inhibitors

Abstract

The present invention relates to the use of inhibitors of the potassium channel Kv1.3 in a method of treatment, in particular the method comprising the step of administering the inhibitor to a subject at specific intervals.

Description

[Technical Field]

[0001] The present invention relates to the use of inhibitors of the potassium channel Kv1.3 in a method of treatment, in particular the method comprising the step of administering the inhibitor to a subject at specific intervals. [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 ion passage 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. Inhibitors of Kv1.3 have been shown to inhibit the 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, for example, 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] Inhibitors 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 required to maintain the calcium influx necessary for T cell activation.

[0007] 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).

[0008] Successful results have been achieved in animal models using Kv1.3 inhibitors, including models of hypersensitivity to ovalbumin and tetanus toxoid (Beeton et al., Mol Pharmacol 67:1369, 2005; Koo et al., Clin Immunol 197:99, 1999), multiple sclerosis models such as the rat adoptive transfer experimental autoimmune encephalomyelitis (ATEAE) model (Beeton et al., Proc Natl Acad Sci USA 103:17414-9, 2006), inflammatory bone resorption models (Valverde et al., J Bone Mineral Res 19:155, 2004), and arthritis models (Beeton et al., Proc Natl Acad Sci 103: 17414, 2006; Tarcha et al., J. Pharmacol. Exp. Ther. 342: 642, 2006). 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). Topical application of Kv1.3 blockers has been proposed for the treatment of inflammation of the skin and mucous membranes.

[0009] Kv1.3 inhibitors therefore have considerable potential for use in the treatment of diseases and disorders, particularly inflammatory disorders such as autoimmune diseases.

[0010] 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.

[0011] Kv1.3 inhibitors 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 a high-fat diet compared with control littermates (Xu et al., Hum. Mol. Genet. 12:551-9, 2003). Furthermore, Kv1.3 inhibitors have been shown to increase the expression of glucose transporter 4 (GLUT4) 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).

[0012] Kv1.3 is also expressed in proliferating human and mouse smooth muscle cells. Kv1.3 inhibitors may be effective in smooth muscle proliferative disorders, such as restenosis in patients after vascular surgery (e.g., angioplasty). Kv1.3 inhibitors 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).

[0013] 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). Therefore, Kv1.3 inhibitors may be beneficial for the treatment of neuroinflammatory diseases, neurodegenerative diseases, and cancer.

[0014] Kv1.3 is part of a closely related subfamily of potassium channels designated Kv1.1 through Kv1.8. When dealing with a large, homologous family, it is always desirable for inhibitors to be as selective and specific as possible for the desired target to improve efficacy and safety and avoid unwanted off-target effects. The most specific Kv1.3 inhibitors identified to date are toxic peptides derived from various types of venomous organisms, including snakes, arachnids (such as scorpions and spiders), and sea anemones. Such Kv1.3 inhibitors include the peptides ShK, Oskl, margatoxin, and kaliotoxin, 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.

[0015] 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. [Prior art documents] [Patent documents]

[0016] [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 Pamphlet [Patent Document 5] International Publication No. 2008 / 088422 Pamphlet [Patent Document 6] International Publication No. 2006 / 116156 Pamphlet [Patent Document 7] International Publication No. 2010 / 105184 Brochure [Patent Document 8] International Publication No. 2014 / 116937 Brochure [Patent Document 9] PCT / EP2020 / 076187 [Non-patent literature]

[0017] [Non-Patent Document 1] Cahalan and Chandy, Immunol.Rev. 231:59-87, 2009 [Non-patent document 2] Rangaraju et al. (Expert Opin. Ther. Targets 13:909-24, 2009) [Non-licensed document 3] Beeton et al. (Proc. Natl. Acad. Sci. US A. 103:17414-9, 2006)

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Non-licensed literature 9

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[0018] The present invention relates to a Kv1.3 inhibitor derived from a venom peptide of the scorpion Parabuthus transvaalicus, which has the amino acid sequence QMDMRCSASVECKQKCLKAIGSIFGKCMNKKCKCYPR (SEQ ID NO: 1).

[0019] Among other desirable properties, this molecule and its derivatives or variants have been found to be highly selective inhibitors of the Kv1.3 potassium ion channel over other voltage-gated potassium channels, and typically also have high potency for blocking the Kv1.3 channel. Peptides that are highly selective for the Kv1.3 channel, such as those derived from PaT1, have the particular advantage of selectively targeting cells that express the Kv1.3 channel, such as specific populations of effector memory T cells. This selectivity therefore confers the potential for targeted therapeutic effects on PaT1-derived Kv1.3 inhibitors.

[0020] Kv1.3 inhibitors based on PaT1 toxin peptides have a short half-life in the subject's body. For example, Example 6 herein shows that such Kv1.3 inhibitors have a half-life of approximately 1 hour in a rat model organism. However, the inventors surprisingly found that despite this short half-life, the Kv1.3 inhibitors described herein have a long-lasting effect on T cells. In particular, Example 8 herein shows that inflammation is reduced in a rat ear inflammation model for up to 7 days after treatment with a Kv1.3 inhibitor. In addition, Example 9 herein shows that treatment with a Kv1.3 inhibitor once every 5 days reduces inflammation in a rat arthritis model. Without wishing to be bound by theory, it is hypothesized that Kv1.3 inhibitors, through their interaction with the Kv1.3 ion channel, somehow "reprogram" T cells to remain in an inactive state for a long period of time, thereby suppressing inflammation for several days.

[0021] These surprising findings support the concept of treating subjects suffering from diseases, conditions, or disorders treatable using the Kv1.3 inhibitors described herein according to a regimen in which the inhibitor is administered at approximately weekly intervals (i.e., once every 2-8 days). Increasing the period between administrations of the active compound as much as possible is advantageous because it saves time and effort (since fewer administrations need to be performed in a given period) and costs (since less active compound needs to be used in a given period). Particularly for active compounds administered by certain routes, such as subcutaneous injection, longer intervals between administrations may also increase patient comfort and thereby patient compliance with the treatment regimen. For example, administering a Kv1.3 inhibitor according to the present invention (i.e., once every 2-8 days) is superior to administering the inhibitor daily for the reasons described above. An unexpected finding described in the present disclosure is that it is possible to administer the Kv1.3 inhibitors described herein once every 2-8 days, despite their short in vivo half-lives, such that the effects of the inhibitors (e.g., reduced inflammation) are observed for several days after administration.

[0022] Accordingly, the present invention provides a Kv1.3 inhibitor, or a pharmaceutically acceptable salt thereof, for use in a method for treating or preventing a disease or disorder in a subject, wherein the Kv1.3 inhibitor comprises or consists of a peptide comprising or consisting of the sequence QMDMRCSASVECKQKCLKAIGSIFGKCMNKKCKCYPR (SEQ ID NO: 1) or a variant thereof, wherein the variant (a) has at least 65% sequence identity to SEQ ID NO: 1 and / or (b) differs from SEQ ID NO: 1 by a total of up to 9 substitutions, insertions and / or deletions, and the method comprises the step of administering the Kv1.3 inhibitor or a pharmaceutically acceptable salt thereof to the subject once every 2 to 8 days. [Brief explanation of the drawings]

[0023] [Figure 1] Figure 1 shows the study design of the keyhole limpet hemocyanin (KLH)-induced delayed-type hypersensitivity (DTH) model to investigate the effect of a Kv1.3 inhibitor (peptide 100) on ear swelling after ear challenge on days 7, 9, 11, or 13. For each ear challenge, ear edema was measured 24 or 48 hours after challenge. [Figure 2] Figure 2 shows the effect of Peptide 100 administered at 300 nmol / kg on day 6 on ear swelling measured 24 hours after ear challenge on days 7, 9, 11, or 13. Data are presented as individual and mean values ​​(n=8 rats / group). Mean values ​​for vehicle- and Peptide 100-treated animals were compared by two-tailed unpaired t-test for each ear challenge time point. [Figure 3]Figure 3 shows the effect of Peptide 100 administered at 300 nmol / kg on day 6 on ear swelling upon ear challenge on days 7, 9, 11, or 13, measured 48 hours after challenge. Data are presented as individual and mean values ​​(n=8 rats / group). Mean values ​​for vehicle- and Peptide 100-treated animals were compared by two-tailed unpaired t-test for each ear challenge time point. [Figure 4] FIG. 4 shows the clinical scores of the forelimbs in the rat collagen-induced arthritis (CIA) model described in Example 9. [Figure 5] Figure 5 shows the study design of the keyhole limpet hemocyanin (KLH)-induced delayed-type hypersensitivity (DTH) model to investigate the effect of different doses of a Kv1.3 inhibitor (peptide 100) on ear swelling after ear exposure on days 7 or 11. For each ear exposure, ear edema was measured 24 or 48 hours after exposure. [Figure 6] Figure 6 shows the effect of Peptide 100 administered at 10, 100, 300, or 700 nmol / kg on day 6 on ear swelling upon ear challenge on days 7 or 11, measured 24 hours after challenge. Data are presented as individual and mean values ​​(n=8 rats / group). Mean values ​​for vehicle- and Peptide 100-treated animals were compared by two-tailed unpaired t-test for each ear challenge time point. [Figure 7] Figure 7 shows the effect of Peptide 100 administered at 10, 100, 300, or 700 nmol / kg on day 6 on ear swelling upon ear challenge on days 7 or 11, measured 48 hours after challenge. Data are presented as individual and mean values ​​(n=8 rats / group). Mean values ​​for vehicle- and Peptide 100-treated animals were compared by two-tailed unpaired t-test for each ear challenge time point. [Figure 8]Figure 8 shows the effect of Peptide 100 administered at 1, 3, 10, 30, or 100 nmol / kg on day 6 on ear swelling upon ear challenge on days 7 or 11, measured 24 hours after challenge. Data are presented as individual and mean values ​​(n=8 rats / group). Mean values ​​for vehicle- and Peptide 100-treated animals were compared by two-tailed unpaired t-test for each ear challenge time point. [Figure 9] Figure 9 shows the effect of Peptide 100 administered at 1, 3, 10, 30, or 100 nmol / kg on day 6 on ear swelling upon ear challenge on days 7 or 11, measured 48 hours after challenge. Data are presented as individual and mean values ​​(n=8 rats / group). Mean values ​​for vehicle- and Peptide 100-treated animals were compared by two-tailed unpaired t-test for each ear challenge time point. DETAILED DESCRIPTION OF THE INVENTION

[0024] The present invention provides a Kv1.3 inhibitor, or a pharmaceutically acceptable salt thereof, for use in a method for treating or preventing a disease or disorder in a subject, wherein the Kv1.3 inhibitor comprises or consists of a peptide comprising or consisting of the sequence QMDMRCSASVECKQKCLKAIGSIFGKCMNKKCKCYPR (SEQ ID NO: 1) or a variant thereof, wherein the variant (a) has at least 70% sequence identity to SEQ ID NO: 1 and / or (b) differs from SEQ ID NO: 1 by a total of up to 9 substitutions, insertions and / or deletions, and the method comprises the step of administering the Kv1.3 inhibitor or a pharmaceutically acceptable salt thereof to the subject once every 2 to 8 days.

[0025] The peptide can be any peptide described herein. The disease or disorder can be any disease or disorder described herein.

[0026] Kv1.3 inhibitors The present invention provides Kv1.3 inhibitors comprising or consisting of peptides.

[0027] The term "Kv1.3 inhibitor" is used herein to refer to a molecule or compound that has inhibitory (or blocking) activity against the Kv1.3 ion channel, i.e., a molecule or compound that can inhibit or eliminate the flow of ions through the Kv1.3 ion channel, such as by binding to the ion channel. As used herein, the term "blocker" is synonymous with the term "inhibitor." Thus, the Kv1.3 inhibitors of the present invention may also be referred to herein as "ion channel blockers." 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 terms "Kv1.3 inhibitor" and "compound" are used interchangeably herein. The term "Kv1.3" is used to refer to potassium voltage-gated channel subfamily A member 3, which is also known as KCNA3, HPCN3, HGK5, HuKIII, and HLK3. "Subfamily A" is sometimes also referred to as the "Shaker-related subfamily." The human amino acid sequence of Kv1.3 is provided in UniProt accession number P22001, version P22001.3(Q5VWN2).

[0029] The 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, the 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 over other ion channels, particularly voltage-gated potassium channels such as Kv1.1, Kv1.2, Kv1.4, Kv1.5, Kv1.6, Kv1.7, and Kv1.8. Further details regarding Kv1.3 and known inhibitors of Kv1.3 can be found in Murray et al. J Med Chem 2015, 58, 17, 6784-6802 and Tanner et al. Clin Immunol 2017, 180, 45-47.

[0030] The Kv1.3 inhibitors or pharmaceutically acceptable salts of the present invention have Kv1.3 inhibitor activity, in other words, the Kv1.3 inhibitors of the present invention (and isolated peptide components of Kv1.3 inhibitors) have inhibitor or blocker activity at the Kv1.3 ion channel, i.e., can inhibit 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 the inhibitor (or blocker). 50 The IC value is a measure of the concentration of inhibitor required for that 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 value may be determined using any suitable assay, such as a fluorescence-based assay that measures ion flux (e.g., thallium ion flux) and a patch clamp assay, which may be performed as described in the Examples herein. For example, a patch clamp assay using a QPatch® system may be preferred.

[0033] In some embodiments, the Kv1.3 inhibitors of the present invention have an IC for the human Kv1.3 potassium channel of about 400 nM or less, e.g., about 300 nM or less, e.g., about 200 nM or less, e.g., about 100 nM or less, e.g., about 50 nM or less, e.g., about 15 nM or less, e.g., about 10 nM or less, e.g., about 5 nM or less. 50 Preferably, the Kv1.3 inhibitors of the present invention have an IC of about 2 nM or less. 50 More preferably, the Kv1.3 inhibitors of the present invention have an IC of about 1 nM or less, e.g., about 0.5 nM or less. 50 It has.

[0034] Selectivity The Kv1.3 inhibitors of the present invention are selective for Kv1.3. In some embodiments, the Kv1.3 inhibitors of the present invention are selective for Kv1.3 over Kv1.1, Kv1.2, Kv1.4, Kv1.5, Kv1.6, Kv1.7, and Kv1.8. In particular, the Kv1.3 inhibitors of the present invention are selective for Kv1.3 over one or more of Kv1.1, Kv1.2, and Kv1.6.

[0035] For example, Kv1.3 inhibitors of the present invention 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, Kv1.3 inhibitors are selective for Kv1.3 over Kv1.1. Furthermore, they may be selective for Kv1.3 over Kv1.2 and / or Kv1.6.

[0036] In this context, "selective" means that Kv1.3 inhibitors have higher inhibitory activity against Kv1.3 than against each of Kv1.1, Kv1.2, and Kv1.6. Therefore, their IC 50 is typically lower than for each of the other ion channels.

[0037] Therefore, selectivity for Kv1.3 over another ion channel X can be expressed as, for example, IC 50 [X] / IC 50 As in [Kv1.3], each IC 50 It can be expressed as a ratio of values.

[0038] Kv1.3 inhibitors of the present invention may have a selectivity for Kv1.3 over Kv1.1 of at least about 10, at least about 100, at least about 1000, or at least about 10,000, and may be up to about 100,000 or more. Typically, Kv1.3 inhibitors of the present invention have a selectivity for Kv1.3 over Kv1.1 of at least about 100, or at least about 1,000.

