AU2015202422A1 - Analogs of ShK toxin and their uses in selective inhibition of Kv1.3 potassium channels - Google Patents

Abstract

Analogs of ShK toxin and methods for using such ShK analogs. The ShK analogs generally comprise ShK toxin attached to a chemical entity (e~g. an atom, molecule, group, residue, 5 compound, moiety, etc.) that has an anionic charge. In some embodiments the chemical entity attached to the ShK toxin may comprise an amino acid residue. The ShK analogs may be administered to human or non-human animal subjects to cause inhibition of potassium channels or to otherwise treat diseases or disorders. In some embodiments, the chemical entity to which the ShK toxin is attached may be chosen to provide selective inhibition of 10 certain potassium channels (e,g., Kv1.3 channels) over other potassium channels (e.g., Kv1,1 channels). In some embodiments, the chemical entity to which the ShK toxin is attached may include a fluorophore, thereby providing a fluorophore tagged ShK analog. Such fluorophore tagged ShK analogs may be used in flow cytometry alone, or in conjunction with class || tetramers that can detect autoreactive cells,

Description

ANALOGS OF ShK TOXIN AND THEIR USES IN SELECTIVE INHIBITION OF Kv1.3 POTASSIUM CHANNELS RELATED APPLICATION 5 This is a divisional of Australian Patent Application No. 2012202994, the entire contents of which are incorporated herein by reference. This patent application claims priority to United States Provisional Patent Application 10 No. 601617,395 filed on October 7, 2004, the entirety of which is expressly incorporated herein by reference. FIELD OF THE INVENTION 15 The present invention provides a) novel compositions of matter, b) methods and kits for in vivo and/or in vitro inhibition of the Kvi.3 channel in T- and B-lymphocytes and other cell types and c) methods for treating autoimmune and other disorders in human or animal subjects. 20 BACKGROUND OF THE INVENTION Cell plasma membranes form the outer surfaces of eukaryotic cells. Various ions (e.g., sodium, potassium, calcium, etc,) move in and out of cells by passive diffusion through the cells' plasma membranes. Such diffusion of ions into and out of cells is facilitated by the 25 presence of "ion channels" within the cell membranes. Ion channels are proteins embedded within the cell membrane that control the selective flux of ions across the membrane, thereby allowing for the formation of concentration gradients between the intracellular contents of the cell and the surrounding extracellular fluid. Because ion concentrations are directly involved in the electrical activity of excitable cells (e.g., neurons), the functioning (or malfunctioning) of ion 30 channels can substantially control the electrical properties and behavior of such cells. Indeed, a variety of disorders, broadly termed "channelopathies," are believed to be linked to ion channel insufficiencies or dysfunctions.

WO 2006/042151 PNT2 3 ion channels are referred to as 'gated" if they can be opened or closed. The basic types of gated ion channels include a ligand gated channels: b) mechanical gated channels and c) voltage gated channels, In particular, voltage gated channels are found in neurons, muscle cells and non-excitable S cells such as lymphocytes. They open or close in response to changes in the charge across the plasma membrane, Kvi,3 Channels and Autoimmune Diseases. Autoimmune diseases such as multiple sclerosis (MS), type-i diabetes mellitus (TIDM), rheumatoid arthritis (RA) and psoriasis affect several 10 hundred million people worldwide. In these disorders specific autoreactive T cells - for instance myelin-specific T cells in MS patents -- are believed to undergo repeated autoantigen stimulation during the course of disease and differentiate into chronically activated memory cells that contribute to pathogenesis by migrating to inflamed tissues and secreting cytokines 15 (Viglietta et al., 2002; Vissers et at, 1002; Wulff et a), 2003b) Therapies that preferentially target chronically activated mahnory T cells would have significant value for autoimmune diseases, Memory T cells are divided into two subsets - central memory (Tc,) and effector memory (Th?) - based on the expression of the