KV1.3 blockers

Abstract

The present invention provides novel blockers of the potassium channel Kv1.3, polynucleotides encoding them, and methods of making and using them.

Description

[Technical field]

[0001] The present invention provides novel blockers of the potassium channel Kv1.3, polynucleotides encoding them, and methods of making and using them. [Background technology]

[0002] Ion channels are membrane proteins that form pores in biological membranes, allowing (and regulating) the flow of ions across the associated membrane. There are many different types of ion channels that can be classified in various ways, such as by the type of ion they provide passage for, the way in which the passage of ions is regulated or "gated" (e.g., "ligand-gated" or "voltage-gated"), and their cellular or subcellular localization.

[0003] Potassium channels are divided into four major classes: voltage-gated potassium channels, calcium-activated potassium channels, inward rectifier potassium channels, and tandem pore domain potassium channels.

[0004] Voltage-gated potassium channels, like other voltage-gated channels, open and close in response to transmembrane voltages. They represent a complex family with diverse biological functions including regulation of neurotransmitter release, heart rate, insulin secretion, neuronal excitability, epithelial electrolyte transport, smooth muscle contraction, and cell volume.

[0005] Kv1.3 (potassium voltage-gated channel subfamily A member 3) channels are expressed on T cells and play a role in regulating T cell activation. Blockers of Kv1.3 have been shown to inhibit proliferation of activated T cells in vitro (reviewed in Cahalan and Chandy, Immunol.Rev. 231:59-87, 2009) and to inhibit T cell-dependent disease progression in various experimental models of autoimmune disease, including experimental autoimmune encephalomyelitis (EAE), experimental arthritis, delayed-type hypersensitivity (DTH), allergic contact dermatitis, and glomerulonephritis. See, e.g., Rangaraju et al. (Expert Opin. Ther. Targets 13:909-24, 2009); Beeton et al. (Proc. Natl. Acad. Sci. US A. 103:17414-9, 2006); Koo et al. (J. Immunol. 158:5120-8, 1997); Hyodo et al. (Am. J. Physiol. Renal Physiol. 299 : F1258-69, 2010). WO 2016 / 112208 describes the topical application of Kv1.3 blockers for the treatment of inflammation of the skin and mucous membranes.

[0006] Therefore, Kv1.3 blockers have considerable potential for use in the treatment of inflammatory diseases, including autoimmune diseases.

[0007] WO 2015 / 013330 proposes the use of Kv1.3 blocking peptides for the treatment of ocular conditions such as dry eye and uveitis, including those caused by autoimmune conditions such as Sjogren's syndrome.

[0008] Blockers of Kv1.3 may also have beneficial metabolic effects, for example, in relation to energy homeostasis, body weight regulation, and glucose control. Kv1.3 knockout (Kv1.3(- / -)) mice exhibit reduced weight gain, higher insulin sensitivity, and reduced plasma glucose levels in response to high-fat diet compared to control littermates (Xu et al., Hum. Mol. Genet. 12:551-9, 2003). Furthermore, Kv1.3 blockers have been shown to increase glucose transporter 4 (GLUT4) expression in skeletal muscle and adipose tissue, increase insulin sensitivity in normal and ob / ob obese mice, and increase glucose uptake in primary adipocytes in vitro (Xu et al., Proc. Natl. Acad. Sci. USA 101:3112-7, 2004). In humans, single nucleotide polymorphisms (SNPs) in the Kv1.3 gene have also been associated with reduced insulin sensitivity and impaired glucose tolerance (Tschritter, Clin Endocrinol Metab 91: 654-8, 2006).

[0009] Kv1.3 is also expressed in proliferating human and mouse smooth muscle cells. Blockers of Kv1.3 may be effective in smooth muscle proliferative disorders, such as restenosis in patients after vascular surgery (e.g., angioplasty). Kv1.3 blockers have been shown to inhibit calcium entry, reduce smooth muscle cell migration, and inhibit neointimal hyperplasia in ex vivo human vein samples (Cheong et al., Cardiovasc. Res. 89:282-9, 2011).

[0010] Further evidence suggests that Kv1.3 channels are involved in the activation and / or proliferation of many types of cells, including tumor cells (Bielanska et al., Curr. Cancer Drug Targets 9:904-14, 2009), microglia (Khanna et al., Am. J. Physiol. Cell Physiol. 280 : C796-806, 2001), and differentiation of neural progenitor cells (Wang et al., J. Neurosci. 30:5020-7, 2010). Thus, Kv1.3 blockers may be beneficial in the treatment of neuroinflammatory diseases, neurodegenerative diseases, and cancer.

[0011] Kv1.3 is part of a subfamily of closely related potassium channels called Kv1.1 to Kv1.8. When dealing with a large homologous family, it is always desirable for the blocker to be as selective and specific as possible for the desired target to improve efficacy and safety and avoid undesirable off-target effects. The most specific Kv1.3 blockers identified to date are toxic peptides derived from various types of venomous organisms, such as snakes, arachnids (such as scorpions and spiders), and sea anemones. Such Kv1.3 blockers include the peptides ShK, Oskl, margatoxin, and karyotoxin, reviewed by Chandy et al., Trends in Pharmacol. Sci. 25:280-9, 2004. See also Abdel-Mottaleb et al., Toxicon 51:1424-30, 2008, and Mouhat et al., Biochem. J. 385(Pt 1):95-104, 2005.

[0012] Various attempts to modify toxin peptides for particular properties, including specificity or potency, have been described, for example, in WO 2006 / 002850, WO 2006 / 042151, WO 2008 / 088422, WO 2006 / 116156, WO 2010 / 105184, and WO 2014 / 116937. International Patent Application No. PCT / EP2020 / 076187 also discloses Kv1.3 blockers.

[0013] However, there remains a need for alternative Kv1.3 blockers. Blockers with improved specificity compared to known blockers would be particularly desirable, although improvements in other properties such as stability and potency may also be useful. [Prior art documents] [Patent documents]

[0014] [Patent Document 1] International Publication No. 2016 / 112208 Brochure [Patent Document 2] International Publication No. 2015 / 013330 Brochure [Patent Document 3] International Publication No. 2006 / 002850 Brochure [Patent Document 4] International Publication No. 2006 / 042151 Brochure [Patent Document 5] International Publication No. 2008 / 088422 Brochure [Patent Document 6] International Publication No. 2006 / 116156 Brochure [Patent Document 7] International Publication No. 2010 / 105184 Brochure [Patent Document 8] International Publication No. 2014 / 116937 Brochure [Patent Document 9] International Patent Application No. PCT / EP2020 / 076187 [Non-patent literature]

[0015] [Non-licensed document 1] Cahalan and Chandy, Immunol.Rev. 231:59-87, 2009

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[0016] The present invention relates to an ion channel blocker derived from a venom peptide of the scorpion Parabuthus transvaalicus, which has the amino acid sequence QMDMRCSASVECKQKCLKAIGSIFGKCMNKKCKCYPR (SEQ ID NO: 1).

[0017] Among other desirable properties, this molecule and its derivatives or variants have been shown to be highly selective blockers of the Kv1.3 potassium ion channel over other voltage-gated potassium channels, and typically also have high potency for blocking Kv1.3 channels.

[0018] The present invention therefore provides an ion channel blocker with Kv1.3 inhibitor activity comprising a variant of the sequence QMDMRCSASVECKQKCLKAIGSIFGKCMNKKCKCYPR (SEQ ID NO: 1), or a pharma- ceutically acceptable salt thereof, in which at least one amino acid at positions 7, 8, 9, 10 or 11 of SEQ ID NO: 1 is substituted with an amino acid having a positively charged side chain and / or an amino acid having an aromatic side chain, said variant not comprising any of the following sequences:

[0019] [Table 1] [Means for solving the problem]

[0020] In one aspect, the present invention provides an ion channel blocker with Kv1.3 inhibitor activity comprising a variant of the sequence QMDMRCSASVECKQKCLKAIGSIFGKCMNKKCKCYPR (SEQ ID NO: 1), or a pharma- ceutically acceptable salt thereof, wherein at least one amino acid at positions 7, 8, 9, 10 or 11 of SEQ ID NO: 1 is substituted with an amino acid having a positively charged side chain and / or an amino acid having an aromatic side chain.

[0021] In other aspects, the invention provides nucleic acids encoding the ion channel blockers of the invention, expression vectors comprising a nucleic acid of the invention, and host cells comprising a nucleic acid of the invention or an expression vector of the invention and capable of expressing an ion channel blocker of the invention.

[0022] The present invention also provides methods of synthesizing the ion channel blockers or pharma- ceutically acceptable salts of the present invention.

[0023] The present invention further provides pharmaceutical compositions comprising an ion channel blocker or a pharma- ceutically acceptable salt of the present invention.

[0024] The present invention further provides pharmaceutical uses of the ion channel blockers, pharma- ceutically acceptable salts, or pharmaceutical compositions of the present invention, as well as methods of treating diseases using the ion channel blockers, pharma- ceutically acceptable salts, or pharmaceutical compositions of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] Ion channel blockers The present invention provides ion channel blockers.

[0026] The ion channel blockers of the present invention may be in the form of a pharma- ceutically acceptable salt. All references herein to "ion channel blockers" should be construed to encompass any pharma- ceutically acceptable salt thereof, whether or not "pharma- ceutically acceptable salt" is explicitly stated.

[0027] The term "ion channel blocker" is used to denote a compound that has inhibitor (or blocking) activity against an ion channel, i.e., a compound that can inhibit or eliminate the flow of ions through the respective ion channel, typically by binding to the ion channel. Similarly, the terms "Kv1.3 inhibitor" and "Kv1.3 inhibitor component" refer to a peptide that can inhibit or eliminate the flow of ions through the Kv1.3 ion channel, typically by binding to the Kv1.3 channel. However, the terms "blocker" and "inhibitor" should not be construed to imply a particular mechanism of action or a particular mode of interaction with the ion channel itself.

[0028] The term Kv1.3 refers to potassium voltage-gated channel subfamily A member 3, also known as KCNA3, HPCN3, HGK5, HuKIII and HLK3. "Subfamily A" is sometimes also known as the "Shaker-related subfamily." The human amino acid sequence is provided in UniProt accession number P22001, version P22001.3(Q5VWN2).

[0029] Kv1.3 channel is expressed in T and B lymphocytes and is involved in T cell activation. Many groups are developing Kv1.3 blockers for inhibiting immune responses and various other indications. However, Kv1.3 channel is part of a complex family of related ion channels, including Kv1.1, Kv1.2, and Kv1.6 channels, which have different physiological roles. Therefore, it is desirable for Kv1.3 inhibitors to be as selective as possible for Kv1.3 in preference to other ion channels, especially other voltage-gated potassium channels such as Kv1.1, Kv1.2, Kv1.4, Kv1.5, Kv1.6, Kv1.7, and Kv1.8.

[0030] The ion channel blockers or pharma- ceutically acceptable salts of the present invention have Kv1.3 inhibitory activity, in other words, the ion channel blockers (and isolated peptide components of the ion channel blockers) of the present invention have inhibitor or blocker activity at the Kv1.3 ion channel, i.e., are capable of inhibiting the flow of ions through the Kv1.3 ion channel.

[0031] I C 50 value I C 50 The IC value can be used as a measure of the activity or potency of an inhibitor (or blocker). 50 The IC value is a measure of the concentration of inhibitor required for the compound to achieve half-maximal inhibition of ion channel activity in a given assay. A compound with a lower IC than a reference compound at a particular ion channel 50 can be considered to be a more active or more potent inhibitor than the reference compound. The terms "activity" and "efficacy" are used interchangeably.