[0039] Kv1.3 inhibitors of the present invention may have a selectivity for Kv1.3 over Kv1.2 of at least about 10, at least about 100, at least about 1000, or at least about 10,000, and may be up to about 100,000 or more. Typically, inhibitors have a selectivity for Kv1.3 over Kv1.2 of at least 10, preferably at least about 50 or at least about 100 or at least about 1,000.

[0040] Kv1.3 inhibitors of the present invention may have a selectivity for Kv1.3 over Kv1.6 of at least about 10, at least about 100, at least about 1000, or at least about 10,000, and may be up to about 100,000 or more. Typically, inhibitors have a selectivity for Kv1.3 over Kv1.6 of at least 100, or at least about 400, or at least about 1,000.

[0041] The Kv1.3 inhibitors of the present invention may have higher selectivity than known Kv1.3 inhibitors such as ShK, mocatoxin (Moka1), Vm24, Odk2, or Osk1. Thus, the Kv1.3 inhibitors 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 Kv1.3 inhibitors 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.

[0042] The Kv1.3 inhibitors of the present invention have lower absolute inhibitory activity (i.e., higher IC ) than known Kv1.3 inhibitors (such as Odk2 or Osk1) at any or all of Kv1.1, Kv1.2, and / or Kv1.6. 50However, it is acceptable for the Kv1.3 inhibitors of the present invention to have lower 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, the Kv1.3 inhibitors of the present invention combine high specificity for Kv1.3 with high potency.

[0043] pharmaceutically acceptable salts The Kv1.3 inhibitors of the present invention may be in the form of pharmaceutically acceptable salts. All references herein to "Kv1.3 inhibitors," "Kv1.3 inhibitors of the present invention," "peptides," or "peptides of the present invention" should be construed to encompass any pharmaceutically acceptable salts thereof, regardless of whether "pharmaceutically acceptable salts" are explicitly stated. Kv1.3 inhibitors may also be referred to as "solvates," which refer to complexes of defined stoichiometry formed between a solute (a Kv1.3 inhibitor of the present invention or a pharmaceutically acceptable salt thereof) and a solvent. Solvents in this context may be, but are not limited to, water, ethanol, or other pharmaceutically acceptable organic species, such as small organic species such as acetic acid or lactic acid. When the solvent in question is water, such solvates are typically referred to as hydrates. In some embodiments, the pharmaceutically acceptable salts of the present invention are acetate salts. In other words, the present invention encompasses salts comprising or consisting of a cation of a Kv1.3 inhibitor and an acetate anion. In some embodiments, the pharmaceutically acceptable salts of the present invention are chloride salts. In other words, the present invention encompasses salts comprising or consisting of a cation of a Kv1.3 inhibitor and a chloride anion.

[0044] peptide The Kv1.3 inhibitors of the present invention comprise or consist of a peptide comprising or consisting of the sequence QMDMRCSASVECKQKCLKAIGSIFGKCMNKKCKCYPR (SEQ ID NO: 1) or a variant thereof, wherein the variant (a) has at least 70% sequence identity to SEQ ID NO: 1 and / or (b) differs from SEQ ID NO: 1 by a total of up to 9 substitutions, insertions and / or deletions.

[0045] SEQ ID NO: 1 is the amino acid sequence of a venom peptide from the Parabuthus transvaalicus scorpion. As described herein, this peptide is a selective Kv1.3 potassium ion channel inhibitor. Thus, it is the peptide portion of the Kv1.3 inhibitors of the present invention that is believed to inhibit Kv1.3. The peptide may be referred to herein as the "peptide component," "peptide element," "Kv1.3 inhibitor component," or "Kv1.3 inhibitor element" of the Kv1.3 inhibitors of the present invention.

[0046] A variant of SEQ ID NO: 1 is a peptide that contains one or more amino acids that are different, additional, or missing from those of SEQ ID NO: 1. In other words, the variant contains one or more amino acid changes compared to SEQ ID NO: 1. Such variants may also be referred to herein as "derivatives," "variant sequences," "sequence variants," "variant peptides," "peptide variants," or simply "peptides."

[0047] In some embodiments, the Kv1.3 inhibitors of the invention comprise a peptide described herein. In other words, in some embodiments, the Kv1.3 inhibitor comprises a peptide along with other features or elements. In some embodiments, the Kv1.3 inhibitors of the invention consist of a peptide described herein. In other words, in some embodiments, the Kv1.3 inhibitor is a peptide (i.e., the Kv1.3 inhibitor consists of a peptide, without other features or elements).

[0048] In some embodiments, the peptide consists of the sequence QMDMRCSASVECKQKCLKAIGSIFGKCMNKKCKCYPR (SEQ ID NO: 1) or a variant thereof, wherein the variant (a) has at least 70% sequence identity to SEQ ID NO: 1, and / or (b) differs from SEQ ID NO: 1 by a total of up to 9 substitutions, insertions, and / or deletions.

[0049] amino acid Throughout this specification and claims, the conventional three-letter and one-letter codes for 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); Also, sarcosine (Sar), norleucine (Nle), α-aminoisobutyric acid (Aib), 2,3-diaminopropanoic acid (Dap), 2,4-diaminobutanoic acid (Dab), 2,5-diaminopentanoic acid (ornithine, Orn), alpha-aminobutyric acid (Abu), Generally accepted codes for other α-amino acids are used, such as F(4-F) (4-fluoro-phenylalanine), F(4-NH) (4-amino-phenylalanine), F(4-NO) (4-nitro-phenylalanine), F(4-CH) (4-methyl-phenylalanine), F(4-F) (4-amino-phenylalanine), F(4-N) (4-amino-phenylalanine), F(4-N) (4-amino-phenylalanine), F(4-N) (4-nitro-phenylalanine), F(4-N) (4-methyl-phenylalanine), F(4-N) (4-amino ...

[0050] The designation [2-amino-5-carboxypentanoyl] refers to a peptide residue of 2-amino-5-carboxypentanoic acid, which therefore has a side chain similar to that of glutamic acid, but with an additional methylene group. [ka]

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

[0052] The designation [2,4-diaminobutanoyl] refers to the peptide residue of 2,4-diaminobutanoic acid, which has the following structure: [ka]

[0053] The designation [2-amino-3-guanidinopropionyl] indicates the peptide residue of 2-amino-3-guanidinopropionic acid, which has the following structure: [ka]

[0054] Such other α-amino acids, when used in general formulas or sequences herein, may be shown in square brackets "[]" (e.g., "[Nle]"), particularly when the remainder of the formula or sequence is shown using single-letter codes. 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, amino acid codes written in lowercase represent the D-configuration of the amino acid in question, for example, "k" represents the D-configuration of lysine (K).

[0055] Amino acid position numbering The amino acid residues of SEQ ID NO: 1 are numbered conventionally from N-terminal to C-terminal from 1 to 37. 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 in the case of 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.

[0056] The residue present at a particular position may be designated by the number of the relevant position along 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, and 2Nle, 2[Nle], Nle2 or [Nle]2 designates a norleucine residue at position 2.

[0057] An asterisk may be used to indicate the location of the deletion relative to the sequence of SEQ ID NO: 1. For example, "1 * " indicates deletion of residue at position 1 compared to SEQ ID NO:1.

[0058] 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.

[0059] In some embodiments, substitutions compared to SEQ ID NO: 1 are conservative substitutions. However, substitutions listed in any of the general formulas provided below may be introduced at each position.

[0060] Cysteine ​​and disulfide bonds The peptide 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. Thus, in the Kv1.3 inhibitors of the present invention or pharmaceutically acceptable salts thereof, the peptide contains a cysteine ​​(C) at each of positions 6, 12, 16, 27, 32, and 34.

[0061] 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 is 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, it should be understood that the active inhibitor compound comprises the appropriate disulfide bonds. In some embodiments, the disulfide bridge pattern differs from the pattern shown in SEQ ID NO: 1 above. In such embodiments, the different disulfide bridge pattern is indicated by the numbering system described above.

[0062] 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 for 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.

[0063] Sequence identity In some embodiments, the variant has at least 70% sequence identity to SEQ ID NO:1.

[0064] The sequences of variants of the Kv1.3 inhibitors of the present invention may be expressed in terms of sequence identity instead of the number of substitutions, insertions and deletions relative to SEQ ID NO:1.

[0065] Accordingly, the present invention provides a Kv1.3 inhibitor, or a pharmaceutically acceptable salt thereof, for use in a method for treating or preventing a disease or disorder in a subject, wherein the Kv1.3 inhibitor comprises or consists of a peptide comprising or consisting of the sequence QMDMRCSASVECKQKCLKAIGSIFGKCMNKKCKCYPR (SEQ ID NO: 1) or a variant thereof, wherein the variant has at least 70% sequence identity to SEQ ID NO: 1, and the method comprises the step of administering the Kv1.3 inhibitor or a pharmaceutically acceptable salt thereof to the subject once every 2 to 8 days.

[0066] In some embodiments, the peptide has at least 75% sequence identity to SEQ ID NO: 1, such as at least 80% sequence identity, for example, at least 85% sequence identity, for example, at least 90% sequence identity, for example, 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, for example, at least 99% sequence identity, for example, 100% identity.

[0067] In some embodiments, the "percentage (%) sequence identity" of a peptide is defined as the percentage of amino acids in the peptide 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 achieve the maximum percentage sequence identity; any conservative substitutions are not considered as part of the sequence identity. Sequence alignment 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, e.g., Altschul et al., Methods in Enzymology 266: 460-480 (1996) or Pearson et al., Genomics 46: 24-36, 1997.

[0068] As used herein in the context of the present invention, percentage sequence identity can be determined using these programs at their 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.

[0069] Substitutions, insertions, and deletions A variant of SEQ ID NO: 1 differs 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.

[0070] 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; 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; thus, for example, the deletion of three consecutive amino acids in SEQ ID NO:1 does not constitute a single deletion, but rather three deletions.

[0071] In some embodiments, a variant differs from SEQ ID NO: 1 by up to a total of 9 substitutions, insertions, and / or deletions. The term "a total of 9" means that a total of up to 9 amino acids can be substituted in, and / or inserted into, and / or deleted from, SEQ ID NO: 1. In other words, the maximum total of all combinations of substitutions, insertions, and deletions in a variant is 9, and within these 9, there can be any combination of substitutions, insertions, and / or deletions. Stated differently, a variant includes any combination of substitutions, insertions, and / or deletions, up to a total of 9 combinations of substitutions, insertions, and deletions.

[0072] The sequences of variants of the Kv1.3 inhibitors of the present invention may be expressed in terms of the number of substitutions, insertions and deletions relative to SEQ ID NO:1 instead of in terms of sequence identity.

[0073] Accordingly, the present invention provides a Kv1.3 inhibitor, or a pharmaceutically acceptable salt thereof, for use in a method for treating or preventing a disease or disorder in a subject, wherein the Kv1.3 inhibitor comprises or consists of a peptide comprising or consisting of the sequence QMDMRCSASVECKQKCLKAIGSIFGKCMNKKCKCYPR (SEQ ID NO: 1) or a variant thereof, which variant differs from SEQ ID NO: 1 by a total of up to 9 substitutions, insertions and / or deletions, and the method comprises the step of administering the Kv1.3 inhibitor or a pharmaceutically acceptable salt thereof to the subject once every 2 to 8 days.

[0074] In some embodiments, a variant of SEQ ID NO: 1 differs from SEQ ID NO: 1 by a total of up to 8 substitutions, insertions, and / or deletions. In some embodiments, a variant differs from SEQ ID NO: 1 by a total of up to 7, 6, 5, 4, 3, or 2 substitutions, insertions, and / or deletions, or by a total of 1 substitution, insertion, or deletion. In some embodiments, the variant comprises a total of 9, 8, 7, 6, 5, 4, 3, 2, or 1 substitutions, insertions, and / or deletions compared to SEQ ID NO: 1. Preferably, the peptide comprises a total of 6 substitutions, insertions, and / or deletions compared to the sequence of SEQ ID NO: 1.

[0075] Amino acids at specific positions In some embodiments of the Kv1.3 inhibitors or pharmaceutically acceptable salts thereof of the present invention, the peptide comprises the following amino acid: the amino acid at position 1 is H, N, P, p, Q, S, V, or Y, or is deleted; the amino acid at position 2 is I, M, Nle, or is deleted; the amino acid at position 3 is D, E, S, or is deleted; the amino acid at position 4 is E, L, M, Nle, S, V, or is deleted; the amino acid at position 5 is R or K or is deleted; the amino acid at position 7 is E, F, H, K, Orn, R, S, Y, 2,3-diaminopropanoyl, 2,4-diaminobutanoyl, or 2-amino-3-guanidinopropionyl; the amino acid at position 8 is A, H, I, L, S, or Y; the amino acid at position 9 is F, L, P, S, Orn, V, Abu, or 2,3-diaminopropanoyl; the amino acid at position 10 is K, P, Q, R, or V; the amino acid at position 11 is E or Q; the amino acid at position 13 is A, E, G, K, L, Q, or V; the amino acid at position 14 is E, K, L, Q, V, or 2-amino-5-carboxypentanoyl; The amino acids at position 15 are K, L, P, and S; the amino acid at position 17 is K, L, R, Y, or is deleted; the amino acid at position 18 is A, D, G, K, Q, hQ, V, Y, or is deleted; the amino acid at position 19 is A, K, R, Y, or is deleted; the amino acid at position 20 is E, I, R, Y, or is deleted; the amino acid at position 21 is E, G, H, or R; the amino acid at position 22 is C, R, or S; the amino acid at position 23 is G, I, K, P, or R; the amino acid at position 26 is K or hK; the amino acid at position 28 is M or Nle; the amino acid at position 30 is G or K; the amino acid at position 33 is H, K, R, or V; The amino acid at position 35 is Y, F(4-F), F(4-CH), F(4-NO), or F(4-NH); the amino acid at position 36 is Q or P or is deleted; and / or The amino acid at position 37 is C, G, R, S, (4-amino-5-hydroxypentyl)guanidine, or is deleted.

[0076] In some embodiments of the Kv1.3 inhibitors or pharmaceutically acceptable salts thereof of the present invention, the peptide comprises the following amino acid: the amino acid at position 1 is N or P 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, K, or deleted; The amino acid at position 7 is S, 2,4-diaminobutanoyl or 2-amino-3-guanidinopropionyl; the amino acid at position 14 is K or Q; the amino acid at position 18 is K or A; the amino acid at position 19 is K, S, or A; the amino acid at position 28 is M or Nle; and / or The amino acid at position 37 is R or S.

[0077] In a preferred embodiment of the Kv1.3 inhibitor or a pharmaceutically acceptable salt thereof of the present invention, the peptide comprises the following amino acid: The amino acid at position 1 is P; The amino acid at position 2 is Nle; The amino acid at position 3 is E; The amino acid at position 4 is Nle; the amino acid at position 18 is A; and / or The amino acid at position 28 is Nle.

[0078] In a preferred embodiment, positions 1 to 5 of the variant are deleted or comprise or consist of an amino acid sequence selected from the following: QMDMR (SEQ ID NO: 155), NMDMR (SEQ ID NO: 156), P[Nle]D[Nle]R, and P[Nle]E[Nle]R.

