> (2)..(8)

<220>

<221> MISC_FEATURE

<222> (3)..(12)

<223> (S) -Bis-homopropargylglycine at position 3 and (S) -3-azidoalanine at position 12 form a triazole bridge ((S) -Bis-homopropargylglycine at position 3 and (S) -3-azido-alanine at position 12 for a triazole bridge)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -bis-homopropargylglycine ((S) -bis-homopropargyl glycine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -3-azido-alanine ((S) -3-azido-alanine)

<400> 39

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Trp Gln Xaa Tyr

1 5 10

<210> 40

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> intermediate conotoxin peptide analog Yj (intermediate conotoxin peptide analog Yj)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -bis-homopropargylglycine ((S) -bis-homopropargyl glycine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -3-azido-alanine ((S) -3-azido-alanine)

<400> 40

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Trp Gln Xaa Tyr

1 5 10

<210> 41

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> intermediate conotoxin peptide analog Zj (intermediate conotoxin peptide analog Zj)

<220>

<221> DISULFID

<222> (2)..(8)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -bis-homopropargylglycine ((S) -bis-homopropargyl glycine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -3-azido-alanine ((S) -3-azido-alanine)

<400> 41

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Trp Gln Xaa Tyr

1 5 10

<210> 42

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> conotoxin peptide analog Ik (Conotoxin peptide analog Ik)

<220>

<221> DISULFID

<222> (2)..(8)

<220>

<221> MISC_FEATURE

<222> (3)..(12)

<223> (S) -3-azidoalanine 3 rd position and (S) -bis-homopropargylglycine 12 th position form a triazole bridge ((S) -3-Azido-alanine at position 3 and (S) -bis-homopropargylglycine at position 12 form a triazole bridge)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -3-azido-alanine ((S) -3-azido-alanine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -bis-homopropargylglycine ((S) -bis-homopropargyl glycine)

<400> 42

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Trp Gln Xaa Tyr

1 5 10

<210> 43

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> intermediate conotoxin peptide analog Yk (intermediate conotoxin peptide analog Yk)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -3-azido-alanine ((S) -3-azido-alanine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -bis-homopropargylglycine ((S) -bis-homopropargyl glycine)

<400> 43

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Trp Gln Xaa Tyr

1 5 10

<210> 44

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> intermediate conotoxin peptide analog Zk (intermediate conotoxin peptide analog Zk)

<220>

<221> DISULFID

<222> (2)..(8)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -3-azido-alanine ((S) -3-azido-alanine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -bis-homopropargylglycine ((S) -bis-homopropargyl glycine)

<400> 44

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Trp Gln Xaa Tyr

1 5 10

<210> 45

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> conotoxin peptide analog il (Conotoxin peptide analog il)

<220>

<221> DISULFID

<222> (2)..(8)

<220>

<221> MISC_FEATURE

<222> (3)..(12)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -propargyl glycine ((S) -propargyl glycine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -5-azidovaline ((S) -5-azidonorrvaline)

<400> 45

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Trp Gln Xaa Phe

1 5 10

<210> 46

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> intermediate conotoxin peptide analog Yl (intermediate conotoxin peptide analog Yl)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -propargyl glycine ((S) -propargyl glycine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -5-azidovaline ((S) -5-azidonorrvaline)

<400> 46

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Trp Gln Xaa Phe

1 5 10

<210> 47

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> intermediate conotoxin peptide analog ZL (intermediate conotoxin peptide analog ZL)

<220>

<221> DISULFID

<222> (2)..(8)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -propargyl glycine ((S) -propargyl glycine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -5-azidovaline ((S) -5-azidonorrvaline)

<400> 47

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Trp Gln Xaa Phe

1 5 10

<210> 48

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> conotoxin peptide analog im (Conotoxin peptide analog im)

<220>

<221> DISULFID

<222> (2)..(8)

<220>

<221> MISC_FEATURE

<222> (3)..(12)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -propargyl glycine ((S) -propargyl glycine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -5-azidovaline ((S) -5-azidonorrvaline)

<220>

<221> MOD_RES

<222> (13)..(13)

<223> Xaa = D-Tyr

<400> 48

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Trp Gln Xaa Xaa

1 5 10

<210> 49

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> intermediate conotoxin peptide analog ym (intermediate conotoxin peptide analog ym)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -propargyl glycine ((S) -propargyl glycine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -5-azidovaline ((S) -5-azidonorrvaline)

<220>

<221> MOD_RES

<222> (13)..(13)

<223> Xaa = D-Tyr

<400> 49

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Trp Gln Xaa Xaa

1 5 10

<210> 50

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> intermediate conotoxin peptide analog zm (intermediate conotoxin peptide analog zm)

<220>

<221> DISULFID

<222> (2)..(8)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -propargyl glycine ((S) -propargyl glycine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -5-azidovaline ((S) -5-azidonorrvaline)

<220>

<221> MOD_RES

<222> (13)..(13)

<223> Xaa = D-Tyr

<400> 50

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Trp Gln Xaa Xaa

1 5 10

<210> 51

<211> 14

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> conotoxin peptide analog in (Conotoxin peptide analog in)

<220>

<221> DISULFID

<222> (2)..(8)

<220>

<221> MISC_FEATURE

<222> (3)..(12)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -propargyl glycine ((S) -propargyl glycine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -5-azidovaline ((S) -5-azidonorrvaline)

<220>

<221> MOD_RES

<222> (14)..(14)

<223> Xaa = N-Me-Gly

<400> 51

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Trp Gln Xaa Tyr Xaa

1 5 10

<210> 52

<211> 14

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> intermediate conotoxin peptide analog Yn (intermediate conotoxin peptide analog Yn)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -propargyl glycine ((S) -propargyl glycine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa c = (S) -5-azidovaline ((S) -5-azidonorrvaline)

<220>

<221> MOD_RES

<222> (14)..(14)

<223> Xaa = N-Me-Gly

<400> 52

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Trp Gln Xaa Tyr Xaa

1 5 10

<210> 53

<211> 14

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> intermediate conotoxin peptide analog Zn (intermediate conotoxin peptide analog Zn)

<220>

<221> DISULFID

<222> (2)..(8)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -propargyl glycine ((S) -propargyl glycine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -5-azidovaline ((S) -5-azidonorrvaline)

<220>

<221> MOD_RES

<222> (14)..(14)

<223> Xaa = N-Me-Gly

<400> 53

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Trp Gln Xaa Tyr Xaa

1 5 10

<210> 54

<211> 14

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> conotoxin peptide analog Io (Conotoxin peptide analog Io)

<220>

<221> DISULFID

<222> (2)..(8)

<220>

<221> MISC_FEATURE

<222> (3)..(12)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -propargyl glycine ((S) -propargyl glycine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -5-azidovaline ((S) -5-azidonorrvaline)

<220>

<221> MOD_RES

<222> (14)..(14)

<223> Xaa = D-Tyr

<400> 54

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Trp Gln Xaa Tyr Xaa

1 5 10

<210> 55

<211> 14

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> intermediate conotoxin peptide analog yo (intermediate conotoxin peptide analog yo)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -propargyl glycine ((S) -propargyl glycine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -5-azidovaline ((S) -5-azidonorrvaline)

<220>

<221> MOD_RES

<222> (14)..(14)

<223> Xaa = D-Tyr

<400> 55

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Trp Gln Xaa Tyr Xaa

1 5 10

<210> 56

<211> 14

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> intermediate conotoxin peptide analog Zo (intermediate conotoxin peptide analog Zo)

<220>

<221> DISULFID

<222> (2)..(8)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -propargyl glycine ((S) -propargyl glycine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -5-azidovaline ((S) -5-azidonorrvaline)

<220>

<221> MOD_RES

<222> (14)..(14)

<223> Xaa = D-Tyr

<400> 56

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Trp Gln Xaa Tyr Xaa

1 5 10

<210> 57

<211> 14

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> conotoxin peptide analog ip (Conotoxin peptide analog ip)

<220>

<221> DISULFID

<222> (2)..(8)

<220>

<221> MISC_FEATURE

<222> (3)..(12)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -propargyl glycine ((S) -propargyl glycine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -5-azidovaline ((S) -5-azidonorrvaline)

<220>

<221> MOD_RES

<222> (14)..(14)

<223> Xaa = N-Me-Tyr

<400> 57

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Trp Gln Xaa Tyr Xaa

1 5 10

<210> 58

<211> 14

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> intermediate conotoxin peptide analog Yp (intermediate conotoxin peptide analog Yp)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -propargyl glycine ((S) -propargyl glycine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -5-azidovaline ((S) -5-azidonorrvaline)

<220>

<221> MOD_RES

<222> (14)..(14)