chemokine 20 receptor GCR7 and the phosphatase CD45RA (Geginat et al.. 2001; Sallusto et at, 1999). Naive and Tc cells home to the lymph node before they migrate to sites of inflammation, whereas The cells home directly to sites of inflammation where they secrete copious amounts of WlN- arid TNF-o and exhibit immediate effector function. It has recently been shown that myelin 25 specific autoreactive T cells in MS patients are predominantly activated T 5 , cells (Wuif et al, 2003b), and adoptive transfer of mylin-specific activated rat T cells into naive recipients induced severe EAE (Beeton et al., 2001a; Beeton et at, 2001b). An exciting new therapeutic target for immunomodulation of TE cells is the voltage-gated Kv13 K channel. T 5 & 30 cells up-regulate Kv13 channels upon activation and their antigendiiven proliferation is exquisitely sensitive to Kv1 .3 blockers (Wulff et al, 2003) Naive and

T

ce cells in contrast are significantly less sensitive to Kvi.3 2 WO 2006/042151 PCTiUS2005/036234 blockers to begin with and rapidly become resistant to Kvl.3 blockade by up regulating the calcium-activated K* channel lK~al (Ghanshani et al., 2000; Wulff et at 2OO3). The dominance of Kv1 3 in TpM cells provides a powerful way to 5 manipulate the activity of this subset with specific Kvl,3 inhibitors. The functionally restricted tissue distribution of the channel and the fact that in vivo Kv1 3 blockade ameliorates TFM-mediated EAE, bone resorption in peridontal disease and delayed type hypersensitivity reactions in animal models without causing obvious side effects has enhanced the attractiveness of Kvl 3 as a 10 therapeutic target (Seeton et al,, 200th; Koo et at, 1997; Valverde et ai, 2004). Although Kv13 blockers would suppress all activated TE cells (for example Tad calls specific for vaccine antigens), a Kvl .3-based therapy would be a significant improvement over current therapies that broadly and indiscriminately modulate the entire immune system. An additional advantage 15 of Kvi.3 blockers is that they are reversible. Thus, one could titrate the therapeuic effect of Kv1 3 blockers When needed and stop therapy in the face of infection, unlike chemotherapeutic agents, which take months to subside. Kv13 Channels and Obesity The Kvl.3 channel was found to play a role in energy homeostasis and 20 energy balance (Hum Ml Genet 2003 12551-) Miice with the Kv1 .3 channel genetically knocked out were able to eat fatty diets without gaining weight, while control mice given the same diet became over-weight, Pharmacological blockade of Kv1 .3 channels recapitulated the effect of genetic knockout of Kvi.3 channels Consequently, KvY.3 blockers are likely 25 to have use in the management of obesity, Kv4.3Channels and Type-2 Djabetes Mellitus Kvl3 channels play a role in regulating insulin-sensitivity In peripheral target organs such as the liver and muscle (Proc Neff Acad Sci U S A. 2004 101:3112-7), Genetic knockout of the Kvi.3 channel in mica enhanced the 30 sensitivity of the liver and muscle to insulin, Consequently, Kvi.3 blockers may have use in the treatment of type-2 diabetes mnelitus by enhancing WO 2006/042151 PCnSJ2005;036234 insulin's peripheral actions and thereby decreasing blood glucose levels. NaturalIV Occrring Polypeptides Known to Inhibit Kvl2 Channels The most potent Kvl .1 inhibitor is the peptide ShK from the Caribbean sea anemone St/chodacyla helianthus. ShK is a 35-residue polypeptide 5 cross-linked by 3 disulfide bridges. ShK blocks Kvl 3 (Kd 11 pM) and suppresses prolferation of TEA cells at picomolar concentrations, and ameliorates experimental autoimurnune encephaomyelitis (EAE) in rats induced by the adoptive transfer of myelin-specific Thu cells A potential drawback of ShK is its low picomolar affinity for the neuronal Kv.1 channel 10 (Kd 2$ pM\ Although no side effects were observed with ShK in EAE trials, ingress of high concentrations of ShK into the brain, as might happen when the blood-brain-barrier is compromised in MS could lead to unwanted neurotoxicity The development of highly specific Kv .3 inhitoOrs is therefore necessary. An extensive effort by the pharmaceutical industry and academic 15 groups has yielded several small molecules that inhibit Kv1,3 in the