[0079] In some embodiments, the variant at position 14 is K or Q. In some embodiments, the variant at position 28 is M or Nle. In some embodiments, the variant at position 37 is R or S. In some embodiments, the amino acid at position 1 of the variant of SEQ ID NO: 1 is not Q. In some embodiments, the amino acid at position 1 of the variant of SEQ ID NO: 1 is N or P or is deleted. In some embodiments, the variant of SEQ ID NO: 1 includes amino acids 22S and 23I.

[0080] In some embodiments of the Kv1.3 inhibitors of the invention, one or more positions of the variant of SEQ ID NO: 1 have the same amino acid as the corresponding position in SEQ ID NO: 1. In other words, the amino acids present at the relevant positions are the same amino acids present at the corresponding positions in SEQ ID NO: 1.

[0081] In some embodiments, positions 6, 12, 16, 27, 32, and 34 of the variant are the same amino acids as the corresponding positions in SEQ ID NO: 1. In other words, in some embodiments, the peptide comprises the following amino acids: 6C, 12C, 16C, 27C, 32C, and 34C.

[0082] In some embodiments, positions 6, 12, 16, 24, 25, 27, 32, and 34 of the variant are the same amino acids as the corresponding positions in SEQ ID NO: 1. In other words, in some embodiments, the peptide comprises the following amino acids: 6C, 12C, 16C, 24F, 25G, 27C, 32C, and 34C.

[0083] In some embodiments, positions 6, 12, 16, 24, 25, 27, 29, 31, 32, and 34 of the variant are the same amino acids as the corresponding positions in SEQ ID NO: 1. In other words, in some embodiments, the peptide comprises the following amino acids: 6C, 12C, 16C, 24F, 25G, 27C, 29N, 31K, 32C, and 34C.

[0084] In some embodiments, the following positions of the variant have the same amino acid as the corresponding positions in SEQ ID NO: 1: 6, 8-13, 15-17, 20-27, and 29-36. In some embodiments, the following positions of the variant have the same amino acid as the corresponding positions in SEQ ID NO: 1: 6-13, 15-17, 20-27, and 29-36.

[0085] 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, 26, 28, 30, 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, 2, 3, 4, 5, 7, 14, 18, 19, 28, and 37 of SEQ ID NO: 1.

[0086] In some embodiments, 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. In some embodiments, at least one amino acid at positions 7, 8, 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. In some embodiments, exactly one amino acid at positions 7, 8, 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. In some embodiments, 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. In some embodiments, the amino acid having an aromatic side chain is Y.

[0087] deletion In some embodiments, when a variant of SEQ ID NO: 1 contains one or more deletions, one of the deletions is at position 1. In some embodiments, when a variant of SEQ ID NO: 1 contains two or more deletions, two of the deletions are at positions 1 and 2.

[0088] In some embodiments, the variant comprises exactly 1, 2, 3, 4, 5, 6, or 7 deletions.

[0089] In some embodiments, the deletion in the peptide variant is selected from the following: Deletion at position 1; Deletions at positions 1 and 2; deletions at positions 1, 2, and 3; Deletions at positions 1, 2, 3, and 4; deletions at positions 1, 2, 3, 4, and 5; deletions at positions 1, 2, 3, 4, 5, and 36; Deletions at positions 1, 2, 3, 4, 5, 36, and 37; deletion at position 17; deletion at position 18; deletion at position 19; deletion at position 19; deletion at position 20; A deletion at position 36; and Deletion at position 37.

[0090] In a preferred embodiment, the deletions in the variant are at positions 1, 2, 3, 4 and 5.

[0091] Insert In some embodiments, the variant comprises up to 4 insertions compared to SEQ ID NO: 1. In some embodiments, the variant comprises up to 3 insertions, up to 2 insertions, or up to 1 insertion compared to SEQ ID NO: 1.

[0092] In some embodiments, the peptide comprises one or more insertions at the N-terminus (i.e., before position 1). In some embodiments, the peptide comprises one or more insertions at the N-terminus only. In some embodiments, the peptide comprises one or more insertions at the C-terminus (i.e., after position 37). In some embodiments, the peptide comprises one or more insertions at the C-terminus only. In some embodiments, the peptide comprises one or more insertions at both termini.

[0093] In some embodiments, the insertion at the N-terminus comprises or consists of the sequence GG or SG. In some embodiments, the insertion at the C-terminus comprises or consists of the sequence RRTA (SEQ ID NO: 158), HRRK (SEQ ID NO: 159), QSKA (SEQ ID NO: 160), AGPR (SEQ ID NO: 161), RSRT (SEQ ID NO: 162), RHKR (SEQ ID NO: 163), GGKR (SEQ ID NO: 164), PKTA (SEQ ID NO: 165), TDAR (SEQ ID NO: 166), HRQQ (SEQ ID NO: 167), RPRH (SEQ ID NO: 168), ARNA (SEQ ID NO: 169), TGRK (SEQ ID NO: 170), HERT (SEQ ID NO: 171), NTRT (SEQ ID NO: 172), GGKR (SEQ ID NO: 173), PKTA (SEQ ID NO: 164), TDAR (SEQ ID NO: 165), HRQQ (SEQ ID NO: 166), RPRH (SEQ ID NO: 167), ARNA (SEQ ID NO: 168), TGRK (SEQ ID NO: 179), HERT (SEQ ID NO: 180), NTRT (SEQ ID NO: 181), GGKR (SEQ ID NO: 182), GGKR (SEQ ID NO: 183), GGKR (SEQ ID NO: 184), GGKR (SEQ ID NO: 185), GGKR (SEQ ID NO: 186), GGKR (SEQ ID NO: 187), GGKR (SEQ ID NO: 188), GGKR (SEQ ID NO: 189), GGKR (SEQ ID NO: 19 No. 172), QRNG (SEQ ID NO: 173), AHRN (SEQ ID NO: 174), PRSA (SEQ ID NO: 175), QRQS (SEQ ID NO: 176), QRRK (SEQ ID NO: 177), ARAK (SEQ ID NO: 178), AKRD (SEQ ID NO: 179), RDKT (SEQ ID NO: 180), RAKR (SEQ ID NO: 182), QRTR (SEQ ID NO: 183), ATRH (SEQ ID NO: 184), ARRS (SEQ ID NO: 185), AKTR (SEQ ID NO: 186), NRQR (SEQ ID NO: 187) or PRNT (SEQ ID NO: 188).

[0094] In some embodiments, the insertion in the variant is selected from: -GG in positions 1 and 0 (i.e. inserted before position 1); -SG at positions 1 and 0 (i.e. inserted before position 1); R at position 38 (i.e. inserted after position 37); Y at position 38 (i.e. inserted after position 37); L at position 38 (i.e., inserted after position 37); H at position 38 (i.e., inserted after position 37); E at position 38 (i.e., inserted after position 37); KS at positions 38 and 39 (i.e., inserted after position 37); FE at positions 38 and 39 (i.e., inserted after position 37); HR at positions 38 and 39 (i.e., inserted after position 37); AK at positions 38 and 39 (i.e., inserted after position 37); 4-amino-5-hydroxypentanamide at position 38 (i.e. inserted after position 37); ST at positions 38 and 39 (i.e., inserted after position 37); RY at positions 38 and 39 (i.e., inserted after position 37); RRTA (SEQ ID NO: 158) at positions 38-41 (i.e., inserted after position 37); HRRK (SEQ ID NO: 159) at positions 38-41 (i.e., inserted after position 37); and RRTK (SEQ ID NO: 157) at positions 38 to 41 (i.e., inserted after position 37).

[0095] In some embodiments, the peptide is a fusion protein comprising SEQ ID NO:1 or a variant thereof as defined herein and one or more heterologous polypeptide sequences. In some embodiments, SEQ ID NO:1 or a variant thereof is inserted into a heterologous scaffold polypeptide. In some embodiments, the peptide has a maximum length of 200 amino acids, 150 amino acids, 125 amino acids, 100 amino acids, 75 amino acids, or 50 amino acids.

[0096] terminal group An "H" (or "Hy-") moiety at the N-terminus of a sequence indicates a hydrogen atom, which corresponds to the presence of a free primary or secondary amino group at the N-terminus. Alternatively, the peptide may contain an alternative N-terminal group (i.e., an N-terminal modification).

[0097] Thus, in some embodiments of the Kv1.3 inhibitors or pharmaceutically acceptable salts of the present invention, the peptide has a C 1-4 It includes groups selected from alkyl, acetyl (Ac), formyl, benzoyl and trifluoroacetyl.

[0098] A "-OH" moiety at the C-terminus of a sequence indicates the presence of a carboxyl group (COOH) at the C-terminus of the molecule. A "-NH2" moiety at the C-terminus of a sequence indicates the presence 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. The CH2OH moiety may be contained in (4-amino-5-hydroxypentyl)guanidine or 4-amino-5-hydroxypentanamide.

[0099] Thus, in some embodiments of the Kv1.3 inhibitors or pharmaceutically acceptable salts thereof of the present invention, the peptide comprises a carboxy group (-COOH), an amino group (-NH2) or a hydroxymethyl group (-CH2OH) at the C-terminus, preferably a carboxy group (-COOH) or an amino group (-NH2).

[0100] array In the context of the Kv1.3 inhibitors of the present invention, the term "sequence" as used herein refers to the order of amino acids in a Kv1.3 inhibitor peptide. Specific sequences are referred to herein using sequence identification numbers (SEQ ID NOs). Each reference herein to a SEQ ID NO refers to the sequence represented by that SEQ ID NO. A peptide that "comprises" a given sequence may include other amino acids at one or both ends of the sequence. A peptide that "consists of" a given sequence does not include any amino acids in its linear sequence other than those of that sequence, but may include other features (e.g., N- or C-terminal chemical groups).

[0101] In some embodiments of the Kv1.3 inhibitors or pharmaceutically acceptable salts thereof of the present invention, the peptide comprises or consists of one of the following sequences: [Table 1] JPEG2025534884000006.jpg251170 JPEG2025534884000007.jpg250170 JPEG2025534884000008.jpg97170

[0102] In some embodiments, the Kv1.3 inhibitor or pharmaceutical salt of the present invention comprises a peptide consisting of the sequence of any one of SEQ ID NOs: 1 to 150. In some embodiments, the Kv1.3 inhibitor or pharmaceutical salt of the present invention comprises a peptide consisting of the sequence of any one of SEQ ID NOs: 1 to 150. In a preferred embodiment, the Kv1.3 inhibitor or pharmaceutical salt of the present invention comprises a peptide consisting of the sequence of SEQ ID NO: 97. In a preferred embodiment, the Kv1.3 inhibitor or pharmaceutical salt of the present invention comprises a peptide consisting of the sequence of SEQ ID NO: 97.

[0103] peptide In the context of the Kv1.3 inhibitors of the present invention, the term "peptide" as used herein refers to the peptide component of the Kv1.3 inhibitor. The term peptide encompasses additional features beyond the peptide's sequence (i.e., the order of amino acids), namely, the chemical groups at the N- and C-termini of the peptide, and the pattern of disulfide bridges within the peptide. For ease of reference, specific peptides are assigned peptide numbers (Ptd no., peptide number) herein. Each reference to a peptide number herein refers to the peptide represented by that peptide number. A Kv1.3 inhibitor that "comprises" a given peptide may include other features. A Kv1.3 inhibitor that "consists of" a given peptide includes only the features of that peptide.

[0104] In some embodiments of the Kv1.3 inhibitor or pharmaceutically acceptable salt of the present invention, the peptide is selected from the following peptides: [Table 2] JPEG2025534884000010.jpg247170 JPEG2025534884000011.jpg254170 JPEG2025534884000012.jpg249170 JPEG2025534884000013.jpg215170

[0105] In some embodiments, the Kv1.3 inhibitor or pharmaceutical salt of the present invention comprises a peptide consisting of any one of peptides 1 to 158. In some embodiments, the Kv1.3 inhibitor or pharmaceutical salt of the present invention consists of any one of peptides 1 to 158. In a preferred embodiment, the Kv1.3 inhibitor or pharmaceutical salt of the present invention comprises peptide 100. In a preferred embodiment, the Kv1.3 inhibitor or pharmaceutical salt of the present invention consists of peptide 100.

[0106] Synthesis of Kv1.3 inhibitors of the present invention The Kv1.3 inhibitors described herein can be synthesized by solid-phase or liquid-phase peptide synthesis. In this context, reference may be made to International Publication No. WO98 / 11125 and, in particular, to Fields, GB et al., 2002, "Principles and Practice of Solid-Phase Peptide Synthesis" in Synthetic Peptides (2nd Edition), and the Examples herein. Alternatively, the Kv1.3 inhibitors described herein can be synthesized by recombinant technology or a combination of recombinant technology and peptide chemistry.

[0107] Exemplary methods for producing the Kv1.3 inhibitors of the present invention include synthesizing the peptide by solid-phase or liquid-phase peptide synthesis and recovering the peptide; or expressing the peptide from a nucleic acid construct encoding the peptide and recovering the expression product; or expressing a precursor peptide from a nucleic acid construct encoding the precursor peptide sequence and recovering the expression product, and modifying the precursor peptide to obtain the Kv1.3 inhibitor.

[0108] The precursor peptide may be prepared by the introduction of one or more non-proteinogenic amino acids (e.g., Nle), the addition of a suitable terminal group R 1 and R2 It may be modified by, for example, the introduction of:

[0109] Expression of a peptide or precursor peptide from a nucleic acid encoding the peptide or precursor peptide can 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 be composed entirely of proteinogenic amino acids (i.e., the 20 amino acids encoded by the standard genetic code).

[0110] 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 purpose and type of application, the vector can take the form of a plasmid, phage, cosmid, minichromosome, or virus, although naked DNA that is only expressed transiently in a particular cell is also an important vector. Preferred cloning and expression vectors (plasmid vectors) are capable of autonomous replication, thereby allowing for high copy numbers for high-level expression or high levels of replication for subsequent cloning.

[0111] In general overview, expression vectors comprise the following features operably linked in a 5' to 3' direction: a promoter for driving expression of the nucleic acid fragment, a nucleic acid sequence encoding an optional leader peptide enabling secretion (to the extracellular phase or optionally the periplasm), a nucleic acid sequence encoding a precursor peptide, and a nucleic acid sequence encoding an optional terminator. They may also contain additional features such as a selectable marker and an origin of replication. When working with expression vectors in producer strains or cell lines, it may be preferable that the vector be capable of integrating into the host cell genome. Those skilled in the art are familiar with suitable vectors and are able to design vectors according to specific requirements.

[0112] Such vectors 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.

[0113] Preferred transformed cells are bacteria (e.g., Escherichia (e.g., E. coli), Bacillus (e.g., Bacillus subtilis), Salmonella, or Mycobacterium (preferably non-pathogenic, e.g., M. bovis BCG)), yeast (e.g., Saccharomyces cerevisiae and Pichia pastoris), and the like. pastoris], as well as microorganisms such as protozoa. Alternatively, the transformed cells may be derived from multicellular organisms, i.e., fungal cells, insect cells, algal cells, plant cells, or animal cells, such as mammalian cells. For purposes of cloning and / or optimized expression, it is preferred that the transformed cells are capable of replicating the nucleic acid fragments of the invention. Cells expressing the nucleic acid fragments may be used for small- or large-scale preparation of the peptides of the invention. When transformed cells are used to produce peptides or precursor peptides, it is convenient, although not at all essential, if the expression product is secreted into the culture medium.