[0032] I C 50The values ​​may be determined using any suitable assay, such as a fluorescence-based assay that measures the flux of ions (e.g., the flux of thallium ions) and a patch clamp assay, which may be performed as described in the Examples below. For example, a patch clamp assay using the QPatch® system may be preferred.

[0033] In some embodiments the ion channel blockers of the invention have an IC for the human Kv1.3 potassium channel of 50 nM or less, such as 20 nM or less, for example 10 nM or less, such as 5 nM or less, for example 2 nM or less, such as 1 nM or less, for example 0.5 nM or less, such as 0.4 nM or less, for example 0.3 nM or less, such as 0.2 nM or less. 50 Preferably, the ion channel blockers of the present invention have an IC for the human Kv1.3 potassium channel of 0.5 nM or less, most preferably 0.2 nM or less. 50 has.

[0034] The ion channel blockers of the present invention include compounds 1 to 12 described herein, which have an IC50 value of 50 nM or less for the human Kv1.3 potassium channel in Example 2 of the present specification. 50 The ion channel blockers of the present invention include compounds 1 to 5 and 7 to 12 described herein, which have been shown in Example 2 of the present specification to have an IC50 value of 5 nM or less for the human Kv1.3 potassium channel. 50 The ion channel blockers of the present invention include compounds 1 to 5, 7, 8, and 10 to 12 described herein, which have an IC50 value of 0.5 nM or less for the human Kv1.3 potassium channel in Example 2 of the present specification. 50 The ion channel blockers of the present invention include compounds 1 to 5, 7, 8, and 11 described herein, which have an IC50 value of 0.3 nM or less for the human Kv1.3 potassium channel in Example 2 of the present specification. 50The ion channel blockers of the present invention include compounds 1 to 5 described herein, which have an IC value for the human Kv1.3 potassium channel of 0.2 nM or less in Example 2 of the present specification. 50 It has been shown that

[0035] Half-life In some embodiments, the ion channel blockers of the present invention have an in vivo half-life of at least 1 hour, such as at least 1.5 hours, such as at least 2 hours, such as at least 2.5 hours.

[0036] Half-life (T 1 / 2 ) can be determined using assays known in the art, such as those described in Example 3 herein.

[0037] Selectivity The ion channel blockers of the present invention are selective for Kv1.3. In one embodiment, the ion channel blockers of the present invention are selective over Kv1.1, Kv1.2, Kv1.4, Kv1.5, Kv1.6, Kv1.7 and Kv1.8. In particular, the ion channel blockers of the present invention are selective for Kv1.3 over one or more of Kv1.1, Kv1.2 and Kv1.6.

[0038] For example, they may be selective for Kv1.3 over Kv1.1; selective for Kv1.3 over Kv1.2; selective for Kv1.3 over Kv1.6; selective for Kv1.3 over Kv1.1 and Kv1.2; selective for Kv1.3 over Kv1.1 and Kv1.6; selective for Kv1.3 over Kv1.2 and Kv1.6; or selective for Kv1.3 over Kv1.1, Kv1.2, and Kv1.6. Typically, ion channel blockers are selective for Kv1.3 over Kv1.1. Furthermore, they may be selective for Kv1.3 over Kv1.2 and / or Kv1.6.

[0039] "Selective" in this context means that the ion channel blockers have higher inhibitory activity against Kv1.3 than against each of Kv1.1, Kv1.2, and Kv1.6. Hence, their IC 50 is typically lower than for each of the other ion channels.

[0040] Thus, selectivity for Kv1.3 over another ion channel X can be expressed as, for example, an IC 50 [X] / IC 50 As in [Kv1.3], each IC 50 It can be expressed as a ratio of values.

[0041] Thus, the ion channel blockers of the invention may have a selectivity for Kv1.3 over Kv1.1 of at least 10, at least 100, at least 1000, or at least 10000, and may be up to 100000 or even more. Typically, the ion channel blockers of the invention have a selectivity for Kv1.3 over Kv1.1 of at least 100, or at least 1000.

[0042] Thus, the ion channel blockers of the invention may have a selectivity for Kv1.3 over Kv1.2 of at least 10, at least 100, at least 1000, or at least 10000, and may be up to 100000 or even more. Typically, they have a selectivity for Kv1.3 over Kv1.2 of at least 10, preferably at least 50, or at least 100, or at least 1000.

[0043] Thus, the ion channel blockers of the invention may have a selectivity for Kv1.3 over Kv1.6 of at least 10, at least 100, at least 1000, or at least 10000, and may be up to 100000 or even more. Typically, they have a selectivity for Kv1.3 over Kv1.6 of at least 100, at least 400, or at least 1000.

[0044] The ion channel blockers of the present invention may have higher selectivity than known ion channel blockers such as ShK, mokatoxin (Moka1), Vm24, Odk2, or Osk1. Thus, the ion channel blockers of the present invention have higher selectivity for Kv1.3 over ion channel X, i.e., IC 50 [X] / IC 50 [Kv1.3], which is higher than the selectivity of the comparison molecule.The selectivity of the two ion channel blockers is determined under the same conditions for each ion channel to allow direct comparison.As mentioned above, any suitable assay can be used, such as fluorescence-based ion flux assay and patch clamp assay.

[0045] The ion channel blockers of the present invention have lower absolute inhibitory activity (i.e., higher IC ) than known ion channel blockers (such as Odk2 or Osk1) at any or all of Kv1.1, Kv1.2 and / or Kv1.6. 50 ) may have a higher absolute inhibitory activity at any or all of these ion channels, provided that their selectivity for Kv1.3 is higher than that of the comparison compound. Typically, however, the compounds of the invention combine high specificity for Kv1.3 with high potency.

[0046] peptide Ion channel blockers of the present invention include variants of the peptide sequence QMDMRCSASVECKQKCLKAIGSIFGKCMNKKCKCYPR (SEQ ID NO:1).

[0047] SEQ ID NO:1 is the amino acid sequence of a venom peptide from the scorpion Parabutus transvaricus. As described herein, this peptide is a selective Kv1.3 potassium ion channel inhibitor.

[0048] amino acid Throughout this specification and claims, the conventional three letter and one letter codes for the naturally occurring amino acids are used, i.e. A(Ala), G(Gly), L(Leu), I(Ile), V(Val), F(Phe), W(Trp), S(Ser), T(Thr), Y(Tyr), N(Asn), Q(Gln), D(Asp), E(Glu), K(Lys), R(Arg), H(His), M(Met), C(Cys) and P(Pro); As well as sarcosine (Sar), norleucine (Nle), α-aminoisobutyric acid (Aib), 2,3-diaminopropanoic acid (Dap), 2,4-diaminobutanoic acid (Dab), 2,5-diaminopentanoic acid (ornithine or Orn), alpha-aminobutyric acid (Abu), Generally accepted codes for other α-amino acids are used, such as h(4-F) (4-fluoro-phenylalanine), F(4-NH) (4-amino-phenylalanine), F(4-NO) (4-nitro-phenylalanine), and F(4-CH) (4-methyl-phenylalanine).

[0049] The designation [2-amino-5-carboxypentanoyl] denotes a peptide residue of 2-amino-5-carboxypentanoic acid, which has a side chain similar to that of glutamic acid, but with an additional methylene group, as shown in the following structure:

[0050] [ka]

[0051] The designation [2,3-diaminopropanoyl] denotes the peptide residue of 2,3-diaminopropanoic acid, which has the following structure:

[0052] [ka]

[0053] The designation [2,4-diaminobutanoyl] denotes the peptide residue of 2,4-diaminobutanoic acid, which has the following structure:

[0054] [ka]

[0055] The designation [2-amino-3-guanidinopropionyl] indicates the peptide residue of 2-amino-3-guanidinopropionic acid, which has the following structure:

[0056] [ka]

[0057] Such other α-amino acids, when used herein in a general formula or sequence, may be shown in square brackets "[]", particularly when the remainder of the formula or sequence is shown using the single letter code (e.g., "[Nle]"). Unless otherwise specified, amino acid residues in the peptides of the invention are in the L-configuration. However, amino acids in the D-configuration may be incorporated. In this context, the amino acid code written in lower case represents the D-configuration of that amino acid, for example, "k" represents the D-configuration of lysine (K).

[0058] The variant of SEQ ID NO:1 contains six cysteine ​​(C) residues that together form three disulfide bonds between residues 6C and 27C, residues 12C and 32C, and residues 16C and 34C.

[0059] The disulfide bonds, with reference to SEQ ID NO:1, are as follows: QMDMRC(1)SASVEC(2)KQKC(3)LKAIGSIFGKC(1)MNKKC(2)KC(3)YPR where a pair of cysteine ​​residues participating together in a disulfide bond are indicated by the same number in parentheses. A similar notation may be applied to any of the other sequences in this application. Unless the context requires otherwise, active inhibitor compounds should be understood to include appropriate disulfide bonds.

[0060] It may be desirable that no other cysteine ​​residues are introduced into the variant of SEQ ID NO: 1 by substitution or insertion. Thus, in some embodiments, the variant contains no other cysteine ​​residues except those at positions corresponding to positions 6, 12, 16, 27, 32, and 34 of SEQ ID NO: 1. In some embodiments, the substitutions or deletions in the variant of SEQ ID NO: 1 are not the amino acids at positions 6, 12, 16, 27, 32, and 34 of SEQ ID NO: 1.

[0061] variant Ion channel blockers of the present invention include variants of SEQ ID NO: 1. In some embodiments, ion channel blockers of the present invention consist of variants of SEQ ID NO: 1. A variant of SEQ ID NO: 1 is a peptide that contains one or more amino acids different from those of SEQ ID NO: 1 (i.e., one or more amino acid changes compared to SEQ ID NO: 1). Such variants may also be referred to as "derivatives", "variant peptides", "peptides" or "compounds".

[0062] A variant of SEQ ID NO: 1 may differ from SEQ ID NO: 1 in that one or more amino acids of SEQ ID NO: 1 have been deleted, and / or one or more amino acids of SEQ ID NO: 1 have been substituted with different amino acids, and / or one or more amino acids have been inserted into the sequence of SEQ ID NO: 1. The amino acids may be inserted at an internal position of SEQ ID NO: 1, at the N-terminus of SEQ ID NO: 1, or at the C-terminus of SEQ ID NO: 1. Thus, a variant of SEQ ID NO: 1 includes one or more substitutions, insertions and / or deletions.

[0063] Unless otherwise specified, a "substitution" refers to the substitution (i.e., replacement) of a single amino acid in SEQ ID NO:1. Thus, for example, the substitution of three consecutive amino acids in SEQ ID NO:1 does not constitute a single substitution, but rather three substitutions. Similarly, an "insertion" refers to the insertion (which may be internal, N-terminal, and / or C-terminal) of a single amino acid into SEQ ID NO:1, such that, for example, the insertion of three consecutive amino acids in SEQ ID NO:1 does not constitute a single insertion, but rather three insertions. A "deletion" refers to the deletion of a single amino acid from SEQ ID NO:1, such that, for example, the deletion of three consecutive amino acids in SEQ ID NO:1 does not constitute a single deletion, but rather three deletions.