<223> Xaa = N-Me-Tyr

<400> 58

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Trp Gln Xaa Tyr Xaa

1 5 10

<210> 59

<211> 14

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> intermediate conotoxin peptide analog zp (intermediate conotoxin peptide analog zp)

<220>

<221> DISULFID

<222> (2)..(8)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -propargyl glycine ((S) -propargyl glycine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -5-azidovaline ((S) -5-azidonorrvaline)

<220>

<221> MOD_RES

<222> (14)..(14)

<223> Xaa = N-Me-Tyr

<400> 59

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Trp Gln Xaa Tyr Xaa

1 5 10

<210> 60

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> conotoxin peptide analog Iq (Conotoxin peptide analog Iq)

<220>

<221> DISULFID

<222> (2)..(8)

<220>

<221> MISC_FEATURE

<222> (3)..(12)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -propargyl glycine ((S) -propargyl glycine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -5-azidovaline ((S) -5-azidonorrvaline)

<400> 60

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Trp Gln Xaa

1 5 10

<210> 61

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> intermediate conotoxin peptide analog Yq (intermediate conotoxin peptide analog Yq)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -propargyl glycine ((S) -propargyl glycine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -5-azidovaline ((S) -5-azidonorrvaline)

<400> 61

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Trp Gln Xaa

1 5 10

<210> 62

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> intermediate conotoxin peptide analog Zq (intermediate conotoxin peptide analog Zq)

<220>

<221> DISULFID

<222> (2)..(8)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -propargyl glycine ((S) -propargyl glycine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -5-azidovaline ((S) -5-azidonorrvaline)

<400> 62

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Trp Gln Xaa

1 5 10

<210> 63

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> conotoxin peptide analog Ir (Conotoxin peptide analog Ir)

<220>

<221> DISULFID

<222> (2)..(8)

<220>

<221> MISC_FEATURE

<222> (3)..(12)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -propargyl glycine ((S) -propargyl glycine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -5-azidovaline ((S) -5-azidonorrvaline)

<220>

<221> MOD_RES

<222> (13)..(13)

<223> Xaa = N-Me-Tyr

<400> 63

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Trp Gln Xaa Xaa

1 5 10

<210> 64

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> intermediate conotoxin peptide analog Yr (intermediate conotoxin peptide analog Yr)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -propargyl glycine ((S) -propargyl glycine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -5-azidovaline ((S) -5-azidonorrvaline)

<220>

<221> MOD_RES

<222> (13)..(13)

<223> Xaa = N-Me-Tyr

<400> 64

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Trp Gln Xaa Xaa

1 5 10

<210> 65

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> intermediate conotoxin peptide analog Zr (intermediate conotoxin peptide analog Zr)

<220>

<221> DISULFID

<222> (2)..(8)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -propargyl glycine ((S) -propargyl glycine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -5-azidovaline ((S) -5-azidonorrvaline)

<220>

<221> MOD_RES

<222> (13)..(13)

<223> Xaa = N-Me-Tyr

<400> 65

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Trp Gln Xaa Xaa

1 5 10

<210> 66

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> peptide analogues of conotoxin is (Conotoxin peptide analogues is)

<220>

<221> DISULFID

<222> (2)..(8)

<220>

<221> MISC_FEATURE

<222> (3)..(12)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -propargyl glycine ((S) -propargyl glycine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -5-azidovaline ((S) -5-azidonorrvaline)

<220>

<221> MOD_RES

<222> (13)..(13)

<223> Xaa = D-Arg

<400> 66

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Trp Gln Xaa Xaa

1 5 10

<210> 67

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> intermediate conotoxin peptide analogues Ys (intermediate conotoxin peptide analogues Ys)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -propargyl glycine ((S) -propargyl glycine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -5-azidovaline ((S) -5-azidonorrvaline)

<220>

<221> MOD_RES

<222> (13)..(13)

<223> Xaa = D-Arg

<400> 67

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Trp Gln Xaa Xaa

1 5 10

<210> 68

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> intermediate conotoxin peptide analogues Zs (intermediate conotoxin peptide analogues Zs)

<220>

<221> DISULFID

<222> (2)..(8)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -propargyl glycine ((S) -propargyl glycine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -5-azidovaline ((S) -5-azidonorrvaline)

<220>

<221> MOD_RES

<222> (13)..(13)

<223> Xaa = D-Arg

<400> 68

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Trp Gln Xaa Xaa

1 5 10

<210> 69

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> conotoxin peptide analog it (Conotoxin peptide analog it)

<220>

<221> DISULFID

<222> (2)..(8)

<220>

<221> MISC_FEATURE

<222> (3)..(12)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -propargyl glycine ((S) -propargyl glycine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -5-azidovaline ((S) -5-azidonorrvaline)

<220>

<221> MOD_RES

<222> (13)..(13)

<223> Xaa = N-Me-D-Tyr

<400> 69

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Trp Gln Xaa Xaa

1 5 10

<210> 70

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> intermediate conotoxin peptide analog Yt (intermediate conotoxin peptide analog Yt)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -propargyl glycine ((S) -propargyl glycine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -5-azidovaline ((S) -5-azidonorrvaline)

<220>

<221> MOD_RES

<222> (13)..(13)

<223> Xaa = N-Me-D-Tyr

<400> 70

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Trp Gln Xaa Xaa

1 5 10

<210> 71

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> intermediate conotoxin peptide analog Zt (intermediate conotoxin peptide analog Zt)

<220>

<221> DISULFID

<222> (2)..(8)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -propargyl glycine ((S) -propargyl glycine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -5-azidovaline ((S) -5-azidonorrvaline)

<220>

<221> MOD_RES

<222> (13)..(13)

<223> Xaa = N-Me-D-Tyr

<400> 71

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Trp Gln Xaa Xaa

1 5 10

<210> 72

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> conotoxin peptide analog iu (Conotoxin peptide analog iu)

<220>

<221> DISULFID

<222> (2)..(8)

<220>

<221> MISC_FEATURE

<222> (3)..(12)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -propargyl glycine ((S) -propargyl glycine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -5-azidovaline ((S) -5-azidonorrvaline)

<220>

<221> MOD_RES

<222> (13)..(13)

<223> Xaa = beta-Tyr

<400> 72

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Trp Gln Xaa Xaa

1 5 10

<210> 73

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> intermediate conotoxin peptide analog yu (intermediate conotoxin peptide analog yu)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -propargyl glycine ((S) -propargyl glycine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -5-azidovaline ((S) -5-azidonorrvaline)

<220>

<221> MOD_RES

<222> (13)..(13)

<223> Xaa = beta-Tyr

<400> 73

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Trp Gln Xaa Xaa

1 5 10

<210> 74

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> intermediate conotoxin peptide analog Zu (intermediate conotoxin peptide analog Zu)

<220>

<221> DISULFID

<222> (2)..(8)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -propargyl glycine ((S) -propargyl glycine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -5-azidovaline ((S) -5-azidonorrvaline)

<220>

<221> MOD_RES

<222> (13)..(13)

<223> Xaa = beta-Tyr

<400> 74

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Trp Gln Xaa Xaa

1 5 10

<210> 75

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> conotoxin peptide analog iv (Conotoxin peptide analog iv)

<220>

<221> DISULFID

<222> (2)..(8)

<220>

<221> MISC_FEATURE

<222> (3)..(12)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -propargyl glycine ((S) -propargyl glycine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -5-azidovaline ((S) -5-azidonorrvaline)

<220>

<221> MOD_RES

<222> (13)..(13)

<223> Xaa = N-Me-Arg

<400> 75

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Trp Gln Xaa Xaa

1 5 10

<210> 76

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> intermediate conotoxin peptide analog Yv (intermediate conotoxin peptide analog Yv)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -propargyl glycine ((S) -propargyl glycine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -5-azidovaline ((S) -5-azidonorrvaline)

<220>

<221> MOD_RES

<222> (13)..(13)

<223> Xaa = N-Me-Arg

<400> 76

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Trp Gln Xaa Xaa

1 5 10

<210> 77

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> intermediate conotoxin peptide analog Zv (intermediate conotoxin peptide analog Zv)

<220>

<221> DISULFID

<222> (2)..(8)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -propargyl glycine ((S) -propargyl glycine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -5-azidovaline ((S) -5-azidonorrvaline)

<220>

<221> MOD_RES

<222> (13)..(13)

<223> Xaa = N-Me-Arg

<400> 77

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Trp Gln Xaa Xaa

1 5 10

<210> 78

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> intermediate conotoxin peptide analog Iw (Conotoxin peptide analog Iw)

<220>

<221> DISULFID

<222> (2)..(8)

<220>

<221> MISC_FEATURE

<222> (3)..(12)