[0114] Treatment or prevention of disease or disorder The present invention provides a Kv1.3 inhibitor or a pharmaceutically acceptable salt thereof for use in a method for treating or preventing a disease or disorder in a subject, as described herein, the method comprising administering the Kv1.3 inhibitor or a pharmaceutically acceptable salt thereof to a subject once every 2 to 8 days.

[0115] Therefore, the present invention also provides a method for treating or preventing a disease or disorder in a subject, the method comprising the step of administering a Kv1.3 inhibitor or a pharmaceutically acceptable salt of the present invention to the subject once every 2 to 8 days.

[0116] The present invention also provides the use of a Kv1.3 inhibitor or a pharmaceutically acceptable salt thereof described herein in the manufacture of a medicament for use in a method for treating or preventing a disease or disorder in a subject, the method comprising administering the Kv1.3 inhibitor to a subject once every 2 to 8 days.

[0117] In other words, the present invention provides medical uses of the Kv1.3 inhibitors or pharmaceutically acceptable salts described herein. The present invention also provides therapeutic, preventative, or prophylactic methods comprising administering to a subject a Kv1.3 inhibitor or pharmaceutically acceptable salt described herein. In all cases, the described medical uses may also be expressed as methods of treating or preventing a disease or disorder, wherein the method comprises administering to a subject a Kv1.3 inhibitor or pharmaceutically acceptable salt of the present invention.

[0118] The term "subject" is used interchangeably herein with "patient" and "individual" and refers to either a human or 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). A subject as described herein is afflicted with a disease or disorder as described herein.

[0119] The terms "disease" and "disorder" refer to a state of bodily dysfunction. The term disease is used herein synonymously with similar terms such as "condition" or "disorder." Thus, the terms "disease," "condition," and "disorder" are used interchangeably herein. In some embodiments, the disease or disorder is one that can be treated or prevented (i.e., treatable or preventable) using a Kv1.3 inhibitor described herein.

[0120] The terms "treating," "treatment," and "treats" refer to alleviating, reducing, or eliminating the symptoms of a disease in a subject. Thus, the term "treating" encompasses curing a disease or disorder, but does not require that the disease or disorder be completely eliminated from the subject. Even a mild reduction in the symptoms of a disease or disorder constitutes "treating" the disease or disorder. The terms "treating," "treatment," and "treat" refer to both the treatment of an existing disease in a subject and the prevention of a disease in a subject, i.e., prophylaxis. Thus, it will be recognized that the treatments referred to herein can be prophylactic in some embodiments.

[0121] The terms "preventing," "prevention," or "prevents" refer to prophylactic treatment, i.e., measures or procedures aimed at preventing the occurrence of a disease or disorder rather than treating an existing disease or disorder. Prevention means achieving a desired pharmacological and / or physiological effect that is prophylactic in that it completely or partially prevents a disease or disorder or its symptoms.

[0122] Treating inflammation As described herein, inhibitors of Kv1.3 can be useful in reducing inflammation. The data presented in the Examples herein directly demonstrate that treating a subject with a Kv1.3 inhibitor reduces inflammation.

[0123] Thus, in some embodiments, the disease or disorder is an inflammatory disease or disorder, which is any disease, condition, or disorder in which a reduction in inflammation is desirable, e.g., one in which inflammation contributes to the symptoms or pathogenesis.

[0124] In some embodiments, the inflammatory condition or disorder is an autoimmune disease, allergy or hypersensitivity, allograft rejection, transplant rejection, graft versus host disease, 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 (e.g., scleroderma, pulmonary fibrosis, cirrhosis), chronic obstructive pulmonary disease (COPD), or a combination thereof. The disease or disorder is selected from: chronic inflammatory demyelinating polyneuropathy, hepatitis, chronic inflammatory demyelinating polyneuropathy, inflammatory bowel disease, colitis (e.g., Crohn's disease and ulcerative colitis), erythema, thyroiditis, psoriasis, atopic dermatitis, allergic contact dermatitis, scleroderma, glomerulonephritis, inflammatory bone resorption, multiple sclerosis, and type 1 diabetes. In a preferred embodiment, the disease or disorder is arthritis, such as rheumatoid arthritis, osteoarthritis, or psoriatic arthritis.

[0125] In some embodiments, the present invention provides an inventive Kv1.3 inhibitor or a pharmaceutically acceptable salt thereof as described herein for use in a method of inhibiting or reducing inflammation.

[0126] In some embodiments, the subject experiences reduced inflammation after administration of a Kv1.3 inhibitor. Inflammation can be measured using techniques known in the art (e.g., by measuring cytokine levels in the subject). In some embodiments, after administration of a Kv1.3 inhibitor, inflammation in the subject is reduced by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%. In some embodiments, after administration of a Kv1.3 inhibitor, inflammation in the subject is reduced by 100% (i.e., inflammation is eliminated).

[0127] Treatment of metabolic diseases Inhibitors of Kv1.3 may also have beneficial metabolic effects, for example, related to energy homeostasis, body weight regulation, and glucose control.

[0128] In some embodiments, the disease or disorder is a metabolic disease or disorder. A metabolic disease or disorder is any disease, condition, or disorder caused by or related to abnormal metabolism (i.e., a disruption in the body's processing of nutrients to sustain life). A metabolic disease or disorder may be characterized by too much or too little of certain chemicals, such as proteins, carbohydrates, lipids, their component molecules (e.g., amino acids, sugars, fatty acids), and other such biomolecules, in the subject's body.

[0129] Given that obese subjects have excess fat and may have abnormal metabolic processes, obesity may be considered a metabolic disorder. Thus, in some embodiments, the disease or disorder is obesity, obesity-related inflammation, obesity-related gallbladder disease, or obesity-induced sleep apnea.

[0130] In some embodiments, the disease or disorder is a disease or disorder caused by or associated with impaired glucose control. Such diseases include metabolic syndrome, insulin resistance, impaired glucose tolerance, prediabetes, elevated fasting blood glucose, and type 2 diabetes. Some of these conditions may be associated with obesity. Its effects on these conditions may be mediated in whole or in part through its effects on body weight, or may be independent of its effects on body weight.

[0131] In some embodiments, the invention provides a KvI .3 inhibitor or pharmaceutically acceptable salt thereof as described herein for use in a method for inhibiting weight gain, promoting weight loss, reducing excess weight, or treating obesity (e.g., by controlling appetite, eating, food intake, calorie intake, and / or energy expenditure). The effect on weight can be therapeutic or cosmetic.

[0132] Treatment of proliferative cells and cancer Kv1.3 is also expressed in proliferating human and mouse smooth muscle cells. Inhibitors of Kv1.3 may be effective in treating smooth muscle proliferative disorders, such as restenosis in patients after vascular surgery (e.g., angioplasty). Thus, in some embodiments, the disease or disorder is a smooth muscle proliferative disorder. In some embodiments, the smooth muscle proliferative disorder is restenosis.

[0133] 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 the differentiation of neural progenitor cells (Wang et al., J. Neurosci. 30:5020-7, 2010). Thus, Kv1.3 inhibitors may be beneficial in the treatment of neuroinflammatory and neurodegenerative disorders. Thus, in some embodiments, the disease or disorder is a neuroinflammatory or neurodegenerative disease or disorder. In some embodiments, the neuroinflammatory or neurodegenerative disease or disorder is Alzheimer's disease, multiple sclerosis (MS), Parkinson's disease, or amyotrophic lateral sclerosis (ALS) (e.g., following a viral infection).

[0134] In some embodiments, the disease or disorder is cancer. In some embodiments, the cancer is breast cancer, prostate cancer, or lymphoma. In some embodiments, the lymphoma is non-Hodgkin's lymphoma (NHL). Non-Hodgkin's 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 Sézary syndrome.

[0135] Administration of Kv1.3 inhibitors The present invention provides a Kv1.3 inhibitor or a pharmaceutically acceptable salt thereof for use in a method for treating or preventing a disease or disorder in a subject, as described herein, the method comprising administering the Kv1.3 inhibitor to the subject once every 2 to 8 days. Thus, the method comprises administering the Kv1.3 inhibitor to the subject once every 2 to 8 days. In other words, the Kv1.3 inhibitor is administered to the subject once every 2 to 8 days.

[0136] The terms "administering" and "administration" refer to the delivery of a Kv1.3 inhibitor to a subject. "An administration" refers to a single event of administering a Kv1.3 inhibitor to a subject. The administration of the Kv1.3 inhibitor can be by any mode of administration common or standard in the art, such as oral, intravenous, intramuscular, subcutaneous, sublingual, intranasal or intradermal administration, by suppository route, or by implantation. In some embodiments, the Kv1.3 inhibitor is administered by injection, preferably by subcutaneous injection.

[0137] Timing of administration According to the present invention, the Kv1.3 inhibitor is administered to a subject once every 2 to 8 days.

[0138] In other words, according to the present invention, the Kv1.3 inhibitor is administered to a subject at a specific dose once every 2 to 8 days. According to some embodiments of the present invention, the Kv1.3 inhibitor is administered to a subject at a specific dose once every 3 to 8 days. According to some embodiments of the present invention, the Kv1.3 inhibitor is administered to a subject at a specific dose once every 3 to 8 days. According to some embodiments of the present invention, the Kv1.3 inhibitor is administered to a subject at a specific dose once every 3 to 7 days. According to some embodiments of the present invention, the Kv1.3 inhibitor is administered to a subject at a specific dose once every 3 to 7 days.

[0139] Kv1.3 inhibitors based on PaT1 toxin peptides have a short half-life in the subject's body. For example, Example 6 herein shows that such Kv1.3 inhibitors have a half-life of approximately 1 hour in a rat model organism. However, despite this short half-life, it has surprisingly been found that the Kv1.3 inhibitors described herein have a long-lasting effect on T cells. In particular, Example 8 herein shows that inflammation is reduced in a rat ear inflammation model for up to 7 days after treatment with a Kv1.3 inhibitor. In addition, Example 9 herein shows that treatment with a Kv1.3 inhibitor once every 5 days reduces inflammation in a rat arthritis model. Without wishing to be bound by theory, it is hypothesized that Kv1.3 inhibitors, through their interaction with the Kv1.3 ion channel, somehow "reprogram" T cells to remain in an inactive state for a long period of time, thereby suppressing inflammation for several days.

[0140] These surprising findings support the concept of treating subjects suffering from diseases, conditions, or disorders treatable using the Kv1.3 inhibitors described herein according to a regimen in which the inhibitor is administered at approximately weekly intervals (i.e., once every 2-8 days). Increasing the period between administrations of the active compound as much as possible is advantageous because it saves time and effort (since fewer administrations need to be performed in a given period) and costs (since less active compound needs to be used in a given period). Particularly for active compounds administered by certain routes, such as subcutaneous injection, longer intervals between administrations may also increase patient comfort and thereby patient compliance with the treatment regimen. For example, administering a Kv1.3 inhibitor according to the present invention (i.e., once every 2-8 days) is superior to administering the inhibitor daily for the reasons described above. An unexpected finding described in the present disclosure is that it is possible to administer the Kv1.3 inhibitors described herein once every 2-8 days, despite their short in vivo half-lives, such that the effects of the inhibitors (e.g., reduced inflammation) are observed for several days after administration.

[0141] Thus, a method for treating a disease or disorder includes administering a Kv1.3 inhibitor to a subject once every 2 to 8 days. In other words, the Kv1.3 inhibitor is administered to a subject at least 2 days after the previous administration of a Kv1.3 inhibitor (if any) to the subject, and at most 8 days after the previous administration of a Kv1.3 inhibitor to the subject. The period between administrations of a Kv1.3 inhibitor to a subject can be referred to as the "interval" between administrations of a Kv1.3 inhibitor. Thus, the method includes administering a Kv1.3 inhibitor to a subject at intervals of 2 to 8 days (i.e., the interval between administrations is 2 to 8 days in duration). The method includes administering a Kv1.3 inhibitor to a subject at intervals of 2 to 8 days.

[0142] The term "day" refers to a period of 24 hours ± 8 hours (i.e., 16 to 32 hours). In other words, a "day" is approximately 24 hours, with a tolerance of 8 hours around the exact 24-hour point. The 8-hour tolerance is not cumulative across multiple days. Thus, "2 days" refers to a period of 48 hours ± 8 hours (i.e., 40-56 hours) rather than a period of 48 hours ± 16 hours; "3 days" refers to a period of 72 hours ± 8 hours (i.e., 64-80 hours); "4 days" refers to a period of 96 hours ± 8 hours (i.e., 88-104 hours); "5 days" refers to a period of 120 hours ± 8 hours (i.e., 112-128 hours); "6 days" refers to a period of 144 hours ± 8 hours (i.e., 136-152 hours); "7 days" refers to a period of 168 hours ± 8 hours (i.e., 160-176 hours); and "8 days" refers to a period of 192 hours ± 8 hours (i.e., 184-200 hours).

[0143] Thus, the phrase "administering a Kv1.3 inhibitor to a subject once every 2 to 8 days" (i.e., once every 2 to 8 days) herein means administering a Kv1.3 inhibitor to a subject once every 40 to 200 hours. Thus, the Kv1.3 inhibitor is administered to a subject 40 to 200 hours after the previous administration (if any). In other words, the Kv1.3 inhibitor is administered to a subject at least 40 hours after the previous administration (if any) of a Kv1.3 inhibitor to a subject, and up to 200 hours after the previous administration (if any) of a Kv1.3 inhibitor to a subject.

[0144] In some embodiments, the method includes administering a Kv1.3 inhibitor to a subject once every 2-7 days, e.g., once every 2-6 days, once every 2-5 days, once every 2-4 days, or once every 2-3 days. In some embodiments, the method includes administering a Kv1.3 inhibitor to a subject once every 3-8 days, e.g., once every 3-7 days, once every 3-6 days, once every 3-5 days, or once every 3-4 days. In some embodiments, the method includes administering a Kv1.3 inhibitor to a subject once every 4-8 days, e.g., once every 4-7 days, once every 4-6 days, or once every 4-5 days. In some embodiments, the method includes administering a Kv1.3 inhibitor to a subject once every 5-8 days, e.g., once every 5-7 days or once every 5-6 days. In some embodiments, the method comprises administering a KvI .3 inhibitor to the subject once every 6-8 days, e.g., once every 6-7 days, hi some embodiments, the method comprises administering a KvI .3 inhibitor to the subject once every 7-8 days.

[0145] In some embodiments, the method comprises administering a KvI.3 inhibitor to a subject once every two days, once every three days, once every four days, once every five days, once every six days, once every seven days, or once every eight days. Preferably, the method comprises administering a KvI.3 inhibitor to a subject once every seven days. Preferably, the method comprises administering a KvI.3 inhibitor to a subject approximately once a week. Preferably, the method comprises administering a KvI.3 inhibitor to a subject once a week.

[0146] The method includes administering a Kv1.3 inhibitor to a subject once every 2 to 8 days during an administration period. An "administration period" refers to the overall period during which a subject is administered the Kv1.3 inhibitor (i.e., the period during which Kv1.3 is administered to the subject at intervals). In other words, an administration period begins with the first administration of a Kv1.3 inhibitor during that administration period and ends with the last administration of the inhibitor during that administration period. The duration of an administration period can depend on various factors, including whether a disease or disorder is being treated or prevented, the type of disease or disorder being treated or prevented, and the characteristics of the subject (e.g., age, weight, or immunological status). The administration period can be determined by the clinician prescribing the Kv1.3 inhibitor to the subject.