mid nanonolar range, but these compounds do not have the selectivity or potency to make then viable drug candidates, Several truncated peptidic analogs of ShK have previously been reported. In one of these ShK analogs the native sequence was truncated 20 and then stabilized by the, introduction of additional covalent links (a non native disuffide and two lactam bridges), in others, non-native structural scaffolds stabilized by disulfide andior lactam bridges were modified to include key amino acid residues from the native toxin. These ShK analogs exhibited varying degrees of kv1 3 inhibItory activity and specificity, Lanigan, 25 M.D. et al.; Designed Peptide Analogues of te potassium Channe/ Blocker ShK Toxin; Biochemistry, 25;40(51)1 5528-37 (December 2001) There remains a need in the art for the development of new analogs of ShK that selectively inhibit Kv .3 channels in lymphocytes wiminimalor no inhibitory effects on Kvi. channels or other potassium channels. 4 \WO( 2006'042151 PCUIJSO00S!036234 SUMMARY OF THE INVENTION The present invention provides novel compositions (referred to herein as "ShK analogs") comprising ShK toxin attached (e.g bound, inked by a 5 lSnker or otherwise associated with) to an organic or inorganic chemical entity (eig. an atom, molecule, group, residue, compound, moiety, etc.) that has an anionic charge. Further in accordance with the present invention, there are provided methods for inhibiting potassium channels and/or treating diseases or 10 disorders in human or animal subjects by administering to the subject an effective amount of an ShK anaog of the present invention, 4n some embodiments, the chemical entity to which the ShK toxin is attached may be chosen to provide selective inhibition of certain potassium channels (eg Kv3 channels) over other potassium channels (e.g., Kl channels). 15 Still further in accordance with the present invention, ShK analogs of the foregoing character may include a fluorophore tag and such fluorophore tagged ShK analogs of the present invention may be used in flow cytometry alone, or in conjunction with class 1 tetramers that can detect autoreactive cels, 20 Further aspects, elements and details of the present invention will be apparent to those of skill in the art upon reading the detailed description and examples set forth herebelow, BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shovs the chemical structures of a number of ShK analogs of 25 the present invention. Figure 2A shows a molecular model of ShK based on the published NR structure wherein the Lys', critica for channe blockade, highlighted in one shade of grey. L-pTyr was attached to the a-amino group of Arg' of ShK (highlighted in a second shade of grey) through: an Aeea linker (right). 30 The structures of the inker and Lopfyr were modeled with AW in WO 2006!042151 PCT/qIS2005/036234 Hyperchem. Figure 23 shows the effect of ShK (top) and ShK(S) (bottom) on Kvl.3 and Kvt1 currents in stably transfected cells, Figure 2C shows dose-dependent inhibition of Kvi.3 (open symbols) 5 and Kvi .1 (closed symbols) by ShK (dark) and ShK(L5) (light). Kds on Kv .3 10 f I pM (ShK) and 69 ± 5 pM (ShK(L5)); Kas on Kv1. = 28 6 pM (ShK) and & 0.0 nM (ShK(L), Figure 2D shows the time course of wash-in and wash-out of ShK(LO) on Kv.S wherein cefis were held at a holding potential of 80 mV and 10 depolarized for 200 msec to 40 mV every 30 secs. Figure 2E shows K values for inhibition of Kvi.3 and Kvtl! by ShK analogs. Kds for ShK-FGCA and ShK-Dap" based on published sources Figure 3A is a graph showing staining intensities of CD45RA and CCR7 as determined by flow cytometry in the CD3tgated population of 15 human PSMCs stained with antibodies against 013, C045RA and OCR7, Figure 3B is a graph showing staining intensities of 0D45RA and CCR7 as determined by flow cytometry in the CD3tgated population in cells of a human TEM line stained WMth antibodies against 00, CD45RA and CCR7. 