[0064] In particularly preferred embodiments, the variant of SEQ ID NO: 1 differs from SEQ ID NO: 1 by a total of up to 10 substitutions, insertions or deletions. In some such embodiments, the variant contains a total of 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 substitutions, insertions or deletions compared to SEQ ID NO: 1. Preferably, the variant differs from SEQ ID NO: 1 by a total of 7 substitutions, insertions or deletions compared to the sequence of SEQ ID NO: 1.

[0065] Numbering The amino acid residues of SEQ ID NO: 1 are conventionally numbered from 1 to 37 in the N-terminal to C-terminal direction. Throughout this specification, amino acid positions in variants of SEQ ID NO: 1 are numbered according to their corresponding positions when optimally aligned with SEQ ID NO: 1. Thus, particularly for variants that contain one or more insertions or deletions compared to SEQ ID NO: 1, the numbering of a given residue reflects the corresponding residue in SEQ ID NO: 1, and not necessarily its linear position in the sequence of the variant.

[0066] The residue present at a particular position may be designated by the number of the relevant position aligned together with the one-letter or three-letter code of the residue present. Thus, 1Q or Q1 (these two forms are interchangeable) designates a glutamine (Q) residue at position 1, 2Nle, 2[Nle], Nle2 or [Nle]2 designates a norleucine residue at position 2.

[0067] An asterisk may be used to denote the location of the deletion relative to the sequence of SEQ ID NO:1. For example, "1 * " indicates a deletion of residue at position 1 compared to SEQ ID NO:1.

[0068] An insertion may be indicated by a series of consecutive residues at a single position, for example "1QA" indicates the insertion of an alanine (A) residue after the glutamine (Q) residue at position 1.

[0069] In some embodiments, the substitutions compared to SEQ ID NO: 1 are conservative substitutions. However, any of the substitutions listed in the general formulae provided below may be introduced at each position.

[0070] In some embodiments, the substitutions or deletions in the variant of SEQ ID NO: 1 are at amino acid positions selected from among positions 1 to 5, 7 to 11, 13 to 15, 17 to 23, 25, 28 to 31, 33, and 35 to 37 of SEQ ID NO: 1. Preferably, the substitutions or deletions in the variant of SEQ ID NO: 1 are at amino acid positions selected from among positions 1 to 5, 7 to 11, 13, 15, 18, 28, and 30 of SEQ ID NO: 1.

[0071] terminal group The "H" (or "Hy-") moiety at the N-terminus of a sequence indicates a hydrogen atom [i.e., R 1 = hydrogen], which corresponds to the presence of a free primary or secondary amino group at the N-terminus. Alternatively, variant peptides may contain alternative N-terminal groups (i.e., N-terminal modifications).

[0072] Thus, in some embodiments of the ion channel blockers or pharma- ceutically acceptable salts of the present invention, the peptide has at its N-terminus a C 1-4 Includes groups selected from alkyl, acetyl (Ac), formyl, benzoyl and trifluoroacetyl.

[0073] A "-OH" moiety at the C-terminus of a sequence indicates the presence of a hydroxy group (OH) as part of a carboxy (COOH) group at the C-terminus of the molecule. A "-NH2" moiety at the C-terminus of a sequence indicates the presence of an amino group (NH2) as part of an amide (CONH2) group at the C-terminus of the molecule. A "CH2OH" moiety at the C-terminus indicates the presence of a hydroxyl group linked to an alkyl group at the C-terminus of the molecule.

[0074] Thus, in some embodiments of the ion channel blockers or pharma- ceutically acceptable salts of the present invention, the peptide comprises an amino group (-NH2), a hydroxyl group (-OH) or a hydroxymethyl group (-CH2OH) at the C-terminus, preferably an amino group (-NH2) or a hydroxyl group (-OH).

[0075] Sequence identity In some embodiments the variant has at least 60% sequence identity, such as at least 65% sequence identity, for example, at least 70% sequence identity, such as at least 75% sequence identity, for example, at least 80% sequence identity, such as at least 85% sequence identity, for example, at least 90% sequence identity, such as at least 95% sequence identity, for example, at least 96% sequence identity, such as at least 97% sequence identity, for example, at least 98% sequence identity, such as at least 99% sequence identity, for example, 100% sequence identity, to SEQ ID NO:1.

[0076] "Percent (%) amino acid sequence identity" herein with respect to a peptide sequence is defined as the percentage of amino acids in a candidate sequence that are identical to the amino acids in the wild-type toxin peptide sequence of SEQ ID NO:1, after aligning the sequences and introducing gaps, if necessary, to maximize the percent sequence identity, and any conservative substitutions are not considered as part of the sequence identity. Alignment of sequences can be performed by one of skill in the art using techniques well known in the art, for example, using publicly available software such as BLAST, BLAST2 or Align software. See, for example, Altschul et al., Methods in Enzymology 266: 460-480 (1996) or Pearson et al., Genomics 46: 24-36, 1997.

[0077] Percentage sequence identity as used herein in the context of the present invention can be determined using these programs with default settings. More generally, those skilled in the art can readily determine appropriate parameters for determining alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0078] Specific peptide variants 7th~11th place Ion channel blockers or pharma- ceutically acceptable salts of the invention include variants of the sequence QMDMRCSASVECKQKCLKAIGSIFGKCMNKKCKCYPR (SEQ ID NO: 1), in which at least one amino acid at positions 7, 8, 9, 10 or 11 of SEQ ID NO: 1 is substituted with an amino acid having a positively charged side chain and / or an amino acid having an aromatic side chain.

[0079] In some embodiments, at least one amino acid at position 7, 8, 9, 10, or 11 of SEQ ID NO:1 is substituted with an amino acid having a positively charged side chain. In some embodiments, the amino acid having a positively charged side chain is selected from the group consisting of H, K, hK, R, Orn, 2,3-diaminopropanoyl, 2,4-diaminobutanoyl, 2-amino-3-guanidinopropionyl, and 4-amino-phenylalanine (F(4-NH2)). Preferably, the amino acid having a positively charged side chain is selected from H, K, R, Orn, 2,3-diaminopropanoyl, 2-amino-3-guanidinopropionyl, and 2,4-diaminobutanoyl. Preferably, the amino acid having a positively charged side chain is selected from H, R, Orn, 2,3-diaminopropanoyl, 2-amino-3-guanidinopropionyl, and 2,4-diaminobutanoyl.

[0080] In some embodiments, at least one amino acid at position 7, 8, 9, 10, or 11 of SEQ ID NO:1 is substituted with an amino acid having an aromatic side chain. In some embodiments, the amino acid having an aromatic side chain is selected from the group consisting of F, W, and Y. Preferably, the amino acid having an aromatic side chain is Y.

[0081] In some embodiments, an ion channel blocker or pharma- ceutically acceptable salt of the invention comprises a variant of the sequence QMDMRCSASVECKQKCLKAIGSIFGKCMNKKCKCYPR (SEQ ID NO:1), in which at least one amino acid at positions 7, 9, or 10 of SEQ ID NO:1 is substituted with an amino acid having a positively charged side chain and / or an amino acid having an aromatic side chain.

[0082] In some embodiments, at least one amino acid at position 7, 9, or 10 of SEQ ID NO:1 is substituted with an amino acid having a positively charged side chain. In some embodiments, the amino acid having a positively charged side chain is selected from the group consisting of H, K, hK, R, Orn, 2,3-diaminopropanoyl, 2,4-diaminobutanoyl, 2-amino-3-guanidinopropionyl, and 4-amino-phenylalanine (F(4-NH2)). Preferably, the amino acid having a positively charged side chain is selected from H, K, R, Orn, 2,3-diaminopropanoyl, 2-amino-3-guanidinopropionyl, and 2,4-diaminobutanoyl. Preferably, the amino acid having a positively charged side chain is selected from H, R, Orn, 2,3-diaminopropanoyl, 2-amino-3-guanidinopropionyl, and 2,4-diaminobutanoyl.

[0083] In some embodiments, at least one amino acid at positions 7, 9, or 10 of SEQ ID NO:1 is substituted with an amino acid having an aromatic side chain. In some embodiments, the amino acid having an aromatic side chain is selected from the group consisting of F, W, and Y. Preferably, the amino acid having an aromatic side chain is Y.

[0084] In some embodiments of the ion channel blockers of the invention, exactly one amino acid at position 7, 8, 9, 10 or 11 of SEQ ID NO:1 is substituted with an amino acid having a positively charged or aromatic side chain. In some embodiments of the ion channel blockers of the invention, exactly one amino acid at position 7, 9 or 10 of SEQ ID NO:1 is substituted with an amino acid having a positively charged or aromatic side chain.

[0085] In some embodiments, the amino acid at position 7 of SEQ ID NO:1 is substituted with an amino acid having a positively charged or aromatic side chain. Preferably, the amino acid at position 7 of SEQ ID NO:1 is substituted with H, K, R, Orn, 2,3-diaminopropanoyl, 2-amino-3-guanidinopropionyl, 2,4-diaminobutanoyl, or Y. Preferably, the amino acid at position 7 of SEQ ID NO:1 is substituted with H, R, Orn, 2,3-diaminopropanoyl, 2-amino-3-guanidinopropionyl, 2,4-diaminobutanoyl, or Y.

[0086] In some embodiments, the amino acid at position 8 of SEQ ID NO:1 is substituted with an amino acid having a positively charged or aromatic side chain, hi some embodiments, the amino acid at position 8 of SEQ ID NO:1 is the same amino acid as position 8 (i.e., A) of SEQ ID NO:1.

[0087] In some embodiments, the amino acid at position 9 of SEQ ID NO:1 is substituted with an amino acid having a positively charged or aromatic side chain. Preferably, the amino acid at position 9 of SEQ ID NO:1 is substituted with K, Orn, or 2,3-diaminopropanoyl. Preferably, the amino acid at position 9 of SEQ ID NO:1 is substituted with Orn or 2,3-diaminopropanoyl.

[0088] In some embodiments, the amino acid at position 10 of SEQ ID NO:1 is substituted with an amino acid having a positively charged or aromatic side chain. Preferably, the amino acid at position 10 of SEQ ID NO:1 is substituted with R.

[0089] In some embodiments, the amino acid at position 11 of SEQ ID NO:1 is substituted with an amino acid having a positively charged or aromatic side chain. Preferably, the amino acid at position 11 of SEQ ID NO:1 is substituted with R. In some embodiments, the amino acid at position 11 of SEQ ID NO:1 is the same amino acid as position 11 (i.e., E) of SEQ ID NO:1.

[0090] Other locations In some embodiments, the residue at position 1 is not Q. Glutamine (Q) residues can be unstable, either in vivo or in vitro, e.g., during storage in aqueous solution, and this may be particularly relevant when the glutamine residue is located at the N-terminus of the molecule, since the side chain can sterically interact with the free alpha amino group, potentially resulting in dehydration to pyroglutamic acid. Preferably, the amino acid at position 1 is N or P, or is deleted (i.e., variants of SEQ ID NO: 1 are 1N, 1P, or 1 * (may include).

[0091] In some embodiments, methionine (M) residues are susceptible to oxidation, so one, two, or all three of the amino acids at positions 2, 4, and 28 are not M. Preferably, one, two, or all three methionine amino acids at positions 2, 4, and 28 are individually substituted with a residue having a non-oxidizable side chain or deleted. Any suitable residue having a non-oxidizable side chain may be used, with Nle, I, V, and L being particularly suitable. Preferably, the amino acids at positions 2, 4, and 28 are all Nle.