<223> the 3 rd position of glycine and the 12 th position of lysine form a lactam bridge (Glu at position 3 and Lys at position 12)

form a lactam bridge)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (10)..(10)

<223> Xaa = 3-I-Tyr

<400> 78

Gly Cys Glu Thr Asp Pro Arg Cys Xaa Xaa Gln Lys Tyr

1 5 10

<210> 79

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Intermediate X1 (Intermediate X1)

<220>

<221> MISC_FEATURE

<222> (13)..(13)

<223> attachment of tyrosine to Rink amide MBHA resin at position 13 (Tyr at position 13 is attached to a. alpha

Rink amide MBHA resin)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (10)..(10)

<223> Xaa = 3-I-Tyr

<400> 79

Gly Cys Glu Thr Asp Pro Arg Cys Xaa Xaa Gln Lys Tyr

1 5 10

<210> 80

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Intermediate X2 (Intermediate X2)

<220>

<221> MISC_FEATURE

<222> (3)..(12)

form a lactam bridge)

<220>

<221> MISC_FEATURE

<222> (13)..(13)

Rink amide MBHA resin)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (10)..(10)

<223> Xaa = 3-I-Tyr

<400> 80

Gly Cys Glu Thr Asp Pro Arg Cys Xaa Xaa Gln Lys Tyr

1 5 10

<210> 81

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Intermediate X3 (Intermediate X3)

<220>

<221> MISC_FEATURE

<222> (3)..(12)

form a lactam bridge)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (10)..(10)

<223> Xaa = 3-I-Tyr

<400> 81

Gly Cys Glu Thr Asp Pro Arg Cys Xaa Xaa Gln Lys Tyr

1 5 10

<210> 82

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> intermediate conotoxin peptide analog Ix (Conotoxin peptide analog Ix)

<220>

<221> DISULFID

<222> (2)..(8)

<220>

<221> MISC_FEATURE

<222> (3)..(12)

form a lactam bridge)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (10)..(10)

<223> Xaa = 3-I-Tyr

<400> 82

Gly Cys Lys Thr Asp Pro Arg Cys Xaa Xaa Gln Glu Tyr

1 5 10

<210> 83

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> PEGylated conotoxin peptide analog IIa (PEGylated conotoxin peptide analog IIa)

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> Glycine attachment to a Linear 30 kDa mPEG Polymer (Gly at position 1 is attached to a linear) Via a pentanoic acid linker

30 kDa mPEG polymer via a valerate linker)

<220>

<221> DISULFID

<222> (2)..(8)

<220>

<221> MISC_FEATURE

<222> (3)..(12)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -propargyl glycine ((S) -propargyl glycine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -5-azidovaline ((S) -5-azidonorrvaline)

<400> 83

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Trp Gln Xaa Tyr

1 5 10

<210> 84

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> PEGylated conotoxin peptide analog IVa (PEGylated conotoxin peptide analog IVa)

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> Glycine attached to a linear 30 kDa mPEG Polymer at position 1 through a butene linker (Gly at position 1 is attached to a linear 30 kDa mPEG Polymer via a butyl linker)

<220>

<221> DISULFID

<222> (2)..(8)

<220>

<221> MISC_FEATURE

<222> (3)..(12)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -propargyl glycine ((S) -propargyl glycine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -5-azidovaline ((S) -5-azidonorrvaline)

<400> 84

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Trp Gln Xaa Tyr

1 5 10

<210> 85

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> PEGylated conotoxin peptide analog Va (PEGylated conotoxin peptide analog Va)

<220>

<221> MOD_RES

<222> (1)..(1)

<223> ACETYLATION (acetalation)

<220>

<221> MISC_FEATURE

<222> (13)..(13)

<223> Glycine 13 position connected to a linear 30 kDa mPEG polymer (Tyr at position 13 is attached to a linear 30 kDa mPEG polymer via an amidopropyl linker)

<220>

<221> DISULFID

<222> (2)..(8)

<220>

<221> MISC_FEATURE

<222> (3)..(12)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -propargyl glycine ((S) -propargyl glycine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -5-azidovaline ((S) -5-azidonorrvaline)

<400> 85

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Trp Gln Xaa Tyr

1 5 10

<210> 86

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> PEGylated conotoxin peptide analog via (PEGylated conotoxin peptide analog via)

<220>

<221> MOD_RES

<222> (1)..(1)

<223> ACETYLATION (acetalation)

<220>

<221> MISC_FEATURE

<222> (7)..(7)

<223> arginine was attached to a linear 30 kDa mPEG polymer at position 7 via a pentanoic acid linker (Arg at position 7 is attached to a linear 30 kDa mPEG polymer via a equivalent linker)

<220>

<221> DISULFID

<222> (2)..(8)

<220>

<221> MISC_FEATURE

<222> (3)..(12)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -propargyl glycine ((S) -propargyl glycine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -5-azidovaline ((S) -5-azidonorrvaline)

<400> 86

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Trp Gln Xaa Tyr

1 5 10

<210> 87

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> PEGylated conotoxin peptide analog VII (PEGylated conotoxin peptide analog VII)

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> Glycine attached to 20 kDa mPEG Polymer linearly through a pentanoic acid linker at position 1 (Gly at position 1 is attached to a linear 20 kDa mPEG Polymer via a equivalent linker)

<220>

<221> DISULFID

<222> (2)..(8)

<220>

<221> DISULFID

<222> (3)..(12)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (10)..(10)

<223> Xaa = 3-I-Tyr

<400> 87

Gly Cys Cys Thr Asp Pro Arg Cys Xaa Xaa Gln Cys Tyr

1 5 10

<210> 88

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> PEGylated conotoxin peptide analog VIII (PEGylated conotoxin peptide analog VIII)

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> Glycine attached to 20 kDa mPEG Polymer linearly through butene linker at position 1 (Gly at position 1 is attached to a linear 20 kDa mPEG Polymer via a butyl linker)

<220>

<221> DISULFID

<222> (2)..(8)

<220>

<221> DISULFID

<222> (3)..(12)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (10)..(10)

<223> Xaa = 3-I-Tyr

<400> 88

Gly Cys Cys Thr Asp Pro Arg Cys Xaa Xaa Gln Cys Tyr

1 5 10

<210> 89

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> PEGylated conotoxin peptide analog IX (PEGylated conotoxin peptide analog IX)

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> 20 kDa mPEG Polymer attached to a branched chain at the 1-position of Glycine by a propylamine linker (Gly at position 1 is attached to a branched 20 kDa mPEG polymer via a propyl amine linker)

<220>

<221> DISULFID

<222> (2)..(8)

<220>

<221> DISULFID

<222> (3)..(12)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (10)..(10)

<223> Xaa = 3-I-Tyr

<400> 89

Gly Cys Cys Thr Asp Pro Arg Cys Xaa Xaa Gln Cys Tyr

1 5 10

<210> 90

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> PEGylated conotoxin peptide analog X (PEGylated conotoxin peptide analog X)

<220>

<221> DISULFID

<222> (2)..(8)

<220>

<221> DISULFID

<222> (3)..(12)

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> Glycine 20 kDa mPEG Polymer attached to a branched chain at position 1 through a carbamate linker (Gly at position 1 is attached to a branched 20 kDa mPEG Polymer via a carbamate linker)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (10)..(10)

<223> Xaa = 3-I-Tyr

<400> 90

Gly Cys Cys Thr Asp Pro Arg Cys Xaa Xaa Gln Cys Tyr

1 5 10

<210> 91

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> PEGylated conotoxin peptide analog XI (PEGylated conotoxin peptide analog XI)

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> Glycine attached to a Linear 30 kDa mPEG Polymer at position 1 through an Aminovaleric acid linker (Gly at position 1 is attached to a linear 30 kDa mPEG Polymer via a equivalent linker)

<220>

<221> DISULFID

<222> (2)..(8)

<220>

<221> DISULFID

<222> (3)..(12)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (10)..(10)

<223> Xaa = 3-I-Tyr

<400> 91

Gly Cys Cys Thr Asp Pro Arg Cys Xaa Xaa Gln Cys Tyr

1 5 10

<210> 92

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> PEGylated conotoxin peptide analog XII (PEGylated conotoxin peptide analog XII)

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> Glycine attached to a linear 40 kDa mPEG Polymer at position 1 through an aminopentanoic acid linker (Gly at position 1 is attached to a linear 40 kDa mPEG Polymer via a equivalent linker)

<220>

<221> DISULFID

<222> (2)..(8)

<220>

<221> DISULFID

<222> (3)..(12)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (10)..(10)

<223> Xaa = 3-I-Tyr

<400> 92

Gly Cys Cys Thr Asp Pro Arg Cys Xaa Xaa Gln Cys Tyr

1 5 10

<210> 93

<211> 14

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Compound I (formula I)