[0147] The administration period may be as long as necessary to treat or prevent a disease or disorder in a subject (i.e., administration of the Kv1.3 inhibitor may continue for as long as necessary). In some embodiments, the administration period is at least one month. In other words, in some embodiments, the method comprises administering a Kv1.3 inhibitor to a subject once every 2-8 days for at least one month. In some embodiments, the administration period is at least two months, e.g., at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months, at least nine months, at least ten months, at least 11 months, or at least 12 months (i.e., at least one year). In some embodiments, the administration period is at least one year. In other words, in some embodiments, the method comprises administering a Kv1.3 inhibitor to a subject once every 2-8 days for at least one year. In some embodiments, the administration period is at least 2 years, e.g., at least 3 years, at least 4 years, at least 5 years, at least 6 years, at least 7 years, at least 8 years, at least 9 years, at least 10 years, at least 11 years, at least 12 years, at least 13 years, at least 14 years, at least 15 years, at least 16 years, at least 17 years, at least 18 years, at least 19 years, or at least 20 years. In some embodiments, the administration period is the subject's lifetime. In other words, in some embodiments, the method comprises administering a KvI.3 inhibitor to a subject once every 2 to 8 days for the subject's lifetime.

[0148] A given subject may undergo multiple (i.e., more than one) administration periods. In other words, a Kv1.3 inhibitor may be administered to a subject for a given administration period, then administration of the Kv1.3 inhibitor may be discontinued, and then the Kv1.3 inhibitor may be administered to the subject again for a further administration period. Over the course of a subject's life, there may be multiple such discontinuations and resumptions of administration of a Kv1.3 inhibitor (i.e., multiple administration periods, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more administration periods). The administration period of the present invention may be any one (or more) of these multiple administration periods, and this is not precluded by the fact that one or more other administration periods (of these multiple administration periods) are not within the scope of the present invention.

[0149] The number of administrations of a Kv1.3 inhibitor to a subject within an administration period (i.e., the number of times a Kv1.3 inhibitor is administered to a subject within an administration period) depends on the duration of the administration period and the time elapsed between administrations (i.e., the interval between administrations). For example, the longer the administration period, the greater the total number of administrations of a Kv1.3 inhibitor to a subject within that administration period is likely to be. The Kv1.3 inhibitor can be administered to a subject any number of times (i.e., the method includes administering a Kv1.3 inhibitor to a subject any number of times). In some embodiments, the method includes administering a Kv1.3 inhibitor to a subject at least twice (i.e., two or more times) within the administration period. In other words, in some embodiments, the method includes administering a Kv1.3 inhibitor to a subject at least twice within the administration period. In some embodiments, the method includes administering a KvI .3 inhibitor to a subject at least three times (i.e., three or more times) within an administration period, e.g., at least four times, at least five times, at least six times, at least seven times, at least eight times, at least nine times, at least ten times, at least fifteen times, at least twenty times, at least thirty times, at least forty times, at least fifty times, at least sixty times, at least seventy times, at least eighty times, at least ninety times, or at least one hundred times.

[0150] Typically, the interval between doses of the Kv1.3 inhibitor (i.e., the amount of time elapsed between a given dose and the next) is the same throughout the dosing period. In other words, in some embodiments, the interval between doses of the Kv1.3 inhibitor is constant or uniform throughout the dosing period. For example, the Kv1.3 inhibitor may be administered once every seven days (i.e., the interval between all doses is seven days). Regular intervals are preferred because administering the inhibitor at regular intervals may be simpler (e.g., it may be easier for the subject / patient to remember to administer the inhibitor), which may improve patient compliance with treatment. However, in some embodiments, the intervals are variable. In other words, in some embodiments, each interval is independently selected to be between two and eight days in duration. For example, the Kv1.3 inhibitor may be administered two days after the previous dose, but the previous dose may have been eight days after the previous dose, which may have been five days after the previous dose, etc. The intervals may be varied in this manner based on the direction of the clinician seeking to achieve a particular clinical result. For example, the interval can be increased during administration to determine the longest interval possible while maintaining treatment or prevention of the disease or disorder.

[0151] According to the present invention, a method includes administering a Kv1.3 inhibitor to a subject once every 2 to 8 days. However, intervals of more than 8 days may be present within a given administration period. It will be understood that the present invention encompasses administration of a Kv1.3 inhibitor once every 2 to 8 days for any portion of the administration period. For example, it is within the scope of the present invention to administer a Kv1.3 inhibitor to a subject once every 2 to 8 days for an initial portion of the administration period, followed by administration of a Kv1.3 inhibitor to the subject at intervals of more than 8 days. Thus, according to the present invention, a method for treating or preventing a disease or disorder includes administering a Kv1.3 inhibitor to a subject at least twice, wherein the interval between at least two administrations of the Kv1.3 inhibitor is 2 to 8 days. In some embodiments, the method includes administering a Kv1.3 inhibitor to a subject with an interval of 2 to 8 days between at least two administrations of the Kv1.3 inhibitor. In some embodiments, the method includes at least one interval (i.e., interval between administrations of the Kv1.3 inhibitor) of 2 to 8 days. In other words, in some embodiments, the method comprises a first administration of a KvI.3 inhibitor to the subject, followed by a second administration of the KvI.3 inhibitor 2-8 days later. The first and second administrations may be preceded or followed by a further administration of the KvI.3 inhibitor to the subject. In some embodiments, the method consists of a first administration of a KvI.3 inhibitor to the subject, followed by a second administration of the KvI.3 inhibitor 2-8 days later (i.e., without a further administration of the KvI.3 inhibitor).

[0152] Kv1.3 inhibitor dose The term "dose" refers to the amount of a Kv1.3 inhibitor administered to a subject in a single administration. For example, in embodiments in which the Kv1.3 inhibitor is administered by subcutaneous injection, "dose" can refer to the amount of the Kv1.3 inhibitor in a single injection of the inhibitor. The terms "dosage" and "dose" are used interchangeably herein.

[0153] In some embodiments, the method comprises administering the Kv1.3 inhibitor in escalating doses.

[0154] In some embodiments, the method comprises administering to the subject a Kv1.3 inhibitor at a dose of between 0.1 mg and 30.0 mg.

[0155] In other words, in some embodiments, the Kv1.3 inhibitor is administered to the subject at a dose of 0.1 mg to 30.0 mg. In other words, in some embodiments, the Kv1.3 inhibitor is administered to the subject at a dose of 0.1 mg or more and 30.0 mg or less. In some embodiments, the Kv1.3 inhibitor is formulated at a dose of 0.1 mg to 30.0 mg. In some embodiments, the method comprises administering the Kv1.3 inhibitor to the subject at a dose of 0.1 mg to 30.0 mg once every 2 to 8 days.

[0156] In some embodiments, each administration of a Kv1.3 inhibitor to a subject is at the same dose as the other administrations. In some embodiments, each administration of a Kv1.3 inhibitor to a subject can be at a different dose than the other administrations. In other words, each administration of a Kv1.3 inhibitor to a subject can be independently selected to be a dose between 0.1 mg and 30.0 mg.

[0157] In some embodiments, the Kv1.3 inhibitor is administered to a subject in a single dose formulation of 0.1 mg to 30.0 mg. This single dose formulation may be administered to a subject one or more times, and each of the multiple dose formulations administered to a subject does not need to contain the same amount of the Kv1.3 inhibitor. In other words, the Kv1.3 inhibitor may be administered to a subject in a series of single doses, but each single dose may not contain the same amount of the Kv1.3 inhibitor.

[0158] In some embodiments, the method includes administering a Kv1.3 inhibitor at a dose of 1.0 mg to 30.0 mg, e.g., 2.0 mg to 30.0 mg, 3.0 mg to 30.0 mg, 4.0 mg to 30.0 mg, 5.0 mg to 30.0 mg, 6.0 mg to 30.0 mg, 7.0 mg to 30.0 mg, 8.0 mg to 30.0 mg, 9.0 mg to 30.0 mg, 10.0 mg to 30.0 mg, 11.0 mg to 30.0 mg, 12.0 mg to 30.0 mg, 13.0 mg to 30.0 mg, 14.0 mg to 30.0 mg, 15.0 mg The method includes administering to the subject a dose of 10.0 mg to 30.0 mg, 16.0 mg to 30.0 mg, 17.0 mg to 30.0 mg, 18.0 mg to 30.0 mg, 19.0 mg to 30.0 mg, 20.0 mg to 30.0 mg, 21.0 mg to 30.0 mg, 22.0 mg to 30.0 mg, 23.0 mg to 30.0 mg, 24.0 mg to 30.0 mg, 25.0 mg to 30.0 mg, 26.0 mg to 30.0 mg, 27.0 mg to 30.0 mg, 28.0 mg to 30.0 mg, or 29.0 mg to 30.0 mg.

[0159] In some embodiments, the method comprises administering a KvI.3 inhibitor in an amount of 1.0 mg to 29.0 mg, e.g., 1.0 mg to 28.0 mg, 1.0 mg to 27.0 mg, 1.0 mg to 26.0 mg, 1.0 mg to 25.0 mg, 1.0 mg to 24.0 mg, 1.0 mg to 23.0 mg, 1.0 mg to 22.0 mg, 1.0 mg to 21.0 mg, 1.0 mg to 20.0 mg, 1.0 mg to 19.0 mg, 1.0 mg to 18.0 mg, 1.0 mg to 1 ...7.0 mg, 1.0 mg to 28.0 mg, 1.0 mg to 29.0 mg, 1.0 mg to 30.0 mg, 1.0 mg to 31.0 mg, 1.0 mg to 32.0 mg, 1.0 mg to 33.0 mg, 1.0 mg to 34.0 mg, 1.0 mg to 35.0 mg, 1.0 mg to 36.0 mg, 1.0 mg to 37.0 mg, 1.0 mg to 38.0 mg, 1.0 mg to 39.0 mg, 1.0 mg The method includes administering to a subject a dose of 1.0 mg to 16.0 mg, 1.0 mg to 15.0 mg, 1.0 mg to 14.0 mg, 1.0 mg to 13.0 mg, 1.0 mg to 12.0 mg, 1.0 mg to 11.0 mg, 1.0 mg to 10.0 mg, 1.0 mg to 9.0 mg, 1.0 mg to 8.0 mg, 1.0 mg to 7.0 mg, 1.0 mg to 6.0 mg, 1.0 mg to 5.0 mg, 1.0 mg to 4.0 mg, 1.0 mg to 3.0 mg, or 1.0 mg to 2.0 mg.

[0160] In some embodiments, the method includes administering to the subject a dose of a KvI.3 inhibitor of 0.1 mg to 15.0 mg, e.g., 0.1 mg to 10.0 mg, 0.1 mg to 9.0 mg, 0.1 mg to 8.0 mg, 0.1 mg to 7.0 mg, 0.1 mg to 6.0 mg, 0.1 mg to 5.0 mg, 0.1 mg to 4.0 mg, 0.1 mg to 3.0 mg, 0.1 mg to 2.0 mg, 0.1 mg to 1.0 mg, or 0.1 mg to 0.5 mg.

[0161] In some embodiments, the method includes administering a KvI.3 inhibitor to a subject at a dose of about 1.0 mg, about 2.0 mg, about 3.0 mg, about 4.0 mg, about 5.0 mg, about 6.0 mg, about 7.0 mg, about 8.0 mg, about 9.0 mg, about 10.0 mg, about 11.0 mg, about 12.0 mg, about 13.0 mg, about 14.0 mg, about 15.0 mg, about 16.0 mg, about 17.0 mg, about 18.0 mg, about 19.0 mg, about 20.0 mg, about 21.0 mg, about 22.0 mg, about 23.0 mg, about 24.0 mg, about 25.0 mg, about 26.0 mg, about 27.0 mg, about 28.0 mg, about 29.0 mg, or about 30.0 mg.

[0162] In some embodiments, the method comprises administering to the subject a Kv1.3 inhibitor at a dose of 10 nmol / kg to 400 nmol / kg.

[0163] In other words, in some embodiments, the Kv1.3 inhibitor is administered to the subject at a dose of 10 nmol / kg to 400 nmol / kg. In other words, in some embodiments, the Kv1.3 inhibitor is administered to the subject at a dose of 10 nmol / kg or more and 400 nmol / kg or less. In some embodiments, the Kv1.3 inhibitor is formulated at a dose of 10 nmol / kg to 400 nmol / kg. In some embodiments, the method comprises administering the Kv1.3 inhibitor to the subject once every 2 to 8 days at a dose of 10 nmol / kg to 400 nmol / kg. In some embodiments, each administration of the Kv1.3 inhibitor to the subject can be independently selected to be a dose of 10 nmol / kg to 400 nmol / kg.

[0164] The unit "nmol / kg" means that the dose of Kv1.3 inhibitor is relative to the subject's body weight / body mass. A given number of nanomoles of Kv1.3 is administered per kilogram of subject's body weight. Therefore, the exact dose of Kv1.3 inhibitor can be determined by clinicians for each subject.

[0165] In some embodiments, the method includes administering a Kv1.3 inhibitor to a subject at a dose of 50 nmol / kg to 400 nmol / kg, e.g., 100 nmol / kg to 400 nmol / kg, 150 nmol / kg to 400 nmol / kg, 200 nmol / kg to 400 nmol / kg, 250 nmol / kg to 400 nmol / kg, 300 nmol / kg to 400 nmol / kg, or 350 nmol / kg to 400 nmol / kg.

[0166] In some embodiments, the method includes administering a Kv1.3 inhibitor to a subject at a dose of 10 nmol / kg to 350 nmol / kg, e.g., 10 nmol / kg to 350 nmol / kg, 10 nmol / kg to 300 nmol / kg, 10 nmol / kg to 250 nmol / kg, 10 nmol / kg to 200 nmol / kg, 10 nmol / kg to 150 nmol / kg, 10 nmol / kg to 100 nmol / kg, or 10 nmol / kg to 50 nmol / kg.

[0167] In some embodiments, the method comprises administering to the subject a KvI .3 inhibitor at a dose of about 3 nmol / kg, about 10 nmol / kg, about 50 nmol / kg, about 100 nmol / kg, about 150 nmol / kg, about 200 nmol / kg, about 250 nmol / kg, about 300 nmol / kg, about 350 nmol / kg, or about 400 nmol / kg. Preferably, the method comprises administering to the subject a KvI .3 inhibitor at a dose of about 300 nmol / kg.

[0168] In some embodiments, the methods include administering to the subject a Kv1.3 inhibitor at a dose of about 3 nmol / kg to about 300 nmol / kg.

[0169] In other words, in some embodiments, the Kv1.3 inhibitor is administered to the subject at a dose of 3 nmol / kg to 300 nmol / kg. In some embodiments, the Kv1.3 inhibitor is administered to the subject at a dose of 3 nmol / kg or more and 300 nmol / kg or less. In some embodiments, the Kv1.3 inhibitor is formulated at a dose of 3 nmol / kg to 300 nmol / kg. In some embodiments, the method comprises administering the Kv1.3 inhibitor to the subject once every 2 to 8 days at a dose of 3 nmol / kg to 300 nmol / kg. In some embodiments, each administration of the Kv1.3 inhibitor to the subject can be independently selected to be a dose of 3 nmol / kg to 300 nmol / kg.