[0092] In some embodiments of the ion channel blockers of the invention, in the variant of SEQ ID NO:1: the amino acid at position 1 is N, P, or Q, or is deleted; the amino acid at position 2 is M or Nle or is deleted; the amino acid at position 3 is D or E or is deleted; the amino acid at position 4 is M or Nle or is deleted; the amino acid at position 5 is R or deleted; the amino acid at position 13 is A or K; the amino acid at position 15 is K or S; the amino acid at position 18 is A or K; the amino acid at position 28 is M or Nle; and / or The amino acid at position 30 is G or K.

[0093] In some embodiments of the ion channel blockers of the invention, positions 1-5 of the variant consist of an amino acid sequence selected from NMDMR (SEQ ID NO: 108), N[Nle]D[Nle]R (SEQ ID NO: 109), N[Nle]E[Nle]R (SEQ ID NO: 110), and P[Nle]E[Nle]R (SEQ ID NO: 111), or positions 1-5 are missing all amino acids. Preferably, positions 1-5 of the variant consist of the amino acid sequence P[Nle]E[Nle]R.

[0094] In some embodiments of the ion channel blockers of the invention, in the variant of SEQ ID NO:1: The amino acids at positions 1 to 5 have the amino acid sequence P[Nle]E[Nle]R; the amino acid at position 13 is A or K; the amino acid at position 15 is K or S; The amino acid at position 18 is A, The amino acid at position 28 is Nle; The amino acid at position 30 is G or K.

[0095] In some embodiments of the ion channel blocker of the present invention, one or more of positions 5, 6, 12, 14, 16, 17, 19-27, 29, and 31-37 are the same amino acid as the corresponding positions in SEQ ID NO: 1. Preferably, positions 5, 6, 12, 14, 16, 17, 19-27, 29, and 31-37 are all the same amino acid as the corresponding positions in SEQ ID NO: 1. In some embodiments of the ion channel blocker of the present invention, one or more of positions 5, 6, 8, 11, 12, 14, 16, 17, 19-27, 29, and 31-37 are the same amino acid as the corresponding positions in SEQ ID NO: 1. Preferably, positions 5, 6, 8, 11, 12, 14, 16, 17, 19-27, 29, and 31-37 are all the same amino acid as the corresponding positions in SEQ ID NO: 1.

[0096] Variants of SEQ ID NO:21 In some embodiments, the ion channel blocker of the present invention has the sequence P[Nle]E[Nle]RCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPR (SEQ ID NO: 21) and The variant differs from SEQ ID NO: 21 by 4, 3, 2 or 1 substitutions, At least one amino acid at positions 7, 8, 9, 10 or 11 of SEQ ID NO:21 is substituted with an amino acid having a positively charged side chain and / or an amino acid having an aromatic side chain.

[0097] SEQ ID NO: 21 is a particular variant of SEQ ID NO: 1. As such, variants of SEQ ID NO: 21 are understood to be variants of SEQ ID NO: 1 as described herein.

[0098] In other words, in some embodiments, a variant of SEQ ID NO:1 comprises or consists of a variant of the sequence P[Nle]E[Nle]RCSASVECKQKCLAAIGSIFGKC[Nle]NKKCKCYPR (SEQ ID NO:21); The variant differs from SEQ ID NO: 21 by 4, 3, 2 or 1 substitutions, At least one amino acid at positions 7, 8, 9, 10 or 11 of SEQ ID NO:21 is substituted with an amino acid having a positively charged side chain and / or an amino acid having an aromatic side chain.

[0099] Thus, all features of the embodiments of variants of SEQ ID NO:1 described herein may be applied to variants of SEQ ID NO:21.

[0100] In some embodiments, the variant differs from SEQ ID NO: 21 by four substitutions or by one substitution. In some embodiments, the variant differs from SEQ ID NO: 21 by one substitution.

[0101] In some embodiments, at least one amino acid at positions 7, 9, or 10 of SEQ ID NO:21 is substituted with an amino acid having a positively charged side chain and / or an amino acid having an aromatic side chain. In some embodiments, exactly one amino acid at positions 7, 9, or 10 of SEQ ID NO:21 is substituted with an amino acid having a positively charged side chain and / or an amino acid having an aromatic side chain.

[0102] In some embodiments, the optional substitutions are at amino acid positions selected from 7, 9, 10, 13, 15 and 30 of SEQ ID NO:21.

[0103] In some embodiments, the variant is: (i) differs from SEQ ID NO:21 by one substitution in which the amino acid at position 7 or 9 of SEQ ID NO:21 is replaced with an amino acid having a positively charged side chain and / or an amino acid having an aromatic side chain; or (ii) differs by four substitutions from SEQ ID NO:21, in which exactly one amino acid at position 7 or 10 of SEQ ID NO:21 is substituted with an amino acid having a positively charged side chain and / or an amino acid having an aromatic side chain.

[0104] In some embodiments, the variant is: (i) differs from SEQ ID NO:21 by one substitution in which the amino acid at position 7 or 9 of SEQ ID NO:21 is replaced with an amino acid having a positively charged side chain and / or an amino acid having an aromatic side chain; or (ii) differs from SEQ ID NO:21 by four substitutions, in which exactly one amino acid at position 7 or 10 of SEQ ID NO:21 is substituted with an amino acid having a positively charged side chain and / or an amino acid having an aromatic side chain, and the other three substitutions are at positions 13, 15 and 30 of SEQ ID NO:21.

[0105] In some embodiments, the variant is: (i) differs from SEQ ID NO:21 by one substitution in which the amino acid at position 7 or 9 of SEQ ID NO:21 is replaced with an amino acid having a positively charged side chain and / or an amino acid having an aromatic side chain; or (ii) differs from SEQ ID NO: 21 by four substitutions; Exactly one amino acid at position 7 or 10 of SEQ ID NO: 21 is substituted with an amino acid having a positively charged side chain and / or an amino acid having an aromatic side chain, The other three substitutions are at positions 13, 15, and 30 of SEQ ID NO:21, (a) the amino acid at position 13 is A; and / or (b) the amino acid at position 15 is S; and / or (c) the amino acid at position 30 is G. In some embodiments, the variant is: (i) differs from SEQ ID NO:21 by one substitution, in which the amino acid at position 7 or 9 of SEQ ID NO:21 is replaced with an amino acid selected from the group consisting of H, R, Orn, 2,3-diaminopropanoyl, 2,4-diaminobutanoyl, 2-amino-3-guanidinopropionyl, and Y; or (ii) differs by four substitutions from SEQ ID NO:21, in which exactly one amino acid at position 7 or 10 of SEQ ID NO:21 is substituted with an amino acid selected from the group consisting of R and 2,4-diaminobutanoyl.

[0106] In some embodiments, the variant is: (i) differs from SEQ ID NO:21 by one substitution, in which the amino acid at position 7 of SEQ ID NO:21 is replaced with an amino acid selected from the group consisting of H, R, Orn, 2,3-diaminopropanoyl, 2,4-diaminobutanoyl, 2-amino-3-guanidinopropionyl, and Y, or the amino acid at position 9 of SEQ ID NO:21 is replaced with Orn, or (ii) differs by four substitutions from SEQ ID NO:21, in which exactly one amino acid at position 7 of SEQ ID NO:21 is substituted with an amino acid selected from the group consisting of R and 2,4-diaminobutanoyl, or exactly one amino acid at position 10 of SEQ ID NO:21 is substituted with R.

[0107] array In a preferred embodiment, the ion channel blocker of the invention comprises or consists of one of the following sequences:

[0108] [Table 2]

[0109] In a preferred embodiment, the ion channel blocker of the invention comprises SEQ ID NO: 10 or SEQ ID NO: 12. In a preferred embodiment, the ion channel blocker of the invention consists of SEQ ID NO: 10 or SEQ ID NO:12.

[0110] compound In a preferred embodiment, the ion channel blocker of the invention comprises or consists of one of the following compounds:

[0111] [Table 3]

[0112] In a preferred embodiment, the ion channel blocker of the present invention comprises compound number 1 or compound number 3. In a preferred embodiment, the ion channel blocker of the present invention consists of compound number 1 or compound number 3.

[0113] Disclaimer International Patent Application No. PCT / EP2020 / 076187 discloses ion channel blockers comprising or consisting of the sequences of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8 or 9 (SEQ ID NOs: 25, 27, 42, 47, 63, 143, 144 and 145, respectively, of International Patent Application No. PCT / EP2020 / 076187).

[0114] International Patent Application No. PCT / EP2020 / 076187 also discloses the following compounds:

[0115] [Table 4]

[0116] International Patent Application No. PCT / EP2020 / 076187 does not disclose any other specific compounds comprising or consisting of the sequences of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8 or 9. For example, International Patent Application No. PCT / EP2020 / 076187 does not disclose SEQ ID NOs: 2, 3, 4, 5 or 6 having a C-terminal hydroxyl (-OH) group, or SEQ ID NOs: 7, 8 or 9 having a C-terminal amino group (-NH2).

[0117] Thus, ion channel blockers of the invention include variants of SEQ ID NO:1 that do not include any of the following sequences:

[0118] [Table 5]

[0119] The ion channel blocker of the present invention is not any of compounds 25, 27, 42, 47, 63, 143, 144 or 145 disclosed in International Patent Application No. PCT / EP2020 / 076187.

[0120] In some embodiments of the ion channel blockers of the invention, the variant of SEQ ID NO: 1 does not comprise or consist of the sequence of SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, or 9. In some embodiments, the ion channel blockers of the invention do not comprise any of the sequences disclosed in International Patent Application No. PCT / EP2020 / 076187. In some embodiments, the ion channel blockers of the invention are not any of the compounds disclosed in International Patent Application No. PCT / EP2020 / 076187.

[0121] Methods for synthesizing ion channel blockers The ion channel blockers described herein may be synthesized by solid-phase or solution-phase peptide synthesis methods. In this context, reference may be made to WO 98 / 11125 and in particular to Fields, GB et al., 2002, "Principles and Practice of solid-phase peptide synthesis" in Synthetic Peptides (2nd edition), and to the Examples herein.

[0122] Alternatively, the ion channel blockers described herein may be synthesized by recombinant techniques, or by a combination of recombinant techniques and peptide chemistry.

[0123] Thus, in one embodiment, the present invention provides a method of synthesizing an ion channel blocker of the present invention comprising: (a) synthesizing a peptide by solid-phase or liquid-phase peptide synthesis and recovering the peptide thus obtained; (b) expressing the peptide from a nucleic acid construct encoding the peptide and recovering the expression product; or (c) expressing the precursor peptide from a nucleic acid construct encoding the precursor peptide sequence, recovering the expression product, and modifying the precursor peptide to obtain an ion channel blocker of the invention.

[0124] The precursor peptide may be prepared by the introduction of one or more non-proteinogenic amino acids (e.g., Nle), a suitable terminal group R 1 and R 2 It may be modified, for example, by the introduction of

[0125] Expression of a peptide or precursor peptide from a nucleic acid encoding the peptide or precursor peptide may be carried out in a cell or cell-free expression system containing such nucleic acid. Such expression typically requires that the peptide or precursor peptide is composed entirely of proteinogenic amino acids (i.e., the 20 amino acids encoded by the standard genetic code).