<220>

<221> DISULFID

<222> (2)..(8)

<220>

<221> MISC_FEATURE

<222> (3)..(12)

<223> Xaa3 and Xaa12 form a triazole bridge (Xaa3 and Xaa12 form a triazine bridge)

<220>

<221> VARIANT

<222> (3)..(3)

<223> Xaa = an unnatural amino acid (an unnatural amino acid that forms a triazole bridge with the residue at position 12 a

triazole bridge with the residue at position 12)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> VARIANT

<222> (12)..(12)

<223> Xaa = an unnatural amino acid which forms a triazole bridge with the residue at position 3 (an unnatural amino acid that is form a triazine bridge)

bridge with the residue at position 3)

<220>

<221> VARIANT

<222> (13)..(13)

<223> Xaa can be Tyr, Phe, Trp, D-Tyr, D-Phe or D-Trp

<220>

<221> VARIANT

<222> (14)..(14)

<223> Xaa can be N-Me-Gly, D-Tyr, N-Me-Tyr or absent

<400> 93

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Trp Gln Xaa Xaa Xaa

1 5 10

<210> 94

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Formula Ia

<220>

<221> DISULFID

<222> (2)..(8)

<220>

<221> MISC_FEATURE

<222> (3)..(12)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -propargyl glycine ((S) -propargyl glycine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -5-azidovaline ((S) -5-azidonorrvaline)

<400> 94

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Trp Gln Xaa Tyr

1 5 10

<210> 95

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> PEGylated conotoxin peptide analog IIg (PEGylated conotoxin peptide analog IIg)

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<223> Glycine attached to a Linear 30 kDa mPEG Polymer at position 1 through a pentanoic acid linker (Gly at position 1 is attached to a linear 30 kDa mPEG Polymer via a equivalent linker)

<220>

<221> DISULFID

<222> (2)..(8)

<220>

<221> MISC_FEATURE

<222> (3)..(12)

<223> (S) -homopropargylglycine at position 3 and (S) -5-azidovaline at position 12 form a triazole bridge ((S) -homopropargylglycine at position 3 and (S) -5-azidinorvaline at position 12 form a triazole bridge)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -homopropargyl glycine ((S) -homopropagyl glycine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -5-azidovaline ((S) -5-azidonorrvaline)

<400> 95

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Trp Gln Xaa Tyr

1 5 10

<210> 96

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> PEGylated conotoxin peptide analog IIh (PEGylated conotoxin peptide analog IIh)

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<220>

<221> DISULFID

<222> (2)..(8)

<220>

<221> MISC_FEATURE

<222> (3)..(12)

<223> (S) -Homopropargyl glycine at position 3 and (S) - γ -azidohomoalanine at position 12 form a triazole bridge ((S) -Homopropargyl glycine at position 3 and (S) -gamma-azido-homoalanine at position 12 form a triazole bridge)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -homopropargyl glycine ((S) -homopropagyl glycine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -gamma-azido-homoalanine ((S) -gamma-azido-homoalanine)

<400> 96

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Trp Gln Xaa Tyr

1 5 10

<210> 97

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> PEGylated conotoxin peptide analog IIi (PEGylated conotoxin peptide analog IIi)

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<220>

<221> DISULFID

<222> (2)..(8)

<220>

<221> MISC_FEATURE

<222> (3)..(12)

<223> (S) -Gamma-azidohomoalanine at position 3 and (S) -homopropynyl glycine at position 12 form a triazole bridge ((S) -Gamma-azido-homoalanine at position 3 and (S) -homopropargyl glycine at position 12 for a triazole bridge)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -gamma-azido-homoalanine ((S) -gamma-azido-homoalanine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -homopropargyl glycine ((S) -homopropagyl glycine)

<400> 97

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Trp Gln Xaa Tyr

1 5 10

<210> 98

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> PEGylated conotoxin peptide analog IIk (PEGylated conotoxin peptide analog IIk)

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<220>

<221> DISULFID

<222> (2)..(8)

<220>

<221> MISC_FEATURE

<222> (3)..(12)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -3-azido-alanine ((S) -3-azido-alanine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -bis-homopropargylglycine ((S) -bis-homopropargyl glycine)

<400> 98

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Trp Gln Xaa Tyr

1 5 10

<210> 99

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> PEGylated conotoxin peptide analog IIl (PEGylated conotoxin peptide analog IIl)

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<220>

<221> DISULFID

<222> (2)..(8)

<220>

<221> MISC_FEATURE

<222> (3)..(12)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -propargyl glycine ((S) -propargyl glycine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -5-azidovaline ((S) -5-azidonorrvaline)

<400> 99

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Trp Gln Xaa Phe

1 5 10

<210> 100

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> PEGylated conotoxin peptide analog IIm (PEGylated conotoxin peptide analog IIm)

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<220>

<221> DISULFID

<222> (2)..(8)

<220>

<221> MISC_FEATURE

<222> (3)..(12)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -propargyl glycine ((S) -propargyl glycine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -5-azidovaline ((S) -5-azidonorrvaline)

<220>

<221> MOD_RES

<222> (13)..(13)

<223> Xaa = D-Tyr

<400> 100

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Trp Gln Xaa Xaa

1 5 10

<210> 101

<211> 14

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> PEGylated conotoxin peptide analog IIn (PEGylated conotoxin peptide analog IIn)

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<220>

<221> DISULFID

<222> (2)..(8)

<220>

<221> MISC_FEATURE

<222> (3)..(12)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -propargyl glycine ((S) -propargyl glycine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -5-azidovaline ((S) -5-azidonorrvaline)

<220>

<221> MOD_RES

<222> (14)..(14)

<223> Xaa = N-Me-Gly

<400> 101

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Trp Gln Xaa Tyr Xaa

1 5 10

<210> 102

<211> 14

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> PEGylated conotoxin peptide analog IIo (PEGylated conotoxin peptide analog IIo)

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<220>

<221> DISULFID

<222> (2)..(8)

<220>

<221> MISC_FEATURE

<222> (3)..(12)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -propargyl glycine ((S) -propargyl glycine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -5-azidovaline ((S) -5-azidonorrvaline)

<220>

<221> MOD_RES

<222> (14)..(14)

<223> Xaa = D-Tyr

<400> 102

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Trp Gln Xaa Tyr Xaa

1 5 10

<210> 103

<211> 14

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> PEGylated conotoxin peptide analog IIp (PEGylated conotoxin peptide analog IIp)

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<220>

<221> DISULFID

<222> (2)..(8)

<220>

<221> MISC_FEATURE

<222> (3)..(12)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -propargyl glycine ((S) -propargyl glycine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -5-azidovaline ((S) -5-azidonorrvaline)

<220>

<221> MOD_RES

<222> (14)..(14)

<223> Xaa = N-Me-Tyr

<400> 103

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Trp Gln Xaa Tyr Xaa

1 5 10

<210> 104

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Formula Ib

<220>

<221> DISULFID

<222> (2)..(8)

<220>

<221> MISC_FEATURE

<222> (3)..(12)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -propargyl glycine ((S) -propargyl glycine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (10)..(10)

<223> Xaa = 3-I-Tyr

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -5-azidovaline ((S) -5-azidonorrvaline)

<400> 104

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Xaa Gln Xaa Tyr

1 5 10

<210> 105

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> PEGylated conotoxin peptide analog IIb (PEGylated conotoxin peptide analog IIb)

<220>

<221> MISC_FEATURE

<222> (1)..(1)

<220>

<221> DISULFID

<222> (2)..(8)

<220>

<221> MISC_FEATURE

<222> (3)..(12)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -propargyl glycine ((S) -propargyl glycine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (10)..(10)

<223> Xaa = 3-I-Tyr

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -5-azidovaline ((S) -5-azidonorrvaline)

<400> 105

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Xaa Gln Xaa Tyr

1 5 10

<210> 106

<211> 14

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Intermediate conotoxin peptide analog

<220>

<221> DISULFID

<222> (2)..(8)

<220>

<221> MISC_FEATURE

<222> (3)..(12)

<220>

<221> MISC_FEATURE

<222> (3)..(12)

<223> When Xaa3 is (S) -propargylglycine, (S) -homopropargylglycine or (S) -homopropargylglycine, Xaa12 is (S) -homopropargylglycine or (S) -5-azidovaline (When Xaa3 is (S) -propargyl glycine, (S) -homopropargyl glycine, or (S) -homopropargyl glycine, Xaa12 is (S) -homopropargyl glycine or (S) -5-azindonorvaline)

<220>

<221> MISC_FEATURE

<222> (3)..(12)