20 Figure 30 is a graph showing the inhibitory effects of ShK (dark grey) and ShK(L5) (light grey) of (OHj thymidine incorporation by PBMCs (open symbols, a mixture of nave/TcN cells) and TaM cells (closed symbols stimulated for 48 hours with anti-03 antibody. Figure 3D is a graphic showing of pre-aotivated human PBMCs 25 (naive/Tef cells) that up-regulate KCa3. expression become resistant to ShK(LS) inhibition when reactivated with anti-CD3 antibody. These cells have previously been reported to become sensitive to the Kc3 specific inhibitor TRAM-34, WO 2006/042151 PCT/ S20010,1634 Figure 4A is a graph showing CD45RC staining of rat splenic T cells (left) and PAS T cells (right) detected by flow cytometry. Figure 4 is a graphic showing of Kvl,3 currents exhibited by quiescent (top) and nelin antigen-activated (bottom) PAS T cells, 5 Figure 4C provides a graphic representation of flow cytornetry profiles of ShK-F6CA-staining in quiescent (top) and myelin antigen-activated (bottom) PAS T cells Unstained cells (black lines) and cells stained with ShK-F6CA (area filled in light grey), Competition of ShK-F6CA staining by unlabeled ShK(L5) is represented by the area filled in dark grey 10 Figure 40 shows confocal manges of Kvl.3 immuncstaining in quiescent (top) and myelin antigen-activated (bottom) PAS T cells. Statistical analysis was carried out using the Mann-Whitney U-test Figure 4E shows dose-dependent inhibition by ShK (dark lines) and ShK(L6) (iight lines) of (3H) thymidine incorporation by rat (left) naive/Tea 15 (open symbols) and ThM (closed symbols) cells activated with Con A (1 pgia). Figure 4F shows dose-dependent inhibition by ShK< (dark lines) and ShK(LS) (light lines) of IL2 secretion by PAS T ceils 7 hours after stimulation with MBP, G, ShK(Lshinduced inhibition of myelin-antigen triggered eli 20 thymidine incorporation by PAS T cells (open symbols) is reversed by the ad-di t ion of 20 uImi lL2 (closed symbols), Figure SA is a graph showing KvI.3 blocking blocking activity of ShK(L5) as determined on Kvl 3 channels stably expressed in L929 cells Figure 58 is a graph showing blood levels of ShK(LG) at various times 25 after a single subcutaneous injection of 200 mg/kg of ShKl5) in four rats. Blood was drawn at the indicated times and serum was tested by patch-amp to determine the amount of ShK(L5), Figure 5C is a graph of the data of Figure 58 fitted to a single exponential decay indicating a half-life of approximately 50 minutes.

WO 2006/042151 PCT/US2005,336234 Figure SD is a graph showing blood levels of ShK(L5) in five Lewis rats receiving single daily subcutaneous Injections of 10 pg/kg/day ShK(LS) for 5 days Blood was drawn each morning (24 hours after the previous injection) and tested for blocking activity on Kv.3 channels by patch-clamp, 5 Figure 5E is a graph showing serum levels of ShK(LS) in rats at various times following a single dose of 10 mg/kg ShK(L5) either subcutaneously (open bars; n = 4) or intravenously (closed bars; n 4) Blood was drawn at the indicated times and serum was tested by patch-clamp to determine the amount of ShK(L5) in blood ShK(LS) maintained a steadystate level of 300 10 pM in the blood almost 24 house after a single subcutaneous injection. This concentration is sufficient to selctively inhibit the function of

T

M cells. Figure :F is a graph showing the % recovery of ShK(L5) after a half blocking dose of ShK(L5) was added to rat plasma or PBS containing 2% rat plasma and incubated at 37*C for varying duration, Aliquots were taken at the 15 indicated times and blocking activity determined on Kv1.3 channels. ShK(LS) is extremely stable in plasma Figure 6A is a graph showing scored prevention of EAE. PAS T cells were activated in vitro, washed, and injected intraperitoneally on day 0, Clinical scoring of EAE: 0= no clinical signs, 0.5 = distal limps tail, I limp tail, 20 2 = mild paraparesis or ataxia, 3 = moderate paraparesls, 4 = complete hind limb paralysis, 5 = 4 + Incontinence, 6 = death, Rats (n = 6/groupi) were injected subcutaneous with vehicle alone (n 6 6) or ShK(LS) (n = 6; 10mg/kg/day) from day 0 to day 5. Figure 68 is a graph showing scored treatment of EAE. PAS T cells 25 were activated in vitro, washed, and injected intrapertoneally on day 0. Treatment with ShK(L5) at 10mg/kg/day was started when rats developed clinical signs of EAE and was continued for 3 days. Figure 60 is a graph showing ear thicknes as an indicator of DTH reaction elicited against ovalbumin in rais. Animals (n 6/group) were heated 30 with ShK(L5) 10 mg/kg/day for 2 days, after which ear sweling was measured. Statistical analysis VWas carried out using the Mann-Whitney U-test VO 20i}/942151 PCTS200S'06234 Figure 7A shows the ShK(L5) structure and a graph showing innibition of Kv1:3 channels in Thm cells as a function of ShK(L5) concentration, Each data-point represents mean of three determinations. Figure 7B is a diagram of Kvt.3-containing signaling complex. 