[0170] In some embodiments, the method includes administering a Kv1.3 inhibitor to the subject at a dose of 50 nmol / kg to 300 nmol / kg, e.g., 100 nmol / kg to 300 nmol / kg, 150 nmol / kg to 300 nmol / kg, 200 nmol / kg to 300 nmol / kg, or 250 nmol / kg to 300 nmol / kg.

[0171] In some embodiments, the method includes administering a Kv1.3 inhibitor to the subject at a dose of 3 nmol / kg to 250 nmol / kg, e.g., 3 nmol / kg to 200 nmol / kg, 3 nmol / kg to 150 nmol / kg, 3 nmol / kg to 100 nmol / kg, or 3 nmol / kg to 50 nmol / kg.

[0172] Pharmaceutical Composition The present invention also provides pharmaceutical compositions comprising a Kv1.3 inhibitor or a pharmaceutically acceptable salt described herein, for use in the methods described herein. Accordingly, the present invention provides a pharmaceutical composition comprising a Kv1.3 inhibitor or a pharmaceutically acceptable salt described herein, for use in a method for treating or preventing a disease or disorder in a subject, the method comprising the step of administering the pharmaceutical composition to the subject once every 2 to 8 days.

[0173] In other words, in some embodiments, the present invention provides a Kv1.3 inhibitor or a pharmaceutically acceptable salt thereof for use as described herein, wherein the Kv1.3 inhibitor or a pharmaceutically acceptable salt thereof is in the form of a composition. Preferably, the composition is a pharmaceutical composition. In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier, excipient, or vehicle. In some embodiments, the pharmaceutical composition of the present invention is one in which the Kv1.3 inhibitor is in the form of a pharmaceutically acceptable acid addition salt.

[0174] In some embodiments, the composition comprises one or more Kv1.3 inhibitors described herein (i.e., more than one Kv1.3 inhibitor). Each of the Kv1.3 inhibitors is independently selected from any of the Kv1.3 inhibitors described herein. In other words, each of the Kv1.3 inhibitors in the composition can be any of the Kv1.3 inhibitors described herein. In some embodiments, the composition comprises one or more peptides. In some embodiments, the composition comprises one or more peptides, each of which comprises or consists of a sequence independently selected from any of the peptide sequences described herein. In some embodiments, the composition comprises one or more peptides, each of which comprises or consists of a peptide independently selected from any of the peptide sequences described herein.

[0175] In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of a Kv1.3 inhibitor or a pharmaceutically acceptable salt thereof. 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 on, among other factors, 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 any concomitant medications, 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, and therefore, the determination of therapeutically effective dosage levels to achieve a desired therapeutic effect is within the realm of those skilled in the art.

[0176] As used herein, the term "therapeutically effective amount" refers to an amount that reduces the symptoms of a given disease, disorder, condition, or pathology, and preferably normalizes the physiological response of an individual with that disease, disorder, condition, or pathology. The reduction of symptoms or normalization of the physiological response can be determined using methods routine in the art and may vary depending on the given disease, disorder, condition, or pathology. In one embodiment, a therapeutically effective amount of a Kv1.3 inhibitor or pharmaceutical composition of the present invention is an amount that restores a measurable physiological parameter to substantially the same value as that parameter in an individual without the disease, disorder, condition, or pathology in question.

[0177] In some embodiments of the present invention, administration of the Kv1.3 inhibitor or pharmaceutical composition of the present invention begins at a lower dosage level and the dosage level is increased until the desired effect of preventing / treating the relevant medical indication is achieved, which defines a therapeutically effective amount. For compounds of the invention, alone or as part of a pharmaceutical composition, such a human dose of active compound can be between about 0.01 pmol / kg and 500 μmol / kg body weight, between about 0.01 pmol / kg and about 300 μmol / kg body weight, between about 0.01 pmol / kg and about 100 μmol / kg body weight, between about 0.1 pmol / kg and about 50 μmol / kg body weight, between about 1 pmol / kg and about 10 μmol / kg body weight, between about 5 pmol / kg and about 5 μmol / kg body weight, between about 10 pmol / kg and about 1 μmol / kg body weight, between about 50 pmol / kg and about 0.1 μmol / kg body weight, between about 100 pmol / kg and about 0.01 μmol / kg body weight, between about 0.001 μmol / kg and about 0.5 μmol / kg body weight, or between about 0.05 μmol / kg and about 0.1 μmol / kg body weight.

[0178] 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. Many 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.

[0179] 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.

[0180] All patents, published patent applications, and non-patent literature referenced in this application are specifically incorporated herein by reference. In case of conflict, the present specification, including its specific definitions, will control.

[0181] 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.

[0182] The present disclosure is not limited by the exemplary methods and materials disclosed herein; any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present 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.

[0183] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0184] 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 integer or component, 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.

[0185] 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 embodiments appended hereto.

[0186] 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.

[0187] Sequence Listing Sequence identification numbers (SEQ ID NOs) are assigned to sequences herein as follows: SEQ ID NOs: 1-150 - Kv1.3 inhibitor peptide sequences SEQ ID NOs: 151-180 and 182-188 - Segments of Kv1.3 inhibitor peptide sequences SEQ ID NO. 181 - Missing [Example]

[0188] [Example 1] General Peptide Synthesis A list of abbreviations and suppliers is provided in Table 1 below.

[0189] [Table 3]

[0190] 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 suitable common protecting groups for side-chain functional groups.

[0191] The polymer support-based resin used was, for example, TentaGel™ The resin was packed into a synthesis apparatus and swollen with DMF before use.

[0192] Coupling CEM Liberty Peptide Synthesizer A solution of Fmoc-protected amino acid (4 equivalents) was added to the resin along with a solution of coupling reagent (4 equivalents) and a solution of base (8 equivalents). The mixture was heated to 70-75°C in a microwave oven for 5 minutes, or for 60 minutes without heating. Nitrogen was bubbled through the mixture during the coupling.

[0193] 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). The coupling time was 10 min at room temperature (RT) unless otherwise stated. The resin was washed with DMF (5 x 0.5 min). When the coupling was repeated, the coupling time was 45 min at room temperature in all cases.

[0194] 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.

[0195] 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).

[0196] Disconnect The dried peptide resin was treated with TFA and appropriate scavengers for approximately 2 hours. The volume of the filtrate was reduced, and diethyl ether was added to precipitate the crude peptide. The crude peptide precipitate was washed several times with diethyl ether and finally dried.

[0197] HPLC purification of crude peptides The crude peptide was purified by preparative reverse-phase HPLC using a conventional HPLC system (e.g., a Gilson GX-281 with a 331 / 332 pump combination) equipped with a column such as a 5 x 25 cm Gemini NX 5u C18 110A column for binary gradient application and a fraction collector using 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.) at a flow rate of 20–40 ml / min. Fractions were analyzed by analytical HPLC and MS, and selected fractions were pooled and lyophilized. The final product was characterized by HPLC and MS.

[0198] Disulfide formation Crude or partially purified linear peptides containing six cysteines were 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 in the open atmosphere. The progress of the reaction was determined by HPLC and was typically estimated 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.

[0199] Analytical HPLC Final purity was determined by analytical HPLC (Agilent 1100 / 1200 series) equipped with an autosampler, degasser, 20 μl flow cell, and Chromeleon software. HPLC was operated at 40 °C with a flow rate of 1.2 ml / min using an analytical column such as a Kinetex 2.6 μm XB-C18 100A 100 x 4.6 mm column. Compounds were detected and quantified at 215 nm. Buffer A (0.1% TFA, aq.) and buffer B (0.1% TFA, 90% MeCN, aq.) were used.

[0200] 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 performed in positive mode using direct injection and a cone voltage of 15 V (1 TOF), 30 V (2 TOF), or 45 V (3 TOF) as specified in the chromatogram. Precision was 5 ppm, and typical resolution was 15,000–20,000.

[0201] The synthesized peptides (i.e., Kv1.3 inhibitors) are shown in Table 2: [Table 4] JPEG2025534884000016.jpg246170 JPEG2025534884000017.jpg250170 JPEG2025534884000018.jpg248170 JPEG2025534884000019.jpg250170 JPEG2025534884000020.jpg246170 [Example 2]

[0202] Selectivity of Kv1.3 inhibitor peptides in patch clamp assays The selectivity of Kv1.3 inhibitor peptides for Kv1.3 over other potassium ion channels (Kv1.1, Kv1.2, and Kv1.6) was determined using patch clamp assays.

[0203] 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.

[0204] To quantify ionic currents, we used an automated, tip-based planar patch clamp device, QPatch®. All recordings were performed in the 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 MgCl, 3 mM CaCl, 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 throughout the experiment. Currents were elicited using a voltage protocol consisting of 30 mV voltage shifts of 500 ms every 15 s from a holding potential of -80 mV.

[0205] Concentration-response relationships were established by cumulatively applying seven increasing concentrations of test compound to individual cells, with a 2-minute recording period for each compound application.

[0206] Efficacy was determined as the mean charge of the last three sweeps from the cursor position at the end of each concentration application period. Percent inhibition for each test dose application period was calculated as the decrease in mean 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 the results shown in Table 3 below. [Table 5] JPEG2025534884000022.jpg255170 JPEG2025534884000023.jpg109170 [Example 3a]

[0207] Inhibitory activity of Kv1.3 inhibitor peptides on human PBMCs Human peripheral blood mononuclear cells (PBMCs) were used to assess the effect of Kv1.3 inhibitor peptides on T cell activation as determined by IL-2 (cytokine) release after stimulation with anti-CD3.

[0208] Human PBMCs were obtained from Precision for Medicine (Frederick, MD). Cells from five donors were used. Bulk T cells in the PBMC preparation were stimulated using plate-bound anti-CD3. Briefly, 96-well plates were coated with anti-CD3 antibody for 2 hours at 37°C using 50 μL of a 0.5 μg / mL anti-CD3 solution diluted in 1x PBS. The plates were then washed twice.

[0209] The peptides shown in Table 4a were diluted in culture medium (RPMI 1640 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). Cyclosporin A (1 μg / ml) and Vm24 peptide (100 nM) were used as positive controls. Finally, 1 × 10 5 PBMCs were added to each well in a volume of 100 μL, for a final volume of 200 μL per well. The plates were incubated in a 37°C / 5% CO2 incubator for 20–24 hours. After centrifugation, 25 μl of the supernatant was transferred to an IL-2 detection plate (MSD Human IL-2 Tissue Culture Kit, catalog number K151AHB-2) and IL-2 was measured according to the manufacturer's instructions (Meso Scale Discovery, Rockville, MD, USA).

[0210] Results are compared with IC obtained from anti-CD3 stimulated human PBMC assays. 50 The geometric means of the values ​​are shown in Table 4a, and all values ​​are from at least four replicates.

[0211] [Table 6]

[0212] Incubation with anti-CD3 antibody activated hPBMCs, and addition of the reference peptide resulted in a dose-dependent decrease in IL-2 secretion. On average, the IC of the peptide 50 The IC values ​​(calculated from IL-2 release) ranged from 0.05 nM to 0.4 nM. This is in line with ShK186 (IC 50 The IC observed was 0.07 nM 50 The IC of Moka1 is comparable to that of IL-2 and is less potent in inhibiting IL-2 secretion. 50 The release of IL-2 was approximately 10-100 times lower than that of Moka1. There was no significant difference between the peptides and ShK186. ShK186 and the peptides were all significantly lower than that of Moka1. Cyclosporine completely blocked CD3-induced IL-2 release in all experiments. Example 3b

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

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

[0215] The peptides were then diluted in medium (RPMI 1640 with Glutamax-I containing 10% v / v fetal bovine serum, 1% v / v penicillin-streptomycin solution) and added in a volume of 50 μl. The peptides shown in Table 4b 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, for a final volume of 100 μl per well. The plates were incubated for 20–24 hours in a 37°C / 5% CO2 incubator. After centrifugation, 25 μl of the supernatant was transferred to an IL-2 detection plate (MSD Human IL-2 Tissue Culture Kit, catalog number 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 4b as the geometric mean of IC50 values ​​obtained from anti-CD3 stimulated human PBMC assays, all values ​​are derived from at least six replicates.

[0218] [Table 7]

[0219] Incubation with anti-CD3 antibody activated hPBMCs, and addition of a Kv1.3 inhibitor peptide resulted in a dose-dependent reduction in IL-2 secretion.

[0220] As shown in Table 4b, the average IC of peptides 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 50The Shk-186 values ​​in the two sets of experiments are not expected to be identical, as this assay was performed using a different donor than that used in Example 3a.

[0221] Cyclosporine completely blocked anti-CD3-induced IL-2 release in all experiments. [Example 4]

[0222] Inhibitory activity of Kv1.3 inhibitor peptides in rat whole blood We used rat whole blood to assess the efficacy of Kv1.3 inhibitor peptides 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 cytokine production, in which the Kv1.3 ion channel plays a key role. Therefore, this experimental system allows us to measure the activity of Kv1.3 inhibitors in primary cells.

[0223] Rat whole blood was obtained by terminal cardiac bleeding from healthy, naive Lewis or Sprague-Dawley rats and collected using sodium heparin blood collection tubes. Peptides were diluted to 4x the final test concentration in assay buffer (DMEM + GlutaMAX), which contains 3.97 mM L-alanine-L-glutamine (Gibco catalog number 61965026) supplemented with 25 mM HEPES buffer, 1 mM sodium pyruvate, 100 units / ml penicillin, 100 μg / ml streptomycin, and 0.05% casein from bovine milk (Sigma-Aldrich). 25 μl of this diluted blood 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. Next, 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 hours at 37°C / 5% CO2 in a humidified box. The assay plate was centrifuged at 300g for 10 minutes at 4°C, and the supernatant was transferred to a new plate. The concentration of IL-17A released into the supernatant was measured using a Rat IL-17A ELISA Kit (Abcam, catalog no. ab214028) according to the manufacturer's recommendations. Samples were diluted 2.5-fold by transferring 20 μl of supernatant to wells of the ELISA plate containing 30 μl of the detection kit's buffer 75BS.

[0224] Data for peptides that induce inhibition of IL-17A were normalized relative to full thapsigargin activation (no inhibitor 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 ex vivo biological effects correlate with the potency of the peptides.

[0226] [Table 8] JPEG2025534884000027.jpg249170 JPEG2025534884000028.jpg130170 [Example 5]

[0227] Inhibitory activity of Kv1.3 inhibitor peptides in human whole blood We used human whole blood to evaluate the efficacy of Kv1.3 inhibitor peptides on T cell activation, as determined by the release of cytokines IFN-γ, IL-2, and IL-17A after stimulation with thapsigargin. Addition of thapsigargin activates a signaling cascade leading to T cell proliferation and cytokine production, in which the Kv1.3 ion channel plays a key role. Therefore, the activity of Kv1.3 inhibitors in human primary T cells can be measured in this experimental system.