<223> When Xaa3 is (S) -3-azido-alanine, (S) - γ -azido-homoalanine or (S) -5-azidovaline, Xa12 is (S) -homopropargylglycine or (S) -bis-homopropargylglycine (When Xaa3 is (S) -3-azido-alanine, (S) -gamma-azido-homoalanine, or (S) -5-azido norvaline, Xa12 is (S) -homopro-peptidyl glycine or (S) -bis-homopro-peptidyl valine)

<220>

<221> VARIANT

<222> (3)..(3)

<223> Xaa can be (S) -propargylglycine, (S) -3-azido-alanine, (S) -homopropargylglycine, (S) - γ -azido-homoalanine, (S) -5-azidovaline or (S) -bis-homopropargylglycine (Xaa can be (S) -propynyl glycine, (S) -3-azido-alanine, (S) -homopropynyl glycine, (S) -gamma-azido-homoalanine, (S) -5-azido-homoalanine or (S) -bis-homopropynyl glycine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> VARIANT

<222> (12)..(12)

<223> Xaa can be (S) -homopropargylglycine, (S) - γ -azidohomoalanine, (S) -5-azidonorvaline or (S) -bis-homopropargylglycine (Xaa can (S) -homoprolylglycine, (S) -gamma-azido-homoalanine, (S) -5-azidinorvaline or (S) -bis-homoprolylglycine)

<220>

<221> VARIANT

<222> (13)..(13)

<223> Xaa can be Tyr, Phe, Trp, D-Tyr, D-Phe or D-Trp

<220>

<221> VARIANT

<222> (14)..(14)

<223> Xaa can be N-Me-Gly, D-Tyr, N-Me-Tyr or absent

<400> 106

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Trp Gln Xaa Xaa Xaa

1 5 10

<210> 107

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Intermediate conotoxin peptide analog

<220>

<221> MOD_RES

<222> (3)..(3)

<223> Xaa = (S) -propargyl glycine ((S) -propargyl glycine)

<220>

<221> MOD_RES

<222> (9)..(9)

<223> Xaa = Citrulline (Citruline)

<220>

<221> MOD_RES

<222> (10)..(10)

<223> Xaa = 3-I-Tyr

<220>

<221> MOD_RES

<222> (12)..(12)

<223> Xaa = (S) -5-azidovaline ((S) -5-azidonorrvaline)

<400> 107

Gly Cys Xaa Thr Asp Pro Arg Cys Xaa Xaa Gln Xaa Tyr

1 5 10

Claims

1. A conotoxin peptide analog of formula (I) (SEQ ID NO: 93):

or a pharmaceutically acceptable salt thereof,

wherein

2. A conotoxin peptide analog or pharmaceutically acceptable salt according to claim 1, wherein the triazole bridge is

Wherein the single wave line

C representing said triazole bridge and said conotoxin peptide analogue¹Carbon tie points, and double wave lines

C representing said triazole bridge and said conotoxin peptide analogue²A point of attachment for carbon; and wherein x is 1, 2, 3 or 4; and y is 2, 3 or 4.

3. A conotoxin peptide analog or pharmaceutically acceptable salt according to claim 2, wherein the triazole bridge is

4. A conotoxin peptide analog or pharmaceutically acceptable salt according to claim 2, wherein the triazole bridge is

5. A conotoxin peptide analog or pharmaceutically acceptable salt according to any one of claims 2-4, wherein x is 1, 2, or 3.

6. A conotoxin peptide analog or pharmaceutically acceptable salt according to claim 5, wherein x is 1.

7. A conotoxin peptide analog or pharmaceutically acceptable salt according to any one of claims 2-6, wherein y is 2 or 3.

8. A conotoxin peptide analog or pharmaceutically acceptable salt according to claim 7, wherein y is 3.

9. A conotoxin peptide analog or pharmaceutically acceptable salt according to claim 2, wherein x is 1, 2 or 3 and y is 2 or 3.

10. A conotoxin peptide analog or pharmaceutically acceptable salt according to claim 3, wherein x is 1 and y is 3.

11. A conotoxin peptide analog or pharmaceutically acceptable salt according to claim 3, wherein x is 2 and y is 3.

12. A conotoxin peptide analog or pharmaceutically acceptable salt according to claim 3, wherein x is 2 and y is 2.

13. A conotoxin peptide analog or pharmaceutically acceptable salt according to claim 4, wherein x is 2 and y is 2.

14. A conotoxin peptide analog or pharmaceutically acceptable salt according to claim 4, wherein x is 1 and y is 3.

15. A conotoxin peptide analog or pharmaceutically acceptable salt 0 of claim 10, wherein the triazole bridge is

Wherein the single wave line

C representing said triazole bridge and said conotoxin peptide analogue²The point of attachment of the carbon.

16. A conotoxin peptide analog or pharmaceutically acceptable salt according to any one of claims 1-15, wherein X is X_AA ¹。

17. A conotoxin peptide analog or pharmaceutically acceptable salt according to any one of claims 1-15, wherein X is X_AA ¹X_AA ²。

18. A conotoxin peptide analog or pharmaceutically acceptable salt according to claim 16 or 17, wherein X_AA ¹Selected from Tyr, D-Tyr and Phe.

19. A conotoxin peptide analog or pharmaceutically acceptable salt according to any one of claims 1-16, wherein X is Tyr.

20. A conotoxin peptide analog or pharmaceutically acceptable salt thereof of any one of claims 1-19, wherein the C-terminus of the conotoxin peptide analog is OH.

21. A conotoxin peptide analog or pharmaceutically acceptable salt thereof of any one of claims 1-19, wherein the C-terminus of the conotoxin peptide analog is NH ₂。

22. A conotoxin peptide analog or a pharmaceutically acceptable salt thereof according to claim 1, wherein the conotoxin peptide analog is of formula (Ia) (SEQ ID NO: 94):

wherein R is¹Is OH or NH₂。

23. A conotoxin peptide analog or pharmaceutically acceptable salt according to claim 22, wherein R¹Is OH.

24. A conotoxin peptide analog or pharmaceutically acceptable salt according to claim 22, wherein R¹Is NH₂。

25. A conotoxin peptide analog or pharmaceutically acceptable salt thereof of claim 1, wherein the conotoxin peptide analog is of formula (Ig) (SEQ ID NO: 30):

26. a conotoxin peptide analog or a pharmaceutically acceptable salt thereof according to claim 1, wherein the conotoxin peptide analog is of formula (Ih) (SEQ ID NO: 33):

27. a conotoxin peptide analog or pharmaceutically acceptable salt thereof of claim 1, wherein the conotoxin peptide analog is of formula (Ii) (SEQ ID NO: 36):

28. a conotoxin peptide analog or pharmaceutically acceptable salt thereof of claim 1, wherein the conotoxin peptide analog is of formula (Ik) (SEQ ID NO: 42):

29. a conotoxin peptide analog or pharmaceutically acceptable salt thereof of claim 1, wherein the conotoxin peptide analog is of formula (Il) (SEQ ID NO: 45):

30. A conotoxin peptide analog or a pharmaceutically acceptable salt thereof according to claim 1, wherein the conotoxin peptide analog is of formula (Im) (SEQ ID NO: 48):

31. a conotoxin peptide analog or a pharmaceutically acceptable salt thereof according to claim 1, wherein the conotoxin peptide analog is of formula (In) (SEQ ID NO: 51):

32. a conotoxin peptide analog or a pharmaceutically acceptable salt thereof according to claim 1, wherein the conotoxin peptide analog is of formula (Io) (SEQ ID NO: 54):

33. a conotoxin peptide analog or a pharmaceutically acceptable salt thereof according to claim 1, wherein the conotoxin peptide analog is of formula (Ip) (SEQ ID NO: 57):

a PEGylated conotoxin peptide analog or a pharmaceutically acceptable salt thereof, wherein the conotoxin peptide analog is of formula (I) (SEQ ID NO: 93):

wherein

35. A pegylated conotoxin peptide analog or pharmaceutically acceptable salt according to claim 34, wherein said triazole bridge is

Wherein the single wave line

36. A pegylated conotoxin peptide analog or pharmaceutically acceptable salt according to claim 35, wherein said triazole bridge is

37. A pegylated conotoxin peptide analog or pharmaceutically acceptable salt according to claim 35, wherein said triazole bridge is

38. A pegylated conotoxin peptide analog or pharmaceutically acceptable salt according to any one of claims 35-37, wherein x is 1, 2 or 3.

39. A pegylated conotoxin peptide analog or pharmaceutically acceptable salt according to claim 38, wherein x is 1.

40. A PEGylated conotoxin peptide analog or pharmaceutically acceptable salt according to any one of claims 35-39, wherein y is 2 or 3.