5 Figure 7C shows co-localization of OD4, Kvl, Kvl2, SAP97, ZIP and p561 at IS. Figure 70 shows C04 and Kv I.3 staying in absence of visible he APO contact. Figure 7E shows CD4 and Kvi3 staining in GAD65-specific T cerls tO exposed to MOP-loaded APCs, Figure 7F shows that ShK(LS) 100 nM does not prevent IS formation. Figure 70 shows that ShK(L5) 100 nM does not disrupt the 1S. Figure 8A is a graphic showing of calcium signaling in GAD-specific TM celis from three TI DM patients triggered by anti-CD3S cossiing 15 secondary antibodies (arrow) in the absence (black) or presence of ShK(LS) 0,1 nM (dark grey 1 nM (medium grey) or 100 nM (light grey Figure 8$ is a graph showing E 3 H--thymidine incorporation by naivecM and ThM cells (left) and naivei'cMreifectors and TrM-effectors from patients with TIDM and RA (right). Thu cells: GAD65-activated T-m clones from three 20 T1DM patients and anti-D3S antibody actvated SF-TM cells from three RA patients. Naive/Tc cells: anti-CD3 antibody-activated PB-naive/TOM cell from the same three RA patients. Figure 80 is a series of bar graphs showing Cytokine production by the Tst and naiveflcu cells used in Figure 811 25 Figure 8D shows the phenotype of disease-relevant and disease irrelevant autoreactive T cells in MS, Ti Di and RA, Figure SE is a diagram showing the manner in which SiiK(L5) inhibits calcium signaing, lymphocyte proliferation and cytolone production but not IS 9Q WO 2006s04211 PCT/U820510 formation. Figure 9 is a diagram representing a rat model of delayed type hypersensitivity (DTH) caused by effector memory T cells, Figure 10 is a diagram showing a treatment protocol for ShK(LS) in a 5 rat model of delayed type hypersensitivity (DTH) caused by effector memory T cells Figure 11 is a diagram represneting specific suppression of effector memory responses in vivo in rats by ShK(L5) without impairing the function of naive and central memory T cells or B cells. 10 Figure 12A shows KvI.3 currents (top) and channel number/cell (bottom) in GADS65-, insulin and myelin-specific T cells from patients with new onset type-I diabetes mellius (TIDM), health controls and patients with multiple sclerosis.. Figure 128 shows Kv1.3 staining (top) and fluorescence intensities of 15 individual T cells (bottom) from these patients. Figure 120 shows graphs of relative cell number vs, CCR7 staining intensity, Cells expressing high levels of Kv1 3 ae COR7-negative ie they are TES-effectors. Cells expressing low levels cf KvI,3 are CCR&-positive i.e. they are either naive or Toe cells 20 Figure 120 shows Kv1 3 number/cel in autoreactive T cells from a patient having TIDM and MS (left), patients having T1DIM for greater than 5 years duration (middle) and patients having non-autoimmune type-2 DM. Figure iE shows Kvi.3 numbers in CD4GAD65-tetramer* T cells from a patient with new-onset TIDI. 25 Figure 1A shows Kv11.3 channel numbers per cellin peripheral T cells blood and synovial fluid T ceilsof RA patients and synovial fid T ceilsof OA patients, Figure 130 shows confocal images of Kv .3 (iight grey) and Kvis2 (darker grey) staining in the cells shown in Figure 13A, WO 2006/042151 P(1/US200503623 4 Figure 13C shows graphs of relative cell number vs. CCR7 staining intensity. Figure 13D shows micrographs (top) and bar graphs of inflammatory index (bottom) of synovium from RA and OA patients stained witn anti-CD or 5 anti-kvt,3 antibodies and counter-stained with hematoxylinleosin (40X), DETAILED DESCRIPTION The following detailed description and the accompanying drawings are intended to describe some, but not necessarily all, examples or embodiments 10 of the invention only. This detailed description and the accompanyng drawings do not limit the scope of the invention in anyway, The present invention provides novel analogs of ShK, methods for making