[0228] Human whole blood was obtained from healthy blood donors and collected after informed consent using sodium heparin blood collection tubes (Becton, Dickinson and Company (BD), catalog number 367876). Test peptides were diluted to 4x the final test concentration in assay buffer (Dulbecco's Modified Eagle's Medium (DMEM) (Gibco catalog number 61965026) containing high glucose and GlutaMAX (3.97 mM L-alanine-L-glutamine), supplemented with 25 mM HEPES buffer, 1 mM sodium pyruvate, 100 units / ml penicillin, 100 μg / ml streptomycin, and 0.05% casein from bovine milk (Sigma-Aldrich catalog number C4765)). 25 μl was added to wells of a 96-well tissue culture plate. 50 μl of human whole blood was then added and incubated at room temperature for a minimum of 5 minutes to allow compound binding. Cells were then activated by adding 25 μl of 40 μM thapsigargin diluted in assay buffer to all wells of the assay plate, followed by incubation for 24 hours at 37°C / 5% CO2 in a humidified chamber. The assay plate was centrifuged at 300 g for 10 minutes at 4°C, and the supernatant was transferred to a new plate. The concentrations of IFN-γ, IL-2, and IL-17A released into the supernatant were measured using a triplex human cytokine detection kit (MSD Human U-Plex IFN-γ, IL-2, and IL-17A kit; Meso Scale Discovery, Inc., catalog number K15067L-2). All three cytokines were measured using an MSD MESO QuickPlex SQ 120 instrument (Meso Scale Discovery, Inc., Rockville, MD, USA) according to the manufacturer's instructions.

[0229] To determine the inhibitory potency of a compound, cytokine concentration data was analyzed using the following equation: Y = Bottom + (Top - Bottom) / (1 + IC 50A three-parameter logistic dose-response model based on the equation (Y / X) was used for fitting, where Y is the measured cytokine concentration, X is the compound concentration, and the top, bottom, and IC 50 are parameters fitted using Graphpad Prism software version 5.04. IC calculated from concentration-response curve 50 Values ​​represent the compound concentration that gives an inhibitory response midway between the basal (Bottom) and maximum (Top) responses.

[0230] The results are shown in Table 6 and IC 50 All values ​​are expressed as . All values ​​are from at least two replicate experiments. All tested Kv1.3 blockers were able to inhibit cytokine production from human T cells present in human whole blood stimulated with thapsigargin. [Table 9] [Example 6]

[0231] Pharmacokinetic characterization of Kv1.3 inhibitors A single subcutaneous (sc) injection of each peptide to be tested was administered to Sprague-Dawley or Wistar rats (male, weighing approximately 250-350 g).

[0232] After sc administration of selected peptides (70 nmol / kg dose, either 2 or 5 mL / kg dose volume), blood samples were collected at 15, 30, 45, 60, 90 minutes, 2 hours, 3 hours, and 4 hours after administration. At each sampling time point, samples were collected from rats by sublingual bleeding or tail amputation. After the final sampling, rats were sacrificed by O2 / CO2 anesthesia. The administration vehicle was 10 mM phosphate, 0.8% NaCl, 0.05% polysorbate 20 (pH 6.0).

[0233] 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 non-compartmental 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.

[0234] The results for some exemplary peptides are shown in Table 7. [Table 10] [Example 7]

[0235] Effect of treatment with Kv1.3 inhibitor peptides in a rat keyhole limpet hemocyanin (KLH) ear inflammation model A classical delayed-type hypersensitivity (DTH) response was elicited in one ear of rats. Briefly, 8-10-week-old male Lewis rats were immunized on day -7 with 200 μL of keyhole limpet hemocyanin (KLH) (Sigma, Catalog No. H7017) (4 mg / mL) emulsified in complete Freund's adjuvant (CFA) (Difco, Catalog No. 263810) subcutaneously administered at the base of the tail. On day 0, rats were challenged intradermally 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.

[0236] The ability of treatment with Kv1.3 inhibitor peptides to reduce DTH ear swelling responses was investigated by comparing the responses of vehicle-treated rats with those of Kv1.3 inhibitor peptides (n = 8–10 per group). 24 h before KLH ear challenge, vehicle or peptide dissolved in vehicle was administered SC (2 mL / kg). Test doses of peptide were 50, 70, or 100 nmol / kg. The test vehicle was 10 mM phosphate, 0.8% w / v NaCl, 0.05% w / v polysorbate 20, pH 6. Cyclosporine (CsA) was included as a positive control in all experiments. Cyclosporine (Sandimmune Neooral® 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.

[0237] As a primary readout of efficacy, the area under the curve (AUC) of Δ ear thickness (mm) was calculated for each animal from 0 to 48 hours after induction of the ear DTH response, and the change (D) was calculated as follows: left ear thickness - right ear thickness. These results were then used to calculate the % inhibition of ear thickness due to Kv1.3 inhibitor treatment: % inhibition: ((1 - (individual ΔAUC Kv1.3 inhibitor / mean ΔAUC vehicle group)) x 100. Results were calculated as % inhibition + / - standard deviation (SD) and are presented in Tables 8 and 9.

[0238] [Table 11] [Table 12] [Example 8]

[0239] Long-lasting effects of treatment with Kv1.3 inhibitor peptide in a KLH-induced delayed-type hypersensitivity (DTH) model animal control committee The animal care facility used was AAALAC-accredited. Female Lewis rats weighing 180 ± 20 g at delivery were used in this study. Upon arrival at the animal facility, all animals underwent a general health assessment. A 1-day acclimation period was allowed before the start of the study.

[0240] Rearing environment Animals were housed under standardized environmental conditions. Rats were housed in open-top cages, six animals per cage. Standard commercially available certified rodent chow was available ad libitum. Tap water was available ad libitum at all times. The chow and water were considered free of known contaminants that would interfere with the objectives of the study. Each cage was identified by its corresponding group and indicated the treatment and the individual identification of the animal housed in the cage.

[0241] The animal room was maintained at a controlled temperature of 20-24°C and relative humidity of 30-70%. A controlled lighting system ensured that the animals received 12 hours of light and 12 hours of darkness per day. Adequate ventilation was maintained at 15 air changes per hour.

[0242] Research details This study was conducted to investigate the longevity of the effect of a Kv1.3 inhibitor (peptide 100) on ear inflammation in a KLH-induced DTH model in rats.

[0243] Healthy male Lewis rats (8-9 weeks old) were immunized on day 0 by subcutaneous injection at the base of the tail with 200 μL of keyhole limpet hemocyanin (KLH) (Sigma, Cat. No. H7017) (4 mg / mL) emulsified with complete Freund's adjuvant (CFA) (Difco, Cat. No. 263810).

[0244] To induce KLH-specific T cell-mediated local inflammation, rats were exposed intradermally to 40 µL of KLH / NaCl 0.9% (2 mg / mL) in the left ear at the indicated time points (days 7, 9, 11, or 13 after immunization). Ear edema was measured 24–48 h later, with the untreated right ear serving as a control. A schematic diagram of the protocol is shown in Figure 1.

[0245] The ability of Kv1.3 inhibitor treatment to reduce DTH ear swelling responses was investigated by comparing responses in vehicle-treated rats (n = 8 / group) with those in rats treated with peptide 100. Vehicle or peptide 100 dissolved in vehicle was administered SC (2 mL / kg) on ​​day 6. The test dose of the Kv1.3 inhibitor was 300 nmol / kg. The test vehicle was 10 mM phosphate, 0.8% w / v NaCl, 0.05% w / v polysorbate 20, pH 6. Cyclosporine A (CsA) was included as a positive control in the study. On day 7, cyclosporine (Sandimmune Neoral® 100 mg / mL oral solution, Novartis) was administered orally (10 mg / kg) 1 hour before and again 6 hours after KLH ear exposure.

[0246] As a readout of efficacy, induced ear thickness was measured 24 and 48 hours after exposure to each ear and compared to the vehicle control. Ear thickness after 24 hours of exposure is shown in Table 10 and Figure 2, and ear thickness after 48 hours of exposure is shown in Table 11 and Figure 3. The results are summarized below.

[0247] For animals challenged on day 7 (i.e., day 1 post-treatment), Peptide 100 and CsA significantly reduced ear swelling compared to vehicle controls when ear swelling was measured 24 hours post-challenge (i.e., day 2 post-treatment) (Peptide 100: 0.676 ± 0.017 mm; Vehicle: 0.796 ± 0.03 mm; CsA: 0.557 ± 0.021 mm; p<0.0001) and when ear swelling was measured 48 hours post-challenge (i.e., day 3 post-treatment) (Peptide 100: 0.623 ± 0.015 mm; Vehicle: 0.733 ± 0.02 mm; CsA: 0.539 ± 0.018 mm; p<0.0001).

[0248] Rats' ears were challenged on day 9 (i.e., day 3 after treatment) and ear swelling was significantly reduced compared to vehicle controls when measured 24 hours after challenge (i.e., day 4 after treatment) (Peptide 100: 0.689 ± 0.031 mm; Vehicle: 0.761 ± 0.04 mm; p = 0.0011) and when measured 48 hours after challenge (i.e., day 5 after treatment) (Peptide 100: 0.659 ± 0.034 mm; Vehicle: 0.729 ± 0.02 mm; p = 0.0002).

[0249] Rats' ears were challenged on day 11 (i.e., day 5 after treatment) and ear swelling was significantly reduced compared to vehicle controls when measured 24 hours after challenge (i.e., day 6 after treatment) (Peptide 100: 0.681±0.023 mm; Vehicle: 0.784±0.025 mm; p<0.0001) and when measured 48 hours after challenge (i.e., day 7 after treatment) (Peptide 100: 0.653±0.013 mm; Vehicle: 0.746±0.41 mm; p<0.0001).

[0250] For ear exposure on day 13 (i.e., day 7 after treatment), the reduction was not significant when ear swelling was measured 24 hours after exposure (i.e., day 8 after treatment) (Peptide 100: 0.729±0.038 mm; Vehicle: 0.767±0.039 mm; p=0.0696) and when ear swelling was measured 48 hours after exposure (i.e., day 9 after treatment) (Peptide 100: 0.699±0.028 mm; Vehicle: 0.720±0.036 mm; p=0.2256).

[0251] In conclusion, peptide 100 administered on day 6 reduced KLH-induced ear swelling after challenge on days 7, 9, and 11 when measured 24 hours after challenge (i.e., reductions in ear swelling were observed on days 2, 4, and 6 after treatment) and when measured 48 hours after challenge (i.e., reductions in ear swelling were measured on days 3, 5, and 7 after treatment). [Table 13] [Table 14] [Example 9]

[0252] Different dosing regimens of Kv1.3 inhibitors in a rat collagen-induced arthritis (CIA) model Peptide and vehicle formulations Vehicle: 10 mM phosphate pH 6 + 0.8% NaCl + 0.05% polysorbate 20

[0253] Peptides: Peptide 100 is formulated at 50 nmol / mL in vehicle. The administration volume was adjusted individually according to the body weight of each animal to reach a target dose of Peptide 100 of 100 nmol / kg.

[0254] animal The animal care facility used was AAALAC-accredited. Female Lewis rats weighing 180 ± 20 g at delivery were used in this study. Upon arrival at the animal facility, all animals underwent a general health assessment. A 1-day acclimation period was allowed before the start of the study.

[0255] Animals were housed under standardized environmental conditions. Rats were housed in open-top cages, six animals per cage. Standard commercially available certified rodent chow was available ad libitum. Tap water was available ad libitum at all times. The chow and water were considered free of known contaminants that would interfere with the objectives of the study. Each cage was identified by its corresponding group and indicated the treatment and the individual identification of the animal housed in the cage.

[0256] The animal room was maintained at a controlled temperature of 20-24°C and relative humidity of 30-70%. A controlled lighting system ensured that the animals received 12 hours of light and 12 hours of darkness per day. Adequate ventilation was maintained at 15 air changes per hour.

[0257] Immunization of rats Animals were divided into groups of 9 animals each based on body weight. All animals were challenged with porcine type II collagen in incomplete Freund's adjuvant (0.2 mg / 0.2 mL / rat, subcutaneously at the base of the tail) on day 1 and boosted (0.1 mg / 0.1 mL / rat, sc) on day 7. Dexamethasone administered PO daily at 0.3 mg / kg was used as a positive control in this study.

[0258] Administration of test peptides and vehicle From day 12 to day 30, Peptide 100 was administered subcutaneously in the flank as a single bolus either daily (QD), every 3 days (Q3D), or every 5 days (Q5D). On days when animals did not receive peptide, they received vehicle only. Peptide and vehicle were administered in a volume of 2 mL / kg. The administration volume was adjusted individually according to the body weight of each animal to reach a target dose of Formulation 1 of 100 nmol / kg.

[0259] Disease Score Disease was assessed using a qualitative severity scoring system (see below, maximum score 16) on days 1, 7, 10, and 12 (pre-dose), on days 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 24, 26, and 28 before peptide treatment, and on day 30, 1 hour after treatment.

[0260] Disease severity scores are based on: [Table 15]

[0261] The ability of Kv1.3 inhibitor treatment to reduce chronic inflammation in arthritis was investigated by comparing the response in rats treated with vehicle (n=9 animals / group) daily, every 3 days, or every 5 days with that of rats treated with Peptide 100. Arthritis severity was determined by summing disease scores for all four limbs from days 12 to 30 for each animal. The results are shown in Table 12. Treatment with Peptide 100 at all dosing regimens resulted in a reduction in the mean clinical score at all dosing regimens. [Table 16]

[0262] The mean clinical scores of the forelimbs were analyzed separately and are shown in Figure 4. All three dosing regimens (daily, every 3 days and every 5 days) were able to delay the disease in the forelimbs. [Example 10]

[0263] Determining the maximally effective dose of Kv1.3 inhibitor peptide treatment in a KLH-induced delayed-type hypersensitivity (DTH) model This study was performed as described in Example 8, except that rats were challenged intradermally with 40 μL of KLH / NaCl 0.9% (2 mg / mL) in the left ear only on days 7 or 11 post-immunization (i.e., days 1 and 5 post-treatment). A schematic diagram of the protocol is shown in Figure 5.

[0264] The dose of Kv1.3 inhibitor treatment capable of reducing the DTH ear swelling response was investigated by comparing responses in vehicle-treated rats (n=8 / group) with those in rats treated with different doses of peptide 100. Vehicle or peptide 100 dissolved in vehicle was administered SC (2 mL / kg) on ​​day 6 post-immunization. The test doses of Kv1.3 inhibitor were 10, 100, 300, or 700 nmol / kg. The test vehicle was 10 mM phosphate, 0.8% w / v NaCl, 0.05% w / v polysorbate 20, pH 6.

[0265] As a readout of efficacy, induced ear thickness was measured 24 and 48 hours after exposure to each ear and compared to the vehicle control. Ear thickness after 24 hours of exposure is shown in Table 13 and Figure 6, and ear thickness after 48 hours of exposure is shown in Table 14 and Figure 7. The results are summarized below.

[0266] For animals exposed on day 7 (i.e., day 1 post-treatment), CsA reduced ear swelling compared to vehicle controls when ear swelling was measured 24 hours post-exposure (i.e., day 2 post-treatment) (Peptide 100: 0.704 ± 0.018 mm, p = 0.1216; Vehicle: 0.721 ± 0.021 mm; CsA: 0.528 ± 0.032 mm, p < 0.0001) and when ear swelling was measured 48 hours post-exposure (i.e., day 3 post-treatment) (Peptide 100: 0.652 ± 0.026 mm, p = 0.0008; Vehicle: 0.694 ± 0.011 mm; CsA: 0.497 ± 0.021 mm, p < 0.0001).