[0126] For recombinant expression, the nucleic acid fragment encoding the precursor peptide will usually be inserted into a suitable vector to form a cloning or expression vector. Depending on the type of purpose and application, the vector can take the form of a plasmid, phage, cosmid, mini-chromosome, or virus, but also naked DNA that is only expressed transiently in a particular cell is an important vector. The preferred cloning and expression vectors (plasmid vectors) are capable of autonomous replication, thereby allowing high copy numbers for high level expression or high levels of replication for subsequent cloning.

[0127] In general overview, expression vectors comprise the following features operably linked in a 5'→3' direction: a promoter for driving expression of the nucleic acid fragment, a nucleic acid sequence encoding an optional leader peptide allowing secretion (to the extracellular phase or optionally the periplasm), a nucleic acid fragment encoding a precursor peptide, and a nucleic acid sequence encoding an optional terminator. They may contain additional features such as selection markers and origins of replication. When working with expression vectors in producer strains or cell lines, it may be preferred that the vector is capable of integrating into the host cell genome. The skilled artisan is familiar with suitable vectors and is able to design vectors according to specific requirements.

[0128] The vectors of the invention are used to transform host cells to produce the peptides or precursor peptides. Such transformed cells can be cultured cells or cell lines used for propagation of the nucleic acid fragments and vectors and / or for recombinant production of the precursor peptides.

[0129] Preferred transformed cells are microorganisms such as bacteria (e.g., Escherichia (e.g., E. coli), Bacillus (e.g., B. subtilis), Salmonella, or Mycobacterium (preferably non-pathogenic, e.g., M. bovis BCG), yeast (e.g., species such as Saccharomyces cerevisiae and Pichia pastoris), and protozoa. Alternatively, the transformed cell may be derived from a multicellular organism, i.e., a fungal cell, an insect cell, an algae cell, a plant cell, or an animal cell, such as a mammalian cell. For purposes of cloning and / or optimized expression, it is preferred that the transformed cell is capable of replicating the nucleic acid fragment of the invention. Cells expressing the nucleic acid fragment may be used for small- or large-scale preparation of the peptide of the invention.

[0130] When transformed cells are used to produce the peptide or precursor peptide, it is convenient, although not at all essential, if the expression product is secreted into the culture medium.

[0131] Pharmaceutical Compositions In another aspect, the present invention provides pharmaceutical compositions comprising an ion channel blocker of the present invention or a pharma- ceutically acceptable salt thereof, hi some embodiments, the pharmaceutical compositions of the present invention comprise an ion channel blocker of the present invention or a pharma- ceutically acceptable salt thereof, and a pharma- ceutically acceptable carrier, excipient, or vehicle.

[0132] Any of the ion channel blockers of the present invention may be in the form of a pharma- ceutically acceptable salt. Any reference to the "ion channel blockers of the present invention" in this specification should be considered to include any pharma- ceutically acceptable salt thereof, regardless of whether "pharma- ceutically acceptable salt" is explicitly stated. The ion channel blockers may also be referred to as "solvates", which means a complex of defined stoichiometry formed between a solute (the peptide according to the present invention or a pharma- ceutically acceptable salt thereof) and a solvent. Solvents in this context are, for example, but are not limited to, water, ethanol, or other pharma- ceutical acceptable, typically small organic species, such as acetic acid or lactic acid. When the solvent in question is water, such solvates are usually referred to as hydrates.

[0133] Thus, a compound of the invention or a salt thereof, particularly a pharma- ceutically acceptable salt thereof, may be prepared for storage or administration and formulated as a composition or pharmaceutical composition comprising a therapeutically effective amount of a compound of the invention or a salt thereof.

[0134] Suitable salts formed with bases include metal salts such as alkali metal salts or alkaline earth metal salts, for example, sodium salts, potassium salts or magnesium salts; ammonia salts and organic amine salts such as those formed with morpholine, thiomorpholine, piperidine, pyrrolidine, lower mono-, di- or tri-alkylamines (e.g., ethyl-tert-butyl-, diethyl-, diisopropyl-, triethyl-, tributyl- or dimethylpropylamine), or lower mono-, di- or tri-(hydroxyalkyl)amines (e.g., mono-, di- or triethanolamine). Internal salts may also be formed. Similarly, when the compound of the present invention contains a basic moiety, salts may be formed using organic or inorganic acids. For example, salts may be formed from the following acids: formic acid, acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, oxalic acid, lactic acid, citric acid, tartaric acid, succinic acid, fumaric acid, maleic acid, malonic acid, mandelic acid, malic acid, phthalic acid, hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid, sulfuric acid, benzoic acid, carbonic acid, uric acid, methanesulfonic acid, naphthalenesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, p-toluenesulfonic acid (i.e., 4-methylbenzenesulfonic acid), camphorsulfonic acid, 2-aminoethanesulfonic acid, aminomethylphosphonic acid, and trifluoromethanesulfonic acid (the latter also referred to as triflic acid), as well as other known pharma- ceutically acceptable acids. Amino acid addition salts may also be formed with amino acids such as lysine, glycine, phenylalanine, and the like.

[0135] In some embodiments, the pharmaceutical compositions of the invention are those in which the compound is in the form of a pharma- ceutically acceptable acid addition salt.

[0136] As will be apparent to those skilled in the medical field, the "therapeutically effective amount" of the compound or pharmaceutical composition of the present invention will vary depending, inter alia, on the age, weight and / or sex of the subject (patient) being treated. Other factors that may be relevant include the physical characteristics of the particular patient under consideration, the patient's diet, the nature of the concomitant medication, the particular compound employed, the particular mode of administration, the desired pharmacological effect, and the particular therapeutic indication. These factors and their relationship in determining this amount are well known in the medical field, so that the determination of therapeutically effective dosage levels to achieve the desired therapeutic effect is within the realm of those skilled in the art.

[0137] The term "therapeutically effective amount" as used herein refers to an amount that reduces the symptoms of a given condition or disease state and preferably normalizes the physiological response of an individual having the condition or disease state. The reduction of symptoms or normalization of physiological response can be determined using methods routine in the art and may vary depending on the given condition or disease state. In one embodiment, a therapeutically effective amount of a compound or pharmaceutical composition of the present invention is an amount that restores a measurable physiological parameter to a value substantially the same as that of an individual not having the condition or disease state in question (preferably within 30%, more preferably within 20%, and even more preferably within 10%).

[0138] In one embodiment of the invention, administration of the compounds or pharmaceutical compositions of the invention begins at lower dosage levels and the dosage levels are increased until the desired effect of preventing / treating the relevant medical indication is achieved, which defines a therapeutically effective amount. For the compounds of the invention alone or as part of a pharmaceutical composition, such human doses of active compound can be between about 0.01 pmol / kg and 500 μmol / kg body weight, between about 0.01 pmol / kg and 300 μmol / kg body weight, between 0.01 pmol / kg and 100 μmol / kg body weight, between 0.1 pmol / kg and 50 μmol / kg body weight, between 1 pmol / kg and 10 μmol / kg body weight, between 5 pmol / kg and 5 μmol / kg body weight, between 10 pmol / kg and 1 μmol / kg body weight, between 50 pmol / kg and 0.1 μmol / kg body weight, between 100 pmol / kg and 0.01 μmol / kg body weight, between 0.001 μmol / kg and 0.5 μmol / kg body weight, between 0.05 μmol / kg and 0.1 μmol / kg body weight.

[0139] Effective dosages and treatment protocols can be determined by conventional means, starting with low doses in experimental animals, then increasing the dosage while monitoring the effects, as well as systematically modifying the dosage regimen. Numerous factors can be considered by the clinician when determining the optimal dosage for a given subject. Such considerations are known to those skilled in the art.

[0140] Medical Uses and Treatment Methods In a further aspect, the present invention provides an ion channel blocker or a pharma- ceutically acceptable salt of the present invention for use in a method of medical treatment, in other words, the present invention provides a method of medical treatment comprising administering to a subject an ion channel blocker or a pharma- ceutically acceptable salt of the present invention.

[0141] The pharmaceutical uses of the ion channel blockers or pharma- ceutically acceptable salts of the invention are described herein. In all cases, the described pharmaceutical uses may also be expressed as methods of treatment, comprising administering to a subject in need of treatment an ion channel blocker or a pharma- ceutically acceptable salt of the invention.

[0142] The terms "patient," "subject," and "individual" may be used interchangeably herein and may refer to either a human or a non-human animal. These terms include mammals such as humans, primates, farm animals (e.g., cows and pigs), companion animals (e.g., dogs and cats), and rodents (e.g., mice and rats).

[0143] As mentioned above, blockers of Kv1.3 have been shown to inhibit the proliferation of activated T cells and have beneficial effects in various experimental disease models. Without wishing to be bound by theory, it is believed that potassium efflux through Kv1.3 channels is necessary to maintain the calcium influx required for T cell activation.

[0144] Kv1.3 is overexpressed in Gad5 / insulin-specific T cells from patients with new-onset type 1 diabetes, myelin-specific T cells from patients with MS, and synovial T cells from patients with rheumatoid arthritis (Beeton et al., Proc Natl Acad Sci USA 103:17414-9, 2006), in breast cancer specimens (Abdul et al., Anticancer Res 23:3347, 2003), and in prostate cancer cell lines (Fraser et al., Pflugers Arch 446:559, 2003).

[0145] Successful results in animal models using Kv1.3 blockers have been reported in models of hypersensitivity to ovalbumin and tetanus toxoid (Beeton et al., Mol Pharmacol 67:1369, 2005; Koo et al., Clin Immunol 197:99, 1999), models of multiple sclerosis such as the rat adoptive transfer experimental autoimmune encephalomyelitis (ATEAE) model (Beeton et al., Proc Natl Acad Sci USA 103:17414-9, 2006), models of inflammatory bone resorption (Valverde et al., J Bone Mineral Res 19:155, 2004), and models of arthritis (Beeton et al., Proc Natl Acad Sci 103: 17414, 2006; Tarcha et al., J. Pharmacol. Exp. Ther. 342: 642, 2012), as well as obesity, diabetes and metabolic disorders (Xu et al., Hum Mol Genet 12:551, 2003; Xu et al., Proc Natl Acad Sci 101: 3112, 2004).

[0146] Topical application of Kv1.3 blockers has been proposed for the treatment of inflammation of the skin and mucous membranes.

[0147] Treating inflammation In some embodiments, the present invention provides an ion channel blocker or a pharma- ceutically acceptable salt of the present invention for use in a method of inhibiting or reducing inflammation.

[0148] In some embodiments, the present invention provides an ion channel blocker or a pharma- ceutically acceptable salt for use in the treatment of an inflammatory condition or disease.

[0149] The inflammatory condition or disease can be any condition or disease in which reduction of inflammation is desirable, for example where inflammation contributes to the symptoms or pathogenesis.

[0150] In some embodiments, the inflammatory condition or disease is an autoimmune disease, an allergy or hypersensitivity disease, an allograft rejection, or a graft-versus-host disease.

[0151] In some embodiments, the inflammatory condition or disease is selected from the group consisting of hay fever, asthma, anaphylaxis, allergic rhinitis, urticaria, eczema, alopecia areata, dermatomyositis, inclusion body myositis, polymyositis, ankylosing spondylitis, vasculitis, arthritis (including rheumatoid arthritis, osteoarthritis, psoriatic arthritis), Sjogren's syndrome, systemic lupus erythematosus (SLE), uveitis, inflammatory fibrosis (including scleroderma, pulmonary fibrosis, cirrhosis of the liver), chronic obstructive pulmonary disease (COPD), and / or chronic myocardial infarction (CHI). disease), hepatitis, chronic inflammatory demyelinating polyneuropathy, inflammatory bowel disease, colitis (including Crohn's disease and ulcerative colitis), erythema, thyroiditis, psoriasis, atopic dermatitis, allergic contact dermatitis, scleroderma, glomerulonephritis, inflammatory bone resorption, multiple sclerosis, type 1 diabetes mellitus, transplant rejection, or graft-versus-host disease.