such compositions and methods for using such compositions to inhibt Kvl,3 channels (or other ion channels) in human or animal cells and for 15 treatment or prevention of diseases and disorders, such as T cell mediated autoimmune disorders. The compositions of the present invention comprise ShK toxin attached (e.g, bound linked by a linker or otherwise associated with) to an organic or inorganic, anionic-charged chemical entity (esg. an atom, moleile, group, residue, compound, moiety, etc.), In at least some 20 embodiments of the invention, the organic or inorganic, anionic-charged chemical entity may be selected to increase or optimize the affinity of the composition for inhibition of Kv1.3 channels over Kvi1 channels Examples of organic or inorganic, anionic-charged molecules or groups that may be linked or bound to ShK in accordance with the present invention include but 25 are not necessarily limited to: amino acids; polypeptides; amino acid residues; unnatural amino acid residues; 30 threonine; threonine derivatives phospho-threonirte; serine; Serine lerivati'ves; phspo-seriie. glutaice acid derivatives, gamnmacarboxy-glutanmc acid; aspartic acid: aspartic acid derivatives; 10C inorganic comnpounds Or groups; organic comrpounds or groups; sucinic anhydride; and phthalic anhydride. 5 n accordance with) the present Invention, some non-limsiting examples of ,om-positions of the present invention, wherein the anioniGc-harged chemical entity comprises an amino acid residue, are shown in Figures 1 and 2C and referred to herein by alphane;umeric designations. as shown in Table. 20 Tabio I DESIGNATION 1 AMINO AmI RESIDUE BOUND -TO ShKI AT' POSI171ON 2 ShK-Ll jAEEAc-L-Pmrpl0H 2 ,) SWi-DI AEEAc,0-PrnpiC?, ) ShK-L3 AEE~kc-L-2mp(EtU ISIRKD! AEEAiO-D- Pmrp(Et-,) ShK-L6 AEEAc-L-Plhe(p--N H, 2 ______------ -- - ----------.--- . I-- WO 200610C151 PCT/U S20053036234 With specific reference to Figure 1, tyrosine or phenyialanine or their charged non-natural derivatives were conjugated to ShK (top left) through a tinker attached on its N terminus (residue Arg1 shown in shaded grey ). The Lys22, required for change blockade, is shown in a darker shade of grey The 5 molecular model of ShK is based on the published NMR structure and the structures of the linker and the new residues were modeled. These embodiments of compositions of the present invention generally comprise the ShK toxin, which is a polypeptide, bound to (e.g., hemicay bound, inked or otherwise associated with) at least one anionic-charged amino acid residue, 10 In embodiments where the amnio acid residue has a chira! center, the D and/or L enantiomer of such amino acid residue may be used The anionic charged amino acid residue may be an unnatural residue and may be attached or linked to an N-terminus of the ShK polypeptide, in some embodiments, the anionic-charged amino acid residue may be linked to an N 15 terminus of ShK through a linker: such as an aminoethyloxyethyloxy-acetyI tinker, These analogs of ShK inhibit the Kv1 3 channel more speciicaly than ShK because they have reduced affinity for other potassium channels (e Kvli) The ShK may be isolated from natural sources as known in the art, or it may be synthesized, 20 Synthesis of ShK Toxin ShK Toxin may be synthesized by any suitable method In one such method, Fmoc-amino acids (Bacherm Feinchemikalien) including Arg(Pmc), Asp(OTBu), Cys(Trt), Girr(T), His(Trt), Lys(Boc), Ser(tBu) and Thr(tBu) are 25 obtained commercialy and assembled to form ShK Toxin. Stepwise assembly of the amino acids may be carried out on an Applied Biosystems 431A peptide synthesizer at the 0.25 mmol scale starting with Fmoc-Cys(Trt)-R. Residues 34 through 22 are single coupled, Thereafter, an aliquot (e.g, half) of the resin is removed to effect better mixing. The remainder of the peptide sequence is then 30 double coupled to the remaining resin aliquot. All couplings are mediated by dicyclohexylcarbodilmide In the presence of 2 eq of 1 -hydroxybenzotriazaOe. The 13 WO 2006/042151 PCTiLS200SS2S4 final two residues are also coupled via HBTU!DIEA chemistry, These residues are Aeea (Fmoc-aminoethyloxyelhyloxyaceti acid) and as the N-termind residue Fmoc-Tyr (P04) monobenzyl ester. Following final removal of the Fmoc-group, the peptide resin (2.42 g) is cleaved from the resin and 5 simultaneously deprotected using reagent K for 2 h at room temperature. Reagent K is known in the art and has been described in the literature. See, King, D.