41. A PEGylated conotoxin peptide analog or pharmaceutically acceptable salt according to claim 40, wherein y is 3.

42. A pegylated conotoxin peptide analog or pharmaceutically acceptable salt according to claim 35, wherein x is 1, 2 or 3 and y is 2 or 3.

43. A pegylated conotoxin peptide analog or pharmaceutically acceptable salt according to claim 36, wherein x is 1 and y is 3.

44. A pegylated conotoxin peptide analog or pharmaceutically acceptable salt according to claim 36, wherein x is 2 and y is 3.

45. A pegylated conotoxin peptide analog or pharmaceutically acceptable salt according to claim 36, wherein x is 2 and y is 2.

46. A pegylated conotoxin peptide analog or pharmaceutically acceptable salt according to claim 37, wherein x is 2 and y is 2.

47. A pegylated conotoxin peptide analog or pharmaceutically acceptable salt according to claim 37, wherein x is 1 and y is 3.

48. A PEGylated conotoxin peptide analog or pharmaceutically acceptable salt according to claim 43, wherein the triazole bridge is

Wherein the single wave line

49. A PEGylated conotoxin peptide analog or pharmaceutically acceptable salt according to any one of claims 34-48, wherein X is X _AA ¹。

50. A PEGylated conotoxin peptide analog or pharmaceutically acceptable salt according to any one of claims 34-48, wherein X is X_AA ¹X_AA ²。

51. A PEGylated conotoxin peptide analog or pharmaceutically acceptable salt according to claim 49 or 50, wherein X_AA ¹Selected from Tyr, D-Tyr and Phe.

52. A PEGylated conotoxin peptide analog or pharmaceutically acceptable salt according to any one of claims 34-49, wherein X is Tyr.

53. A PEGylated conotoxin peptide analog or pharmaceutically acceptable salt according to any one of claims 34-52, wherein the C-terminus of the conotoxin peptide analog is OH.

54. A PEGylated conotoxin peptide analog or pharmaceutically acceptable salt according to any one of claims 34-52, wherein the C-terminus of the conotoxin peptide analog is NH₂。

55. A PEGylated conotoxin peptide analog or pharmaceutically acceptable salt thereof according to claim 34, wherein the conotoxin peptide analog is of formula (Ia) (SEQ ID NO: 94)

Wherein R is¹Is OH or NH₂。

56. A PEGylated conotoxin peptide analog or pharmaceutically acceptable salt according to claim 55, wherein R¹Is OH.

57. A PEGylated conotoxin peptide analog or pharmaceutically acceptable salt according to claim 55, wherein R ¹Is NH₂。

58. A PEGylated conotoxin peptide analog or pharmaceutically acceptable salt according to claim 34, wherein said conotoxin peptide analog is of formula (Ig) (SEQ ID NO: 30):

59. a PEGylated conotoxin peptide analog or pharmaceutically acceptable salt according to claim 34, wherein said conotoxin peptide analog is of formula (Ih) (SEQ ID NO: 33):

60. a pegylated conotoxin peptide analog or pharmaceutically acceptable salt according to claim 34, wherein said conotoxin peptide analog is of formula (Ii) (SEQ ID NO: 36):

61. a PEGylated conotoxin peptide analog or pharmaceutically acceptable salt according to claim 34, wherein said conotoxin peptide analog is of formula (Ik) (SEQ ID NO: 42):

62. a PEGylated conotoxin peptide analog or pharmaceutically acceptable salt according to claim 33, wherein said conotoxin peptide analog is of formula (Il) (SEQ ID NO: 45):

63. a PEGylated conotoxin peptide analog or pharmaceutically acceptable salt according to claim 34, wherein said conotoxin peptide analog is of formula (Im) (SEQ ID NO: 48):

64. a pegylated conotoxin peptide analog or pharmaceutically acceptable salt according to claim 34, wherein said conotoxin peptide analog is of formula (In) (SEQ ID NO: 51):

65. A PEGylated conotoxin peptide analog or pharmaceutically acceptable salt according to claim 34, wherein said conotoxin peptide analog is of formula (Io) (SEQ ID NO: 54):

66. a PEGylated conotoxin peptide analog or pharmaceutically acceptable salt according to claim 34, wherein said conotoxin peptide analog is of formula (Ip) (SEQ ID NO: 57):

67. a PEGylated conotoxin peptide analog or pharmaceutically acceptable salt according to any one of claims 34-66, wherein the conotoxin peptide analog is covalently attached to a PEG polymer.

68. A PEGylated conotoxin peptide analog or pharmaceutically acceptable salt according to claim 67, wherein the PEG polymer is covalently attached to the N-terminus of the conotoxin peptide analog.

69. A PEGylated conotoxin peptide analog or pharmaceutically acceptable salt according to claim 67, wherein the PEG polymer is covalently attached to the C-terminus of the conotoxin peptide analog.

70. A PEGylated conotoxin peptide analog or pharmaceutically acceptable salt according to claim 67, wherein the PEG polymer is covalently attached to an amino acid residue position other than the N-terminus or C-terminus of the conotoxin peptide analog.

71. A PEGylated conotoxin peptide analog or pharmaceutically acceptable salt according to any one of claims 67-70, wherein the PEG polymer is covalently attached to the conotoxin peptide analog via a linking group.

72. A PEGylated conotoxin peptide analog or pharmaceutically acceptable salt according to claim 71, wherein said linking group is a valerate linker having the formula:

73. a PEGylated conotoxin peptide analog or pharmaceutically acceptable salt according to claim 71, wherein said linking group is butylene.

74. A PEGylated conotoxin peptide analog or pharmaceutically acceptable salt according to claim 71, wherein said linking group is a carbonyl group.

75. A PEGylated conotoxin peptide analog or pharmaceutically acceptable salt according to any one of claims 67-74, wherein the PEG polymer is a linear or branched PEG polymer.

76. A PEGylated conotoxin peptide analog or pharmaceutically acceptable salt according to claim 75, wherein the PEG polymer is a linear PEG polymer.

77. A pegylated conotoxin peptide analog or pharmaceutically acceptable salt according to claim 76, wherein said PEG polymer has a molecular weight in the range of from 10kDa to 40 kDa.

78. A PEGylated conotoxin peptide analog or pharmaceutically acceptable salt according to claim 77, wherein the PEG polymer is a linear 30kDa PEG polymer.

79. A PEGylated conotoxin peptide analog or pharmaceutically acceptable salt according to claim 78, wherein the PEG polymer is a linear 30kDa mPEG polymer.

80. A pegylated conotoxin peptide analog or pharmaceutically acceptable salt according to claim 34, wherein said pegylated conotoxin peptide analog has the formula (IIa) (SEQ ID NO: 83):

81. a pegylated conotoxin peptide analog or pharmaceutically acceptable salt according to claim 34, wherein said pegylated conotoxin peptide analog has the formula (IIg) (SEQ ID NO: 95):

82. a pegylated conotoxin peptide analog or pharmaceutically acceptable salt according to claim 34, wherein said pegylated conotoxin peptide analog has the formula (IIh) (SEQ ID NO: 96):

83. a pegylated conotoxin peptide analog or pharmaceutically acceptable salt according to claim 34, wherein said pegylated conotoxin peptide analog has formula (IIi) (SEQ ID NO: 97):

84. a pegylated conotoxin peptide analog or pharmaceutically acceptable salt according to claim 34, wherein said pegylated conotoxin peptide analog has the formula (IIk) (SEQ ID NO: 98):

85. A pegylated conotoxin peptide analog or pharmaceutically acceptable salt according to claim 34, wherein said pegylated conotoxin peptide analog has the formula (IIl) (SEQ ID NO: 99):

86. a pegylated conotoxin peptide analog or pharmaceutically acceptable salt according to claim 34, wherein said pegylated conotoxin peptide analog has the formula (IIm) (SEQ ID NO: 100):

87. a pegylated conotoxin peptide analog or pharmaceutically acceptable salt according to claim 34, wherein said pegylated conotoxin peptide analog has the formula (IIn) (SEQ ID NO: 101):

88. a pegylated conotoxin peptide analog or pharmaceutically acceptable salt according to claim 34, wherein said pegylated conotoxin peptide analog has the formula (IIo) (SEQ ID NO: 102):

89. a pegylated conotoxin peptide analog or pharmaceutically acceptable salt according to claim 34, wherein said pegylated conotoxin peptide analog has the formula (IIp) (SEQ ID NO: 103):

90. a conotoxin peptide analog of formula (Ib) (SEQ ID NO: 104):

or a pharmaceutically acceptable salt thereof,

wherein R is²Is OH or NH₂。

91. A conotoxin peptide analog or pharmaceutically acceptable salt according to claim 90, wherein R ²Is OH.

92. A conotoxin peptide analog or pharmaceutically acceptable salt according to claim 90, wherein R²Is NH₂。

93. A PEGylated conotoxin peptide analog or a pharmaceutically acceptable salt thereof, wherein the conotoxin peptide analog is of formula (Ib) (SEQ ID NO: 104):

wherein R is²Is OH or NH₂(ii) a And

94. A PEGylated conotoxin peptide analog or pharmaceutically acceptable salt according to claim 93, wherein R is²Is OH.

95. A PEGylated conotoxin peptide analog or pharmaceutically acceptable salt according to claim 93, wherein R is²Is NH₂。

96. A pegylated conotoxin peptide analog or pharmaceutically acceptable salt according to any one of claims 93-95, wherein the conotoxin peptide analog is covalently attached to a PEG polymer.