[0152] metabolic effect Blockers of Kv1.3 may also have beneficial metabolic effects, for example related to energy homeostasis, body weight regulation, and glucose control.

[0153] Thus, the ion channel blockers described herein may be used to inhibit weight gain, promote weight loss, reduce excess weight, or treat obesity (e.g., by controlling appetite, feeding, food intake, caloric intake, and / or energy expenditure), as well as in the treatment of related diseases and conditions, including obesity-related inflammation, obesity-related gallbladder disease, and obesity-induced sleep apnea.

[0154] The effect on body weight can be therapeutic or cosmetic.

[0155] Ion channel blockers may also be used to treat conditions resulting from or associated with impaired glucose control, including metabolic syndrome, insulin resistance, glucose intolerance, prediabetes, elevated fasting glucose, and type 2 diabetes. Some of these conditions may be associated with obesity. Their effects on these conditions may be mediated in whole or in part through their effects on body weight, or may be independent of their effects on body weight.

[0156] Treatment of proliferative cells and cancer Kv1.3 is also expressed in proliferating human and mouse smooth muscle cells. Blockers of Kv1.3 may be effective in smooth muscle proliferative disorders, such as restenosis in patients following vascular surgery (e.g. angioplasty).

[0157] In some embodiments, the present invention provides an ion channel blocker or a pharma- ceutically acceptable salt according to the present invention for use in the treatment of a smooth muscle proliferative disorder. In some embodiments, the disorder is restenosis.

[0158] Further evidence suggests that Kv1.3 channels are involved in the activation and / or proliferation of many types of cells, including tumor cells (Bielanska et al., Curr. Cancer Drug Targets 9:904-14, 2009), microglia (Khanna et al., Am. J. Physiol. Cell Physiol. 280: C796-806, 2001), and differentiation of neural progenitor cells (Wang et al., J. Neurosci. 30:5020-7, 2010). Thus, Kv1.3 blockers may be beneficial in the treatment of neuroinflammatory and neurodegenerative diseases, such as Alzheimer's disease, multiple sclerosis (MS), Parkinson's disease, and amyotrophic lateral sclerosis (ALS) (e.g., following viral infection).

[0159] In some embodiments, the present invention provides an ion channel blocker or a pharma- ceutically acceptable salt of the present invention for use in the treatment of cancer. In some embodiments, the cancer is breast cancer, prostate cancer, or lymphoma. In some embodiments, the lymphoma is non-Hodgkin lymphoma (NHL). Non-Hodgkin lymphoma includes T-cell NHL and B-cell NHL. Forms of B-cell NHL include diffuse large B-cell lymphoma, follicular lymphoma, Burkitt's lymphoma, immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, and mantle cell lymphoma. Forms of T-cell NHL include mycosis fungoides, anaplastic large cell lymphoma, peripheral T-cell lymphoma, precursor T-lymphoblastic lymphoma, and Sezary syndrome.

[0160] Patent Terminology Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings commonly understood by those of ordinary skill in the art. Generally, the nomenclature used in connection with and techniques of chemistry, molecular biology, cell and cancer biology, immunology, microbiology, pharmacology, and protein and nucleic acid chemistry described herein are those well known and commonly used in the art.

[0161] All patents, published patent applications, and non-patent literature referenced in this application are specifically incorporated herein by reference. In the case of conflict, the present specification, including its specific definitions, shall control.

[0162] Each embodiment of the present invention described herein may be employed alone or in combination with one or more other embodiments of the present invention.

[0163] The disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the disclosure. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, any nucleic acid sequence is written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.

[0164] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0165] Throughout this specification, the word "comprise" and its grammatical variations such as "comprises" or "comprising" are understood to imply the inclusion of a stated integer or component, or group of integers or components, but not the exclusion of any other integers or components, or group of integers or components. As used herein, the terms "comprising", "comprises", and "comprised of" are synonymous with "including", "includes", or "containing", "contains", and are inclusive or open-ended, and do not exclude additional, unrecited components, elements, or method steps. The terms "comprising", "comprises", and "comprised of" also encompass the term "consisting of". The term "including" is used to mean "including but not limited to". "Including" and "including but not limited to" may be used interchangeably.

[0166] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application, and nothing herein should be construed as an admission that such publications constitute prior art with respect to the aspects attached hereto.

[0167] The present invention will now be further illustrated by examples which are meant to aid those skilled in the art in practicing the invention and are not intended to limit the scope of the invention in any way. [Example] [Example 1]

[0168] General Peptide Synthesis A list of abbreviations and suppliers is provided in the table below.

[0169] [Table 6]

[0170] Apparatus and synthesis strategy Peptides were synthesized batchwise on a peptide synthesizer such as a CEM Liberty Peptide Synthesizer or Symphony X Synthesizer according to solid-phase peptide synthesis procedures using 9-fluorenylmethyloxycarbonyl (Fmoc) as the N-α-amino protecting group and appropriate common protecting groups for side chain functionalities.

[0171] As a polymer support-based resin, for example, TentaGel™ was used. The resin was filled into a synthesis apparatus and swollen with DMF before use.

[0172] Coupling CEM Liberty Peptide Synthesizer A solution of Fmoc-protected amino acid (4 eq.) was added to the resin along with a solution of coupling reagent (4 eq.) and a solution of base (8 eq.). The mixture was heated to 70-75° C. in a microwave and allowed to couple for 5 min or coupled without heating for 60 min. Nitrogen was bubbled through the mixture during coupling.

[0173] Symphony X Synthesizer The coupling solutions were transferred to the reaction vessel in the following order: amino acid (4 eq.), HATU (4 eq.), DIPEA (8 eq.). Coupling time was 10 min at room temperature (RT) unless otherwise stated. The resin was washed with DMF (5×0.5 min). When couplings were repeated, the coupling time was 45 min at room temperature in all cases.

[0174] Deprotection CEM Liberty Peptide Synthesizer The Fmoc group was deprotected using piperidine in DMF or other suitable solvent. The deprotection solution was added to the reaction vessel and the mixture was heated for 30 seconds until it reached approximately 40° C. The reaction vessel was drained and fresh deprotection solution was added, followed by heating to 70-75° C. for 3 minutes. After draining the reaction vessel, the resin was washed with DMF or other suitable solvent.

[0175] Symphony X Synthesizer Fmoc deprotection was carried out using 40% piperidine in DMF for 2.5 min and repeated using the same conditions. The resin was washed with DMF (5 x 0.5 min).

[0176] Disconnect The dried peptide resin was treated with TFA and appropriate scavengers for approximately 2 hours. The volume of the filtrate was reduced and the crude peptide was precipitated after adding diethyl ether. The crude peptide precipitate was washed several times with diethyl ether and finally dried.

[0177] HPLC purification of crude peptides The crude peptides were purified by preparative reversed-phase HPLC using a conventional HPLC system (such as a Gilson GX-281 with a 331 / 332 pump combination) for binary gradient application with a column such as a 5 x 25 cm Gemini NX 5u C18 110A column and a fraction collector using a flow rate of 20-40 ml / min with a suitable gradient of buffer A (0.1% formic acid, aq.) or A (0.1% TFA, aq.) and buffer B (0.1% formic acid, 90% MeCN, aq.) or B (0.1% TFA, 90% MeCN, aq.). Fractions were analyzed by analytical HPLC and MS, and selected fractions were pooled and lyophilized. The final product was characterized by HPLC and MS.

[0178] Disulfide formation The crude or partially purified linear peptide with six cysteines was dissolved in a buffer such as sodium bicarbonate (NaHCO3) or ammonium acetate (NH4Ac) to a final concentration of approximately 0.1 mg / ml or 25 μM. The pH of the buffer was adjusted to pH 8.0 and the solution was stirred under magnetic stirring at room temperature open to air. The progress of the reaction was determined by HPLC and was typically assessed to be complete overnight. The solution was quenched by lowering the pH of the solution (pH<4) with an organic acid such as acetic acid or trifluoroacetic acid. The solution was filtered and loaded directly onto a preparative HPLC column for purification.

[0179] AnalyticalHPLC Final purity was determined by analytical HPLC (Agilent 1100 / 1200 series) equipped with an autosampler, degasser, 20 μl flow cell, and Chromeleon software. The HPLC was operated at 40° C. with a flow rate of 1.2 ml / min using analytical columns such as Kinetex 2.6 μm XB-C18 100A 100 x 4.6 mm columns. Compounds were detected and quantified at 215 nm. Buffer A (0.1% TFA, aq.) and Buffer B (0.1% TFA, 90% MeCN, aq.).

[0180] mass spectrometry Final MS analysis was performed using a conventional mass spectrometer, e.g., a Waters Xevo G2 Tof equipped with an electrospray detector and lock mass calibration and MassLynx software. Operation was in positive mode using direct injection and cone voltages of 15 V (1TOF), 30 V (2TOF) or 45 V (3TOF) as specified in the chromatogram. Precision was 5 ppm, typical resolution was 15,000-20,000.

[0181] compound The synthesized compounds are shown in Table 1.

[0182] [Table 7]

[0183] The following compounds were also synthesized for use as reference compounds:

[0184] [Table 8] JPEG2024511449000013.jpg196141JPEG2024511449000014.jpg214146 [Example 2]

[0185] Kv1.3 blocker activity in the FLIPR thallium assay The human Kv1.3 voltage-gated K+ channel cell line was purchased from Perkin Elmer (#TDS-AX-010-C-1). This cell line is based on CHO-DUKX cells stably transfected with the human Kv1.3 voltage-gated K+ channel.

[0186] This cell line was grown in MEMα containing nucleotides, GlutaMAX (Gibco #32571028), 10% Foetal Bovine Serum (FBS), 0.4 mg / ml Geneticin, 100 units / ml penicillin, and 100 μg / ml streptomycin, and seeded at 10,000 cells / well in black poly-D-lysine coated 96-well plates.

[0187] FluxOR™ Potassium Ion Channel Assay (Invitrogen #F10016) was used to quantify the influx of thallium ions into cells (thereby generating a fluorescent signal proportional to channel activity) in response to Kv1.3 activation by stimulation buffer, which causes cell membrane depolarization. The assay was performed as described by the assay kit manufacturer, including a pre-incubation step of compounds in assay buffer containing 0.02% w / V casein from bovine milk (Sigma #C4765) prior to cell membrane depolarization using a final stimulation buffer composition corresponding to 0.2X FluxOR™ chloride-free buffer, 5 mM K2SO4, 1 mM Tl2SO4 (30 min, 37° C., 5% CO2). Fluorescence responses were recorded and quantified (maximal response at 50 s, 37° C.) using a FLIPR® Tetra High Throughput Screening System (Molecular Devices, Inc.).