&, Fields, 0.G. and Fields, G.B. (1990) mt , Peptide Protein Res 36, 255-265. Following cleavage, the peptide is filtered to remove the spent resin beads and precipitated with ice cold diethyl ether. The peptide is then 10 collected on a fine filter funnel, washed with ice cold ether and finally extracted with 20% AcOH in H20. The peptide extract is subsequently diluted ito 2 liters of H20, the pH is adjusted to 8.0 with NH 4 OH and allowed to air oxidize at room temperature for 36 hours, Following oxidation of the disulfide bonds with a 2:1 ratic of reduced to oxidized glutathione, the peptide solution is acidified to pH 15 2.5 and pumped onto a Rainin Dynamax Ci column (5.0 x 30 cm). The sample is eluted with a linear gradient frorn 5-30% acetonitrile into H20 containing 016% TFA The resulting fractions are analyzed using two analytical RP-HPLC systems TFA and TEAP. Pure fractions are pooled and lyophiized. (See, Pennington, MW, Byrnes, MA.E., Zaydenberg, L: Khaytin, I., de Chastonay J. 20 Krafte, D., Hilt, R, Mahnir, V., Volberg, WA, GoraycA, W and Kem, WR, (1995) /n . Peptide Protein Res. 46, 354-358.) Aliematively, solid-phase peptide synthesis employing a Boc-3zi protecting group strategy may be utilized to assemble the primary structure as well as analogs of the peptide. The peptide could then be cleaved from the 25 solid-phase by anhydrous HF, yielding the linear peptide ready for folding as described above for the Fmoc synthesized pepide. (See: Stewart J.M and Young JD. (1984) Solid Phase Peptide Synthesis. 2 " Edition Pierce Chemical Company. Rockford, II.) 14 WO 2006/042151 PCTU52005/03Q?34 Alternatively, other synthetic methods to assemble the primary structure of ShK or analogs could include chemical ligation technology where the peptide is prepared as a series of designed fragments with C-terminal thioester peptides The the thicester peptide can react with another peptide containing an 5 N-terminal Cys residue to form a peptide containing a native pepide bond. By using this technology, one could effectively assemble the primary structure of ShK (See, (4) Wilken, J and Kent SRB-H, (1998) Chemical protein synthesis Current Op7. Biofoch 9, 412-426.) Alternatively, another synthetic method that may be employed to 0 assemble the primary structure of ShK would utilize a protected peptide fragment convergent approach as described in Albericio F,, Lloyd-Williams, P., and Giralt, E. (1997) Convergent peptide synthesis: in Methods in Enzymol. Ed G, Fields, Academic Press, New York, NY. pp 313-335. In this method, linear protected fragments are assembled as fully side chair protected 15 fragments, These fragments can then be coupled together in a convergent manner to assemble the primary sequence of ShK or one of its anaog Assembly of the fragments could also utilize a solid-phase resin to facilitate coupling and wash steps Alternatively, recombinant methods may be used wherein the cDNA 20 coding sequence for ShK could be generated for exPression in either a prokaryotic or eucaryotic expression system. Recombinant ShK analogs containing unnatural amino acids are also possible by utilizing preload tRNA molecules which utilize non-standard condones. The oCNA construct can be engineered to use one of these unused codons to add the phosphotyrosine 25 residue as wel as the Aeea residue. Folding of the recombinantly produced 0SK analog could then be accomplished in a similar method to that used for the synthetic peptides. (See, Pennington, M.W., Byrnes, M.Fr., Zaydenberg, I., Khaytin. I., de Chastonay. J,, Krafte, D, Hill, ,Mahnir, V, Volberg A, Gorczyca: W. and Kem, WIR (1995) Int J. Peptide Protein Res, 46 354 30 358,) 15 WO 206y042151 PCTILUS2005036234 Attaching Anionic Amino Acid Residues To ShK and Optional Modifications to ShK Anionic amino acid residues may be attached to the N terminus of natural or synthetic ShK Toxin by way of a liner, such as an aminoethyoxyethyioxy 5 acetyl linker, or bay any other suitable means. In this example, the nine (9) ShK