[0267] Peptide 100 was administered to rat ears at doses of 10, 100, 300, or 700 nmol / kg on day 11 (i.e., day 5 after treatment), and ear swelling was measured 24 hours after exposure (i.e., day 6 after treatment) (10 nmol / kg Peptide 100: 0.807 ± 0.025 mm, p = 0.0495; 100 nmol / kg: 0.777 ± 0.019 mm, p = 0.0015; 300 nmol / kg: 0.772 ± 0.009 mm, p = 0.0005; 700 nmol / kg: 0.771 ± 0.018 mm, p = 0.0007; bilateral Ear swelling was significantly reduced compared to vehicle controls (10 nmol / kg peptide 100: 0.761 ± 0.025 mm, p = 0.0009; 100 nmol / kg: 0.713 ± 0.034 mm, p < 0.0001; 300 nmol / kg: 0.684 ± 0.016 mm, p < 0.0001; 700 nmol / kg: 0.671 ± 0.011 mm, p < 0.0001; vehicle: 0.819 ± 0.03 mm) and when ear swelling was measured 48 hours after exposure (i.e., 7 days after treatment) (10 nmol / kg peptide 100: 0.761 ± 0.025 mm, p = 0.0009; 100 nmol / kg: 0.713 ± 0.034 mm, p < 0.0001; 300 nmol / kg: 0.684 ± 0.016 mm, p < 0.0001; 700 nmol / kg: 0.671 ± 0.011 mm, p < 0.0001; vehicle: 0.819 ± 0.03 mm).

[0268] In conclusion, a dose of 300 nmol / kg of peptide 100 provides the maximal effect in the KLH-induced DTH model. [Table 17] [Table 18] [Example 11]

[0269] Determining the minimal effective dose of Kv1.3 inhibitor peptide treatment in a KLH-induced delayed-type hypersensitivity (DTH) model This study was performed as described in Example 8, except that rats were challenged intradermally with 40 μL of KLH / NaCl 0.9% (2 mg / mL) in the left ear only on days 7 or 11 post-immunization (i.e., days 1 and 5 post-treatment). A schematic diagram of the protocol is shown in Figure 5.

[0270] The dose of Kv1.3 inhibitor treatment capable of reducing the DTH ear swelling response was investigated by comparing responses in vehicle-treated rats (n=8 / group) with those in rats treated with different doses of peptide 100. Vehicle or peptide 100 dissolved in vehicle was administered SC (2 mL / kg) on ​​day 6. The test doses of Kv1.3 inhibitor were 1, 3, 10, 30, or 100 nmol / kg. The test vehicle was 10 mM phosphate, 0.8% w / v NaCl, 0.05% w / v polysorbate 20, pH 6.

[0271] As a readout of efficacy, induced ear thickness was measured 24 and 48 hours after exposure to each ear and compared to the vehicle control. Ear thickness after 24 hours of exposure is shown in Table 15 and Figure 8, and ear thickness after 48 hours of exposure is shown in Table 16 and Figure 9. The results are summarized below.

[0272] For animals exposed on day 7 (i.e., day 1 post-treatment), CsA significantly reduced ear swelling compared to vehicle controls when ear swelling was measured 24 hours post-exposure (i.e., day 2 post-treatment) (Peptide 100: 0.736 ± 0.026 mm, p<0.0001; Vehicle: 0.849 ± 0.032 mm; CsA: 0.601 ± 0.033 mm, p<0.0001) and when ear swelling was measured 48 hours post-exposure (i.e., day 3 post-treatment) (Peptide 100: 0.671 ± 0.022 mm, p<0.0001; Vehicle: 0.777 ± 0.036 mm; CsA: 0.551 ± 0.021 mm, p<0.0001).

[0273] Rats were exposed to Peptide 100 at doses of 1, 3, 10, 30, or 100 nmol / kg on day 11 (i.e., day 5 after treatment) and ear swelling was measured 24 hours after exposure (i.e., day 6 after treatment) (1 nmol / kg Peptide 100: 0.872 ± 0.037 mm, p = 0.0872; 3 nmol / kg: 0.814 ± 0.014 mm, p < 0.0001; 10 nmol / kg: 0.796 ± 0.043 mm, p < 0.0001; 30 nmol / kg: 0.754 ± 0.017 mm, p < 0.0001; 100 nmol / kg: 0.751 ± 0.009 mm, p < 0.0001; vehicle). Ear swelling was significantly reduced compared to vehicle controls (1 nmol / kg peptide 100: 0.854 ± 0.038 mm, p = 0.1953; 3 nmol / kg: 0.814 ± 0.019 mm, p = 0.0001; 10 nmol / kg: 0.794 ± 0.036 mm, p = 0.0001; 30 nmol / kg: 0.736 ± 0.03 mm, p < 0.0001; 100 nmol / kg: 0.731 ± 0.016 mm, p < 0.0001; vehicle: 0.876 ± 0.027 mm) and when ear swelling was measured 48 hours after exposure (i.e., 7 days after treatment) (1 nmol / kg peptide 100: 0.854 ± 0.038 mm, p = 0.1953; 3 nmol / kg: 0.814 ± 0.019 mm, p = 0.0001; 10 nmol / kg: 0.794 ± 0.036 mm, p = 0.0001; 30 nmol / kg: 0.736 ± 0.03 mm, p < 0.0001; 100 nmol / kg: 0.731 ± 0.016 mm, p < 0.0001; vehicle: 0.876 ± 0.027 mm).

[0274] In conclusion, a dose of 3 nmol / kg of peptide 100 is the minimal dose to obtain efficacy in the KLH-induced DTH model. [Table 19] [Table 20]

[0275] All publications mentioned in the above specification are herein incorporated 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 covered in the following aspects.

Claims

1. 1. A KvI.3 inhibitor, or a pharmaceutically acceptable salt thereof, for use in a method for treating or preventing a disease or disorder in a subject, wherein the KvI.3 inhibitor comprises or consists of a peptide comprising or consisting of the sequence QMDMRCSASVECKQKCLKAIGSIFGKCMNKKCKCYPR (SEQ ID NO: 1) or a variant thereof, wherein the variant (a) has at least 70% sequence identity to SEQ ID NO: 1 and / or (b) differs from SEQ ID NO: 1 by a total of up to 9 substitutions, insertions and / or deletions, and the method comprises the step of administering the KvI.3 inhibitor to the subject once every 2 to 8 days.

2. The Kv1.3 inhibitor has an IC50 for the human Kv1.3 potassium channel of 400 nM or less, preferably 300 nM or less, preferably 50 nM or less, preferably 15 nM or less, preferably 10 nM or less, preferably 5 nM or less, preferably 2 nM or less. ５０ 2. The Kv1.3 inhibitor or pharmaceutically acceptable salt thereof for use according to claim 1, wherein

3. 3. The KvI.3 inhibitor or pharmaceutically acceptable salt for use according to claim 1 or 2, wherein the variant has at least 70% sequence identity to SEQ ID NO:1, preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:

1.

4. 1. The peptide comprising the following amino acids: The amino acid at position 6 is C; Amino acid at position 12 is C; Amino acid at position 16 is C; Amino acid at position 27 is C; The amino acid at position 32 is C; and Amino acid at position 34 is C; and preferably: The amino acid at position 24 is F; The amino acid at position 25 is G; The amino acid at position 29 is N; and The amino acid at position 31 is K; The Kv1.3 inhibitor or a pharmaceutically acceptable salt thereof for use according to any one of claims 1 to 3, further comprising:

5. 5. The KvI.3 inhibitor or pharmaceutically acceptable salt for use according to any of claims 1 to 4, wherein the variant differs from SEQ ID NO: 1 by a total of up to 9 substitutions, insertions and / or deletions, preferably by a total of up to 8, 7, 6, 5, 4, 3 or 2 substitutions, insertions and / or deletions, or by a total of 1 substitution, insertion or deletion.

6. 6. The KvI.3 inhibitor or pharmaceutically acceptable salt for use according to any one of claims 1 to 5, wherein the substitution or deletion in the variant of SEQ ID NO: 1 is at an amino acid position selected from among positions 1 to 5, 7 to 11, 13 to 15, 17 to 23, 26, 28, 30, 33 and 35 to 37 of SEQ ID NO: 1, preferably at an amino acid position selected from among positions 1, 2, 3, 4, 5, 7, 14, 18, 19, 28 and 37 of SEQ ID NO:

1.

7. 1. The peptide comprising the following amino acids: the amino acid at position 1 is H, N, P, p, Q, S, V, or Y, or a deletion; the amino acid at position 2 is I, M, or Nle, or is deleted; the amino acid at position 3 is D, E, or S, or is deleted; the amino acid at position 4 is E, L, M, Nle, S, or V, or a deletion; the amino acid at position 5 is R or K or deleted; The amino acid at position 7 is E, F, H, K, Orn, R, S, Y, 2,3-diaminopropanoyl, 2,4-diaminobutanoyl, or 2-amino-3-guanidinopropionyl; The amino acid at position 8 is A, H, I, L, S, or Y; The amino acid at position 9 is F, L, P, S, Orn, V, Abu, or 2,3-diaminopropanoyl; The amino acid at position 10 is K, P, Q, R, or V; The amino acid at position 11 is E or Q; The amino acid at position 13 is A, E, G, K, L, Q, or V; The amino acid at position 14 is E, K, L, Q, V, or 2-amino-5-carboxypentanoyl; The amino acid at position 15 is K, L, P, or S; the amino acid at position 17 is K, L, R, or Y, or is deleted; the amino acid at position 18 is A, D, G, K, Q, hQ, V, or Y, or is deleted; the amino acid at position 19 is A, K, R, or Y, or is deleted; the amino acid at position 20 is E, I, R, or Y, or is deleted; The amino acid at position 21 is E, G, H, or R; The amino acid at position 22 is C, R, or S; The amino acid at position 23 is G, I, K, P, or R; The amino acid at position 26 is K or hK; The amino acid at position 28 is M or Nle; The amino acid at position 30 is G or K; The amino acid at position 33 is H, K, R, or V; The amino acid at position 35 is Y, F(4-F), or F(4-CH ３ ), F(4-NO ２ ) or F(4-NH ２ ); the amino acid at position 36 is Q or P or is deleted; and / or the amino acid at position 37 is C, G, R, S, or (4-amino-5-hydroxypentyl)guanidine, or a deletion; 7. A Kv1.3 inhibitor or a pharmaceutically acceptable salt thereof for use according to any one of claims 1 to 6, comprising:

8. The peptide has the following sequence: 【Table 1】 8. A Kv1.3 inhibitor or a pharmaceutically acceptable salt thereof for use according to any one of claims 1 to 7, comprising or consisting of one of:

9. The peptide is: 【Table 2】 9. The Kv1.3 inhibitor or a pharmaceutically acceptable salt thereof for use according to any one of claims 1 to 8, selected from:

10. The disease or disorder is (a) inflammatory diseases or disorders, preferably autoimmune diseases, allergies or hypersensitivity diseases, allograft rejection, transplant rejection, graft versus host disease, 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 (e.g., scleroderma, pulmonary fibrosis, cirrhosis of the liver), chronic obstructive pulmonary disease (COPD), hepatitis, chronic inflammatory demyelinating polyneuropathy, inflammatory bowel disease, colitis (e.g., Crohn's disease and ulcerative colitis), erythema, thyroiditis, psoriasis, atopic dermatitis, allergic contact dermatitis, scleroderma, glomerulonephritis, inflammatory bone resorption, multiple sclerosis, and type 1 diabetes; or (b) a metabolic disease or disorder; or (c) selected from obesity, obesity-related inflammation, obesity-related gallbladder disease, and obesity-induced sleep apnea; or (d) a disease or disorder caused by or associated with impaired glucose control, preferably selected from metabolic syndrome, insulin resistance, impaired glucose tolerance, prediabetes, elevated fasting blood glucose, and type 2 diabetes; or (e) a smooth muscle proliferative disorder, preferably restenosis; or (f) a neuroinflammatory or neurodegenerative disease or disorder, preferably a neuroinflammatory or neurodegenerative disease or disorder selected from Alzheimer's disease, multiple sclerosis (MS), Parkinson's disease, and amyotrophic lateral sclerosis (ALS); or (g) cancer, preferably breast cancer, prostate cancer or lymphoma, preferably said lymphoma is non-Hodgkin's lymphoma (NHL), preferably said NHL is selected from diffuse large B-cell lymphoma, follicular lymphoma, Burkitt's lymphoma, immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, mantle cell lymphoma, mycosis fungoides, anaplastic large cell lymphoma, peripheral T-cell lymphoma, precursor T-lymphoblastic lymphoma and Sézary syndrome; A Kv1.3 inhibitor or a pharmaceutically acceptable salt thereof for use according to any one of claims 1 to 9.

11. (a) inhibiting or reducing inflammation; or (b) A Kv1.3 inhibitor or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 9 for use in a method for inhibiting weight gain, promoting weight loss, reducing excess weight, or treating obesity, the method comprising administering a Kv1.3 inhibitor to a subject once every 2 to 8 days; The Kv1.3 inhibitor or a pharmaceutically acceptable salt thereof.

12. 12. The KvI.3 inhibitor or a pharmaceutically acceptable salt thereof for use according to any of claims 1 to 11, wherein the method comprises administering the KvI.3 inhibitor to a subject once every 2 to 7 days, once every 2 to 6 days, once every 2 to 5 days, once every 2 to 4 days, once every 2 to 3 days, once every 3 to 8 days, once every 3 to 7 days, once every 3 to 6 days, once every 3 to 5 days, once every 3 to 4 days, once every 4 to 8 days, once every 4 to 7 days, once every 4 to 6 days, once every 4 to 5 days, once every 5 to 8 days, once every 5 to 7 days, once every 5 to 6 days, once every 6 to 8 days, once every 6 to 7 days, or once every 7 to 8 days.

13. 13. The KvI.3 inhibitor or a pharmaceutically acceptable salt thereof for use according to any of claims 1 to 12, wherein the method comprises administering the KvI.3 inhibitor to a subject once every 2 days, once every 3 days, once every 4 days, once every 5 days, once every 6 days, once every 7 days, or once every 8 days, preferably once every 7 days.

14. 14. The Kv1.3 inhibitor or a pharmaceutically acceptable salt thereof for use according to any of claims 1 to 13, wherein the method comprises administering the Kv1.3 inhibitor to a subject by injection, preferably by subcutaneous injection.

15. 15. The Kv1.3 inhibitor or a pharmaceutically acceptable salt thereof for use according to any one of claims 1 to 14, wherein the method comprises administering the Kv1.3 inhibitor to a subject at a dose of 0.1 mg to 30.0 mg.

16. 16. The Kv1.3 inhibitor or a pharmaceutically acceptable salt thereof for use according to any one of claims 1 to 15, wherein the method comprises administering the Kv1.3 inhibitor to a subject at a dose of 10 nmol / kg to 400 nmol / kg, or at a dose of 3 nmol / kg to 300 nmol / kg.

17. 17. The Kv1.3 inhibitor or a pharmaceutically acceptable salt for use according to any one of claims 1 to 16, wherein the Kv1.3 inhibitor or a pharmaceutically acceptable salt is in the form of a composition, preferably said composition is a pharmaceutical composition, preferably said pharmaceutical composition comprises a pharmaceutically acceptable carrier, excipient or vehicle.

18. The composition comprises one or more peptides, each of which has the following sequence: 【Table 3】 18. The KvI.3 inhibitor or pharmaceutically acceptable salt thereof for use according to claim 17, comprising or consisting of a sequence independently selected from:

19. The composition comprises one or more peptides, said peptides having the following peptide sequence: 【Table 4】 19. The KvI.3 inhibitor or a pharmaceutically acceptable salt thereof for use according to claim 17 or 18, independently selected from:

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