[0188] Data from test compounds inducing inhibition of thallium influx into cells were normalized to the positive control (ShK, 10 nM) and negative control (vehicle), and IC values ​​were calculated from the concentration-response curves using a four-parameter logistic (4PL) nonlinear concentration-response model based on the equation Y = Bottom + (Top-Bottom) / (1+10^((LogIC50-X)*HillSlope)). 50was calculated (where Y is the percent inhibition, X is the compound concentration, and Top, Bottom, Hill Slope, and IC50 are the fitted parameters). The results are shown in Table 3 (n≧2). IC 50 can be taken as a measure of the inhibitory potency for the respective compound. IC 50 The IC value is a measure of the concentration of inhibitor required for the compound to achieve half-maximal inhibition of ion channel activity in a given assay. A compound with a lower IC than a reference compound at a particular ion channel 50 Compounds having a value can be considered to be more active or more potent inhibitors than the reference compound.

[0189] [Table 9] [Example 3]

[0190] Pharmacokinetic characterization method Sprague-Dawley mice (male, weighing approximately 250-350 g) were given a single subcutaneous (sc) injection of each peptide being tested.

[0191] After sc administration of the selected compounds (either at a dose of 100 nmol / kg, or at a dose volume of 2 mL / kg), blood samples were taken at 5, 15, 30, 60 min, 2, 3, 4, 6, and 8 h after dosing. At each sampling time point, samples were taken from the rats by sublingual bleeding. After the last sampling, the rats were sacrificed by O2 / CO2 anesthesia. The dosing vehicle was 10 mM phosphate, 0.8% NaCl, 0.05% polysorbate 20 (pH 6.0).

[0192] Plasma samples were analyzed by liquid chromatography mass spectrometry (LC-MS / MS) after solid phase extraction (SPE). Mean plasma concentrations were used to calculate pharmacokinetic parameters using a noncompartmental approach in Phoenix WinNonlin 6.4 or later versions. Plasma terminal elimination half-life (T 1 / 2 AUC is determined as ln(2) / λz, where λz is the slope of the log-linear regression of the terminal log concentration versus time profile. inf is the area under the plasma concentration-time curve extrapolated to infinity (AUC inf = AUC last + C last / λz, where C last is the last observed plasma concentration). Cmax is the maximum observed concentration occurring at Tmax. Results for selected compounds are shown in Table 4.

[0193] [Table 10]

[0194] Pharmacokinetic characterization of compound 14 was performed according to the methods described in Example 6 of International Patent Application No. PCT / EP2020 / 076187. The results for compound 13 are shown in Table 5 below.

[0195] [Table 11] [Example 4]

[0196] Kv1.3 selectivity in patch clamp assays Chinese Hamster Ovary (CHO) cell lines stably expressing exogenous human α-subunits of each potassium ion channel were grown and passaged under standard culture conditions.

[0197] To quantify ionic currents, we used the QPatch®, an automated, tip-based planar patch clamp device. All recordings were made in a conventional whole-cell configuration after establishing a gigaohm seal. The external recording solution contained (150 mM NaCl, 10 mM KCl, 10 mM HEPES, 1 mM MgCl2, 3 mM CaCl2, 10 mM glucose, pH 7.4 adjusted with NaOH), and the internal recording solution contained (20 mM KCl, 120 mM KF, 10 mM HEPES, 10 mM EGTA, 5 mM NaATP, pH 7.2 adjusted with KOH). All external recording solutions contained 0.1% (v / v) BSA as vehicle during the experiments. Currents were elicited using a voltage protocol that shifted the voltage to 30 mV for 500 ms every 15 seconds from a holding potential of -80 mV.

[0198] Concentration-response relationships were established by cumulatively applying seven increasing concentrations of test compounds to individual cells, with a 2 min recording period for each compound application.

[0199] Efficacy was determined as the average charge of the last three sweeps at the end of each concentration application period from the cursor position. The percent inhibition of each test dose application period was calculated as the decrease in the average cursor value (charge) relative to the cursor value measured at the end of the vehicle period and was calculated from the concentration-response curve as the IC 50 was used to calculate

[0200] The results are shown in Table 6.

[0201] [Table 12]

[0202] Both compounds 1 and 3 exhibited extremely high selectivity for Kv1.3, with observed selectivity ratios for Kv1.1, Kv1.2, and Kv1.6 of greater than 6000. Thus, no detectable inhibition of these ion channels is expected at therapeutically relevant concentrations of compounds 1 or 3.

[0203] The selectivity of the reference compounds was also determined using the same method, and the results are shown in Table 7.

[0204] [Table 13] JPEG2024511449000020.jpg172132 [Example 5a]

[0205] Inhibitory activity of Kv1.3 blocker reference compounds on human PBMC Human peripheral blood mononuclear cells (PBMC) were used to assess the effect of Kv1.3 blocker reference compounds on T cell activation as determined by IL-2 (a cytokine) release after stimulation with anti-CD3.

[0206] Human PBMCs were obtained from Precision for Medicine (Frederick, MD). Cells from five donors were used. Plate-bound anti-CD3 was used to stimulate bulk T cells in the PBMC preparation. Briefly, 96-well plates were coated with anti-CD3 antibody for 2 hours at 37°C using 50 μL of 0.5 μg / mL anti-CD3 solution diluted in 1× PBS. Plates were then washed twice.

[0207] Kv1.3 blockers shown in Table 8a were diluted in culture medium (RPMI1640 with Glutamax-I containing 10% v / v fetal bovine serum, 1% v / v penicillin-streptomycin solution) and added in a volume of 100 μl at concentrations ranging from 0.01 pM to 100 nM (10-fold dilutions). Cyclosporine A (1 ug / ml) and Vm24 peptide (100 nM) were used as positive controls. Finally, 1 × 10 5PBMCs were added to each well in a volume of 100 μL, resulting in a final volume of 200 μL per well. Plates were incubated for 20–24 h in a 37°C / 5% CO2 incubator. After centrifugation of the plates, 25 μl of the supernatant was transferred to an IL-2 detection plate (MSD Human IL-2 Tissue Culture Kit, Cat. No. K151AHB-2) and IL-2 was measured according to the manufacturer's instructions (Meso Scale Discovery, Rockville, MD, USA).

[0208] Results are summarized in IC obtained from anti-CD3 stimulated human PBMC assays. 50 The geometric means of the values ​​are shown in Table 8a. All values ​​are from at least four replicates.

[0209] [Table 14]

[0210] Incubation with anti-CD3 antibody activated hPBMCs, and addition of Kv1.3 blockade resulted in a dose-dependent reduction in IL-2 secretion. On average, the IC 50 The IC values ​​(calculated from IL-2 release) ranged from 0.05 nM to 0.4 nM. 50 The IC observed was 0.07 nM. 50 and the IC of Moka1, which is less potent in inhibiting IL-2 secretion 50 was approximately 10 to 100 times lower than that

[0211] There was no significant difference between the test compounds and ShK186. ShK186 and the test compounds were all significantly lower than Moka1.

[0212] Cyclosporine completely blocked CD3-induced IL-2 release in all experiments. [Example 5b]

[0213] Inhibitory activity of Kv1.3 blocker reference compounds on human PBMC Human peripheral blood mononuclear cells (PBMCs) were used to assess the effect of Kv1.3 blocker reference compounds on T cell activation as determined by IL-2 release following stimulation with anti-CD3.

[0214] Human PBMCs were obtained from Precision for Medicine (Frederick, MD). Cells from five donors were used. Plate-bound anti-CD3 was used to stimulate bulk T cells in the PBMC preparation. Briefly, 96-well plates were coated with anti-CD3 antibody for approximately 16 hours at 5°C using 50 μl of 1 μg / ml anti-CD3 solution diluted in PBS. Plates were then washed twice.

[0215] Subsequently, Kv1.3 blockers were diluted in culture medium (RPMI1640 with Glutamax-I containing 10% v / v fetal bovine serum, 1% v / v penicillin-streptomycin solution) and added in a volume of 50 μl. Compounds shown in Table 8b were used at concentrations ranging from 0.3 pM to 1000 nM (half log dilutions, starting concentrations varied). Cyclosporin A (1 μg / ml) and Vm24 peptide (100 nM) were used as positive controls.

[0216] Finally, 50,000 PBMCs in the same medium were added to each well in a volume of 50 μl, resulting in a final volume of 100 μl per well. The plates were incubated for 20–24 h in a 37°C / 5% CO2 incubator. After centrifugation of the plates, 25 μl of the supernatant was transferred to an IL-2 detection plate (MSD Human IL-2 Tissue Culture Kit, Cat. No. K151AHB-2) and IL-2 was measured according to the manufacturer's instructions (Meso Scale Discovery, Rockville, MD, USA).

[0217] Results are shown in Table 8b as the geometric mean of IC50 values ​​obtained from anti-CD3 stimulated human PBMC assays, all values ​​are from at least six replicates.

[0218] [Table 15]

[0219] Incubation with anti-CD3 antibody activated hPBMCs and addition of a compound of the invention resulted in a dose-dependent reduction in IL-2 secretion.

[0220] As shown in Table 8b, the mean IC 50 The IC values ​​(calculated from IL-2 release) ranged from 0.01 nM to 0.09 nM, which is comparable to the IC observed with ShK186. 50 (I C 50 The Shk-186 values ​​in the two sets of experiments are not expected to be identical, since this assay was performed using a different donor than that used in Example 5a.

[0221] Cyclosporine completely blocked anti-CD3-induced IL-2 release in all experiments. [Example 6]

[0222] Inhibitory activity of Kv1.3 blocker reference compounds in rat whole blood Rat whole blood was used to assess the potency of Kv1.3 blocker reference compounds on T cell activation as determined by IL-17A release after thapsigargin stimulation. Addition of thapsigargin leads to activation of a signaling cascade, ultimately resulting in T cell proliferation and activation of cytokine production, in which the Kv1.3 ion channel plays a key role, and therefore the activity of Kv1.3 blockers in primary cells can be measured in this experimental system.

[0223] Rat whole blood was obtained by terminally bleeding healthy naïve Lewis or Sprague-Dawley rats from the heart and using sodium heparin blood collection tubes for collection. Test compounds were diluted to 4x the final test concentration in assay buffer (DMEM+GlutaMAX), a medium containing 25 mM HEPES buffer, 1 mM sodium pyruvate, 100 units / ml penicillin, 100 μg / ml streptomycin, and 3.97 mM L-alanine-L-glutamine (Gibco catalog number 61965026) supplemented with 0.05% casein from bovine milk (Sigma-Aldrich), and 25 μl was added to wells of a 96-well plate. 50 μl of rat whole blood was then added and incubated at room temperature for a minimum of 5 minutes to allow compound binding. Then, 25 μl of 40 μM thapsigargin diluted in assay buffer was added to all wells of the assay plate to activate the cells, followed by incubation for 24 h at 37° C. / 5% CO2 in a humidified box. The assay plate was centrifuged at 300 g for 10 min at 4° C. and the supernatant was transferred to a new plate. The concentration of IL-17A released into the supernatant was measured using the Rat IL-17A ELISA Kit (Abcam Cat. No. ab214028) according to the manufacturer's recommendations. Samples were diluted 2.5-fold by transferring 20 μl of supernatant into wells of the ELISA plate containing 30 μl of detection kit buffer 75BS.

[0224] Data for test compounds inducing inhibition of IL-17A were normalized relative to full thapsigargin activation (no blocker added) and to a no-activation control (assay buffer added instead of thapsigargin), and IC values ​​were calculated from concentration-response curves. 50 was calculated.

[0225] The results are expressed as standard deviations (IC 50 IC with _SD) 50 The results are shown in Table 9 as mean ± S. All values ​​are from at least two replicates. The ex vivo biological effects correlate with compound potency.