97. A pegylated conotoxin peptide analog or pharmaceutically acceptable salt according to claim 96, wherein said PEG polymer is attached to the N-terminus of said conotoxin peptide analog.

98. A pegylated conotoxin peptide analog or pharmaceutically acceptable salt according to claim 96, wherein said PEG polymer is attached to the C-terminus of said conotoxin peptide analog.

99. A pegylated conotoxin peptide analog or pharmaceutically acceptable salt according to claim 96, wherein said PEG is attached to an amino acid residue position other than the N-terminus or C-terminus of said conotoxin peptide analog.

100. A pegylated conotoxin peptide analog or pharmaceutically acceptable salt according to any one of claims 96-99, wherein the PEG polymer is covalently attached to the conotoxin peptide analog via a linking group.

101. A pegylated conotoxin peptide analog or pharmaceutically acceptable salt according to claim 100, wherein said linking group is a valerate ester linker having the formula:

102. a pegylated conotoxin peptide analog or pharmaceutically acceptable salt according to claim 100, wherein said linking group is butylene.

103. A pegylated conotoxin peptide analog or pharmaceutically acceptable salt according to claim 100, wherein said linking group is a carbonyl group.

104. A pegylated conotoxin peptide analog or pharmaceutically acceptable salt according to any one of claims 96-103, wherein the PEG polymer is a linear or branched PEG polymer.

105. A pegylated conotoxin peptide analog or pharmaceutically acceptable salt according to claim 104, wherein said PEG polymer is a linear PEG polymer.

106. A pegylated conotoxin peptide analog or pharmaceutically acceptable salt according to claim 105, wherein said PEG polymer has a molecular weight in the range of from 10kDa to 40 kDa.

107. A pegylated conotoxin peptide analog or pharmaceutically acceptable salt according to claim 106, wherein said PEG polymer is a linear 30kDa PEG polymer.

108. A pegylated conotoxin peptide analog or pharmaceutically acceptable salt according to claim 107, wherein said PEG polymer is a linear 30kDa mPEG polymer.

109. A pegylated conotoxin peptide analog or pharmaceutically acceptable salt according to claim 93, wherein said pegylated conotoxin peptide analog has formula (IIb) (SEQ ID NO: 105):

110. a conotoxin peptide analog selected from conotoxin peptide analogs Ia, Ia ', Ib', Ic, Id, Ie, If, Ig, Ih, Ii, Ij, Ik, Il, Im, In, Io, Ip, Iq, Ir, Is, It, Iu and Iv or pharmaceutically acceptable salts thereof.

111. A conotoxin peptide analog or pharmaceutically acceptable salt 10 of claim 1, wherein the conotoxin peptide analog is selected from conotoxin peptide analogs Ia, Ia ', Ib', Ig, Ih, Ii, Ik, Il, Im, In, Io and Ip.

112. A conotoxin peptide analog or pharmaceutically acceptable salt 10 of claim 1, wherein the conotoxin peptide analog is selected from conotoxin peptide analogs Ia, Ia ', Ib and Ib'.

113. A pharmaceutical composition comprising a conotoxin peptide analog or a pharmaceutically acceptable salt thereof according to any one of claims 1-33, 90-92 and 110-112, or a pegylated conotoxin peptide analog or a pharmaceutically acceptable salt thereof according to any one of claims 34-89 and 93-109, and optionally a pharmaceutically acceptable carrier.

114. A method of treating or preventing a condition in a subject that is conducive to treatment or prevention by inhibition of α 9-containing nicotinic acetylcholine receptors (nachrs), comprising administering to the subject a therapeutically effective amount of a conotoxin peptide analog or pharmaceutically acceptable salt thereof as described in any one of claims 1-33, 90-92 and 110-112, or a pegylated conotoxin peptide analog or pharmaceutically acceptable salt thereof as described in any one of claims 34-89 and 93-109, or a pharmaceutical composition as described in claim 113.

115. A method of treating or preventing a condition associated with activation of α 9-containing nicotinic acetylcholine receptors (nachrs) in a subject, comprising administering to the subject a therapeutically effective amount of a conotoxin peptide analog or pharmaceutically acceptable salt thereof as described in any one of claims 1-33, 90-92 and 110-112, or a pegylated conotoxin peptide analog or pharmaceutically acceptable salt thereof as described in any one of claims 34-89 and 93-109, or a pharmaceutical composition as described in claim 113.

116. The method of claim 114, wherein a condition that contributes to treatment or prevention by inhibiting α 9-containing nachrs is pain or inflammation.

117. The method of claim 116, wherein the condition is pain.

118. The method of claim 117, wherein the pain is selected from the group consisting of systemic pain, chronic pain, neuropathic pain, nociceptive pain, inflammatory pain, visceral pain, somatic pain, pain caused by peripheral nerve injury, pain caused by an inflammatory disorder, pain caused by a metabolic disorder, pain caused by cancer, pain caused by chemotherapy, pain caused by a surgical procedure, and pain caused by a burn.

119. The method of claim 117, wherein the pain is cancer-related chronic pain.

120. The method of claim 114, wherein a condition that contributes to treatment or prevention by inhibiting α 9-containing nachrs is an inflammatory condition.

121. The method of claim 120, wherein the inflammatory condition is selected from the group consisting of inflammation, chronic inflammation, rheumatic diseases, sepsis, fibromyalgia, inflammatory bowel disease, sarcoidosis, endometriosis, uterine fibroids, inflammatory skin diseases, inflammatory lung diseases, diseases associated with nervous system inflammation, periodontal disease, and cardiovascular disease.

122. The method of claim 120, wherein the inflammatory condition is mediated by immune cells.

123. The method of claim 120, wherein the inflammatory condition is long-term inflammation and/or peripheral neuropathy after injury.

124. The method of claim 114, wherein conditions conducive to treatment or prevention by inhibition of α 9-containing nachrs are pain and inflammation.

125. The method of claim 116, wherein conditions conducive to treatment or prevention by inhibition of α 9-containing nachrs are inflammation and neuropathy.

126. The method of any one of claims 114-125, wherein the condition that contributes to treatment or prevention by inhibition of α 9-containing nicotinic acetylcholine receptors (nachrs) is a condition that contributes to treatment or prevention by inhibition of the α 9 α 10 subtype of nachrs.

127. The method of any one of claims 114-126, wherein the subject is a human.

128. 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 or pharmaceutically acceptable salt thereof according to any one of claims 1-33, 90-92 and 110-112, or a pegylated conotoxin peptide analog or pharmaceutically acceptable salt thereof according to any one of claims 34-89 and 93-109, or a pharmaceutical composition according to claim 113.

129. A conotoxin peptide analog or a pharmaceutically acceptable salt thereof according to any one of claims 1-33, 90-92 and 110-112, or a pegylated conotoxin peptide analog or a pharmaceutically acceptable salt thereof according to any one of claims 34-89 and 93-109, or a pharmaceutical composition thereof according to claim 113, for use in treating or preventing a condition in a subject that facilitates treatment or prevention by inhibition of α 9-containing nicotinic acetylcholine receptors (nachrs), preferably the α 9 α 10 subtype of nachrs, or for use in treating or preventing pain or inflammation.

130. Use of a pharmaceutical composition comprising a conotoxin peptide analog or a pharmaceutically acceptable salt thereof according to any one of claims 1-33, 90-92 and 110-112, or a pegylated conotoxin peptide analog or a pharmaceutically acceptable salt thereof according to any one of claims 34-89 and 93-109, for treating or preventing a condition in a subject that is conducive to treatment or prevention by inhibition of a 9-containing nicotinic acetylcholine receptor (nAChR), preferably the a 9 α 10 subtype of the nAChR, or for treating or preventing pain or inflammation.