[0226] [Table 16] JPEG2024511449000024.jpg225135 [Example 7]

[0227] Effect of Kv1.3 blocker reference compound treatment in the rat keyhole limpet hemocyanin (KLH) ear inflammation model Classical delayed-type hypersensitivity (DTH) responses were elicited in one ear of rats. Briefly, 8-10 week-old male Lewis rats were immunized on day -7 subcutaneously at the base of the tail with 200 μL of keyhole limpet hemocyanin (KLH) (Sigma, Cat. No. H7017) (4 mg / mL) emulsified in complete Freund's adjuvant (CFA, Difco, Cat. No. 263810). On day 0, rats were challenged intradermally in the left ear with 40 μL of KLH / NaCl 0.9% (2 mg / mL). Following ear challenge, rats developed T cell-dependent inflammation in the KLH-challenged left ear. The right ear remained uninflamed and served as a control.

[0228] The ability of Kv1.3 blocker reference compound treatment to reduce the DTH ear swelling response was investigated by comparing the responses of rats treated with vehicle and Kv1.3 blocker (n=8-10 / group). Vehicle or Kv1.3 blocker dissolved in vehicle was administered SC (2mL / kg) 24 hours prior to KLH ear exposure. The test doses of Kv1.3 blocker were 50, 70, or 100 nmol / kg. Test vehicle was 10 mM phosphate, 0.8% w / v NaCl, 0.05% w / v polysorbate 20, pH 6. In all experiments, cyclosporine (CsA) was included as a positive control in the study. Cyclosporine (Sandimmune Neoral® 100 mg / mL oral solution, Novartis) was administered orally (10 mg / kg) 1 hour before KLH ear exposure and again 6 hours after KLH ear exposure.

[0229] As a primary readout of efficacy, the Area Under Curve (AUC) of ΔEar Thickness (mm) was calculated for each animal 0-48 hours after induction of the ear DTH response and the change (D) was calculated as: Left Ear Thickness - Right Ear Thickness. These results were then used to calculate the % inhibition of ear thickness due to Kv1.3 blocker treatment: % Inhibition: ((1-(individual ΔAUC Kv1.3 blocker / mean ΔAUC vehicle group)) x 100. Results were calculated as % inhibition + / - standard deviation (SD) and are shown in Tables 10 and 11.

[0230] [Table 17]

[0231] [Table 18]

[0232] All publications mentioned in the above specification are incorporated herein by reference. Various modifications and variations of the described methods and systems of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in biochemistry, molecular biology, or related fields are intended to be within the scope of the following claims.

Claims

1. comprising a variant of the sequence QMDMRCSASVECKQKCLKAIGSIFGKCMNKKCKCYPR (SEQ ID NO: 1), said variant differs from SEQ ID NO: 1 by a total of up to 10 substitutions, insertions or deletions, At least one amino acid at positions 7, 8, 9, 10 or 11 of SEQ ID NO:1 is substituted with an amino acid having a positively charged side chain and / or an amino acid having an aromatic side chain, said variant having the following sequence: 【Table 1】 An ion channel blocker having Kv1.3 inhibitor activity, or a pharma- ceutically acceptable salt thereof, which does not include any of the following:

2. The ion channel blocker of claim 1, wherein any substitution or deletion in the variant of SEQ ID NO:1 is at an amino acid position selected from 1 to 5, 7 to 11, 13 to 15, 17 to 23, 25, 28 to 31, 33 and 35 to 37 of SEQ ID NO:1, preferably at an amino acid position selected from 1 to 5, 7 to 11, 13, 15, 18, 28 and 30 of SEQ ID NO:

1.

3. IC for human Kv1.3 potassium channel of 50 nM or less, such as 20 nM or less, for example 10 nM or less, such as 5 nM or less, for example 2 nM or less, such as 1 nM or less, for example 0.5 nM or less, such as 0.4 nM or less, for example 0.3 nM or less, such as 0.2 nM or less ５０ The ion channel blocker of claim 1 or 2, having the formula:

4. Amino acids with positively charged side chains are H, K, hK, R, Orn, 2,3-diaminopropanoyl, 2,4-diaminobutanoyl, 2-amino-3-guanidinopropionyl, and 4-amino-phenylalanine (F(4-NH ２ )), preferably selected from the group consisting of H, K, R, Orn, 2,3-diaminopropanoyl, 2-amino-3-guanidinopropionyl, and 2,4-diaminobutanoyl; and / or The amino acid having an aromatic side chain is selected from the group consisting of F, W and Y, preferably Y; The ion channel blocker according to any one of claims 1 to 3.

5. The amino acid at position 7 of SEQ ID NO:1 is substituted with an amino acid having a positively charged or aromatic side chain, preferably H, K, R, Orn, 2,3-diaminopropanoyl, 2-amino-3-guanidinopropionyl, 2,4-diaminobutanoyl, or Y; the amino acid at position 8 of SEQ ID NO:1 is substituted with an amino acid having a positively charged or aromatic side chain, or the amino acid at position 8 of the variant is the same as the amino acid at position 8 of SEQ ID NO:1, i.e., A; The amino acid at position 9 of SEQ ID NO:1 is substituted with an amino acid having a positively charged or aromatic side chain, preferably K, Orn, or 2,3-diaminopropanoyl; The amino acid at position 10 of SEQ ID NO:1 is substituted with an amino acid having a positively charged or aromatic side chain, preferably R; and / or The amino acid at position 11 of SEQ ID NO:1 is substituted with an amino acid having a positively charged or aromatic side chain, preferably R; The ion channel blocker according to any one of claims 1 to 4.

6. In the variant of SEQ ID NO: 1, the amino acid at position 1 is N, P, or Q, or is deleted; the amino acid at position 2 is M or Nle or is deleted; the amino acid at position 3 is D or E or is deleted; the amino acid at position 4 is M or Nle or is deleted; the amino acid at position 5 is R or deleted; The amino acid at position 13 is A or K; the amino acid at position 15 is K or S; The amino acid at position 18 is A or K; the amino acid at position 28 is M or Nle; and / or The amino acid at position 30 is G or K; The ion channel blocker according to any one of claims 1 to 5.

7. In the variant of SEQ ID NO: 1, the amino acids at positions 1 to 5 consist of the amino acid sequence P[Nle]E[Nle]R; the amino acid at position 13 is A or K; the amino acid at position 15 is K or S; the amino acid at position 18 is A; the amino acid at position 28 is Nle; and The amino acid at position 30 is G or K; The ion channel blocker according to any one of claims 1 to 6.

8. A variant of SEQ ID NO:1 has the following sequence: 【Table 2】 The ion channel blocker according to any one of claims 1 to 7, comprising or consisting of one of:

9. The variant is the compound: 【Table 3】 The ion channel blocker according to any one of claims 1 to 8, comprising or consisting of one of:

10. An ion channel blocker which is H-P[Nle]E[Nle]RC(1)[2,4-diaminobutanoyl]ASVEC(2)KQKC(3)LAAIGSIFGKC(1)[Nle]NKKC(2)KC(3)YPR-OH(Cpd.1) (SEQ ID NO:10), or a pharma- ceutically acceptable salt thereof, wherein a pair of cysteine ​​residues which are both involved in a disulfide bond are indicated by the same number in parentheses.

11. An ion channel blocker which is H-P[Nle]E[Nle]RC(1)[2-amino-3-guanidinopropionyl]ASVEC(2)KQKC(3)LAAIGSIFGKC(1)[Nle]NKKC(2)KC(3)YPR-OH(Cpd.3) (SEQ ID NO:12), or a pharma- ceutically acceptable salt thereof, wherein a pair of cysteine ​​residues which are both involved in a disulfide bond are indicated by the same number in parentheses.

12. An ion channel blocker which is H-P[Nle]E[Nle]RC(1)HASVEC(2)KQKC(3)LAAIGSIFGKC(1)[Nle]NKKC(2)KC(3)YPR-OH(Cpd.4) (SEQ ID NO:13), or a pharma- ceutically acceptable salt thereof, wherein a pair of cysteine ​​residues which are both involved in a disulfide bond are indicated by the same number in parentheses.

13. An ion channel blocker which is H-P[Nle]E[Nle]RC(1)[2,3-diaminopropanoyl]ASVEC(2)KQKC(3)LAAIGSIFGKC(1)[Nle]NKKC(2)KC(3)YPR-OH(Cpd.7) (SEQ ID NO:16), or a pharma- ceutically acceptable salt thereof, wherein a pair of cysteine ​​residues which are both involved in a disulfide bond are indicated by the same number in parentheses.

14. A pharmaceutical composition comprising the ion channel blocker or a pharma- ceutically acceptable salt thereof according to any one of claims 1 to 13.

15. A pharmaceutical composition according to claim 14 for use in the treatment or prevention of a disease.

16. Use of an ion channel blocker or a pharma- ceutically acceptable salt thereof according to any one of claims 1 to 13, or a pharmaceutical composition according to claim 14, in the manufacture of a medicament for treating a disease.

17. The disease is (a) an inflammatory condition or disease, preferably the inflammatory condition or disease is an autoimmune disease, an allergy or hypersensitivity, an allograft rejection, a graft versus host disease, hay fever, asthma, anaphylaxis, allergic rhinitis, urticaria, eczema, alopecia areata, dermatomyositis, inclusion body myositis, polymyositis, ankylosing spondylitis, vasculitis, arthritis, rheumatoid arthritis, osteoarthritis, psoriatic arthritis, Sjogren's syndrome group, systemic lupus erythematosus (SLE), uveitis, inflammatory fibrosis, scleroderma, pulmonary fibrosis, liver cirrhosis, chronic obstructive pulmonary disease (COPD), hepatitis, chronic inflammatory demyelinating polyneuropathy, inflammatory bowel disease, colitis, Crohn's disease, ulcerative colitis, erythema, thyroiditis, psoriasis, atopic dermatitis, allergic contact dermatitis, scleroderma, glomerulonephritis, inflammatory bone resorption, multiple sclerosis, type 1 diabetes, or transplant rejection; (b) obesity, obesity-related inflammation, obesity-related gallbladder disease, or obesity-induced sleep apnea; (c) a condition caused by or associated with impaired glucose regulation, preferably said condition caused by or associated with impaired glucose regulation is metabolic syndrome, insulin resistance, glucose intolerance, prediabetes, elevated fasting glucose, or type 2 diabetes; (d) a smooth muscle proliferative disorder, preferably wherein the smooth muscle proliferative disorder is restenosis or angioplasty; (e) a neuroinflammatory or neurodegenerative disease, preferably wherein the neuroinflammatory or neurodegenerative disease is Alzheimer's disease, multiple sclerosis (MS), Parkinson's disease, or amyotrophic lateral sclerosis (ALS); and / or (f) cancer, preferably said cancer is breast cancer, prostate cancer, or lymphoma, preferably said lymphoma is non-Hodgkin's lymphoma (NHL), preferably said NHL is T-cell NHL or B-cell NHL, preferably said T-cell NHL is mycosis fungoides, anaplastic large cell lymphoma, peripheral T-cell lymphoma, precursor T-lymphoblastic lymphoma, or Sézary syndrome, preferably said B-cell NHL is diffuse large B-cell lymphoma, follicular lymphoma, Burkitt's lymphoma, immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, or mantle cell lymphoma; A pharmaceutical composition for the use according to claim 15 or the use according to claim 16.

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