131. Use of a conotoxin peptide analog or a pharmaceutically acceptable salt thereof according to any one of claims 1-33, 90-92 and 110-112, or a pegylated conotoxin peptide analog or a pharmaceutically acceptable salt thereof according to any one of claims 34-89 and 93-109, or a pharmaceutical composition thereof according to claim 113, for the manufacture of a medicament for the treatment or prevention of a condition in a subject that facilitates treatment or prevention by inhibition of the α 9-containing nicotinic acetylcholine receptor (nAChR), preferably the α 9 α 10 subtype of the nAChR, or for the treatment or prevention of pain or inflammation.

132. A conotoxin peptide analogue or a salt thereof, wherein the amino acid sequence of the conotoxin peptide analogue is Gly-Cys-X_AA ³-Thr-Asp-Pro-Arg-Cys-X_AA ⁹-Trp-Gln-X_AA ¹²-X(SEQ ID NO：106)，

Wherein

X_AA ⁹is citrulline;

133. A conotoxin peptide analog or salt according to claim 132,wherein X_AA ³Is (S) -propargyl glycine or (S) -azido alanine.

134. A conotoxin peptide analog or salt according to claim 132, wherein X_AA ¹²Is (S) -azidovaline or (S) -bishomopropargyl glycine.

135. A conotoxin peptide analog or salt according to claim 132, wherein X_AA ³Is (S) -propargylglycine and X_AA ¹²Is (S) -azidovaline.

136. A conotoxin peptide analog or salt according to claim 132, wherein X_AA ³Is (S) -homopropargylglycine and X_AA ¹²Is (S) -azidovaline.

137. A conotoxin peptide analog or salt according to claim 132, wherein X_AA ³Is (S) -homopropargylglycine and X_AA ¹²Is (S) -azidohomoalanine.

138. A conotoxin peptide analog or salt according to claim 132, wherein X_AA ³Is (S) -azidohomoalanine and X_AA ¹²Is (S) -homopropargyl glycine.

139. A conotoxin peptide analog or salt according to claim 132, wherein X_AA ³Is (S) -azidoalanine and X_AA ¹²Is (S) -di-homopropargyl glycine.

140. A conotoxin peptide analog or salt according to any one of claims 132-139, wherein X is X_AA ¹。

141. The method as set forth in any one of claims 132-139Conotoxin peptide analogs or salts wherein X is X _AA ¹X_AA ²。

142. A conotoxin peptide analog or salt according to claim 140 or 141, wherein X_AA ¹Selected from Tyr, D-Tyr and Phe.

143. A conotoxin peptide analog or salt according to any one of claims 132-140, wherein X is Tyr.

144. A conotoxin peptide analog or salt according to any one of claims 132-143, wherein the C-terminus of the conotoxin peptide analog is OH.

145. A conotoxin peptide analog or salt according to any one of claims 132-143, wherein the C-terminus of the conotoxin peptide analog is NH₂。

146. A conotoxin peptide analog or salt according to claim 132, wherein X_AA ³Is (S) -propargylglycine, X_AA ¹²Is (S) -azidovaline, and X is Tyr.

147. A conotoxin peptide analog or salt according to claim 146, wherein the C-terminus of the conotoxin peptide analog is a carboxyl group.

148. A conotoxin peptide analog or salt according to claim 146, wherein the C-terminal end of the conotoxin peptide analog is an amide group.

149. A conotoxin peptide analog or salt according to claim 132, wherein X_AA ³Is (S) -homopropargylglycine, X_AA ¹²Is (S) -azidovaline, X is Tyr; and wherein the C-terminus of the conotoxin peptide analog is a carboxyl group.

150. A conotoxin peptide analog or salt according to claim 132, wherein X_AA ³Is (S) -homopropargylglycine, X_AA ¹²Is (S) -azidohomoalanine, X is Tyr; and wherein the C-terminus of the conotoxin peptide analog is a carboxyl group.

151. A conotoxin peptide analog or salt according to claim 132, wherein X_AA ³Is (S) -azidohomoalanine, X_AA ¹²Is (S) -homopropargyl glycine, and X is Tyr; and wherein the C-terminus of the conotoxin peptide analog is a carboxyl group.

152. A conotoxin peptide analog or salt according to claim 132, wherein X_AA ³Is (S) -azidoalanine, X_AA ¹²Is (S) -di-homopropargyl glycine, and X is Tyr; and wherein the C-terminus of the conotoxin peptide analog is a carboxyl group.

153. A conotoxin peptide analog or salt according to claim 132, wherein X_AA ³Is (S) -propargylglycine, X_AA ¹²Is (S) -azidovaline, X is Phe; and wherein the C-terminus of the conotoxin peptide analog is a carboxyl group.

154. A conotoxin peptide analog or salt according to claim 132, wherein X_AA ³Is (S) -propargylglycine, X_AA ¹²Is (S) -azidovaline, and X is D-Tyr; and wherein the C-terminus of the conotoxin peptide analog is a carboxyl group.

155. A conotoxin peptide analog or salt according to claim 132, wherein X_AA ³Is (S) -propargylglycine, X_AA ¹²Is (S) -azidovaline, and X is Tyr-N-Me-Gly; and wherein the C-terminus of the conotoxin peptide analog is carboxyl。

156. A conotoxin peptide analog or salt according to claim 132, wherein X_AA ³Is (S) -propargylglycine, X_AA ¹²Is (S) -azidovaline, and X is Tyr-D-Tyr; and wherein the C-terminus of the conotoxin peptide analog is a carboxyl group.

157. A conotoxin peptide analog or salt according to claim 132, wherein X_AA ³Is (S) -propargylglycine, X_AA ¹²Is (S) -azidovaline, and X is Tyr-N-Me-Tyr; and wherein the C-terminus of the conotoxin peptide analog is a carboxyl group.

158. A conotoxin peptide analogue or a salt thereof, wherein the amino acid sequence of the conotoxin peptide analogue is Gly-Cys-X_AA ³-Thr-Asp-Pro-Arg-Cys-X_AA ⁹-X_AA ¹⁰-Gln-X_AA ¹²-Tyr(SEQ ID NO：107)，

Wherein

X_AA ³Is (S) -propargylglycine;

X_AA ⁹is citrulline;

X_AA ¹⁰is 3-iodo-Tyr;

X_AA ¹²is (S) -azidovaline;

159. A conotoxin peptide analog or salt of claim 158, wherein the C-terminus of the conotoxin peptide analog is a carboxyl group.

160. A conotoxin peptide analog or salt according to claim 158, wherein the C-terminal end of the conotoxin peptide analog is an amide group.

161. A method for preparing conotoxin peptide analogue (SEQ ID NO: 93) shown in chemical formula (I) or pharmaceutically usable salt thereof,

wherein

Wherein

X_AA ⁹is citrulline;

wherein when X is_AA ³Selected from (S) -propargylglycine, (S) -homopropargylglycine and (S) -bishomopropargylglycine, X _AA ¹²Is (S) -azidohomoalanine or (S) -azidovaline; and when X_AA ³Selected from (S) -azidoalanine, (S) -azidohomoalanine and (S) -azidovaline, X_AA ¹²Is (S) -homopropargylglycine or (S) -bishomopropargylglycine;

x is as defined above for the conotoxin peptide analogue of formula (I); and

162. A method for preparing conotoxin peptide analogue (SEQ ID NO: 104) shown in chemical formula (Ib) or pharmaceutically acceptable salt thereof,

Wherein

X_AA ³Is (S) -propargylglycine;

X_AA ⁹is citrulline;

X_AA ¹⁰is 3-iodo-Tyr;

X_AA ¹²is (S) -azidovaline; and

Wherein under the triazole forming conditions, X_AA ³And X_AA ¹²Reacted to form a compound of formula (Ib)The triazole bridge shown in the conotoxin peptide analogs of (a).

163. A method of preparing a PEGylated conotoxin peptide analog or a pharmaceutically acceptable salt thereof, comprising contacting a conotoxin peptide analog or a salt thereof with one or more reactive polyethylene glycol (PEG) polymers under reaction conditions to form a PEGylated conotoxin peptide analog, wherein the reactive PEG polymers each comprise a reactive group covalently attached to a PEG polymer, optionally through a linking group, 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 to the one or more PEG polymers, either directly or through a linking group,

Wherein

X_AA ⁹is citrulline;

164. A method of preparing a PEGylated conotoxin peptide analog or a pharmaceutically acceptable salt thereof, comprising contacting a conotoxin peptide analog or a salt thereof with one or more reactive polyethylene glycol (PEG) polymers under reaction conditions to form a PEGylated conotoxin peptide analog, wherein the reactive PEG polymers each comprise a reactive group covalently attached to a PEG polymer, optionally through a linking group, 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 to the one or more PEG polymers, either directly or through a linking group,

Wherein

X_AA ³Is (S) -propargylglycine;

X_AA ⁹is citrulline;

X_AA ¹⁰is 3-iodo-Tyr;

X_AA ¹²is (S) -azidovaline;