analogs shown in Figure 1 are prepared, Initially, Fmoc-Aeea-OH is coupled to the N-terminus of synthetic ShK Toxin assembled as described above The resin is then divided into 9 aliquots. Either FmocTyr(PO 4 Bz) OH, Fmoc-d-Tyr(PO 4 zBfriOH, Fmoc-Tyr(PO 4 Me )-OH, Fmoc-Pmp-OH, Fmoc 10 d-Pmp-OH, Fmoc-Pmp(Et)-OH, Fmoc-Pmp(Et) 2 -OH, Fmoc-Tyr(tBu)-OH. or Fmoc-Amp(Boc)-OH is then coupled using DIG and HOST to one of the resin aliquots. The deblocked peptide resin is then cleaved and deprotecied with reagent K (King et al., 1990) containing 5% triisopropylsilane for 2 h at RT Met(O) is reduced by addition of solid NH 4 I to the cleavage cocktail at t-1 5 15 min. (Nicolas et at, 1995). For the peptide containing Tyr(PO 4 Me2/CH a cleavage cocktail containing I MA TMSBr in TFA containing thicanisole as a scavenger for 18 hr at 44 C was used (Tian et a,993) Incomplete removal of the methyl protecting groups Is common when using this method and 'o of the species (Tyr(PO 4 ) and Tyr(PO 4 Me)) are easily purified by RP-HPLC The 20 Tyr(PO 4 Me) containing analog is cleaved via standard Reagent K cleavage keeping both Me groups intact. In each case, the cleavage mixture is filtered and the crude peptide is precipitated into ice-cold diethyl ether. The precipitate is collected yielding approximately 75 rng of peptide from 200 rug of resin. The crude product is dissolved in 20 ml of 50% aqueous AcOH and 25 diluted into 0.75 I of H20. The pH of the solution is adjusted vith NH40H to 8.2, and it was allowed to fold overnight with the addition of glutathione (2rM:1mM) (reduced:oxidized), All analogs are purified using RP-HPLC as described previously (Pennington et al., 1995; Pennington et 1996a; Pennington et al., 1996b), Pure f-actions are pooled and lyophilized Each 30 sample is confirmed by RP-HPLC, AAA and MALDI-TOF MS and adjusted to account for peptide content prior to bloassay, In some embodiments of the invention, to improve the PK/PD properties of the ShK structure, residues which are sensitive to degradation is WO 2006/042151 PCTIgI25/03234 properties may be replaced or substituted. Thus, subsituian of the Met residue at position 21 may be carried out to impart a stabilizing effect to oxidation. Additionally, substitution of the C-temrinal acid function with an amide will impart stability to C-terminal corboxypeptidase enzymes These 5 two substitions to the primary structure of ShK combined with the anionic moiety at the N-torminus have been synthesized to generate the most stable and selective KvI,3 blocker. Nonhydrolyzable phosphate substitutions will also impart a stabizing effect versus acid and basic hydrolysis of the phosphate as well as stability against phosphatase enzymes. The 10 substitutions are summarized below. The acronyms used are defined as follows: Pmp=p-phosphonomethyl-Phenylatanine; PpapPhosphatity Pheny!alanine and Nle=Norieucine. Substitutions: 15 p-phospho~Tyr.Aeea.-ShK-Nle21-Cys35-amide p-Phosphono-mrethyl-Phenyalanine-AeeaShK'1e2 Cys35amide (Pmp) p-Phosphatityl-Phe-Aeea-ShK-Nie21-Cys35-amide (Ppa) 20 p-phospho-Tyr-Aeea-ShK-Nle21 -Cys35-acid p-Phosphono-methjl-Phenylalanine-Aeea-ShK-Nle2t Cys35acid (Pmp) p-PhosphatitylaPhe-Aeea-ShNle21-Cys35cid (Ppa) In addition to the nonhydrolyzable Pmp and Ppa, substitution of p 25 Phosphono(difiuoro-rnethyl)-Phenylalanine (Pfp) and p-Phosphono methylketo-Phenylalanine (Pkp) are also anionic substituionsrovidig the following: Pfp-Aeea-Shk-Nle21 Oys35 amide Pkp-Aeea.-S hK-Nle2I-Cys35 amide 30 Pfp-Aeea-Shk-Nle21 Cys35 acid PiKp-Aeea-ShK-le2 1-ys35 acid

V?

WO 2006/042151 PCT/U 8200036234 Structures of the N-terminal substitutions are set forth in Appendix B. Other strucIuMs that are within the scope of the present invention are published in Beeton, C. et al., Targeting Effector Memory T Ce/is with a Selective Peptide Inhibitor of Kv1.3 Chennels for Therapy of Auto nmune Diseases, Molecular 5 Pharmacology, Vol. 67. No4, 1369- (2005), the entirety of which is expressly incorporated herein by reference and a complete copy of which is appended hereto as Appendix C, Therapeutic Uses of ShK Analoos of the Present Invention The present invention provides methods for treating or preventing 10 cerain disorders or diseases, such as T cell mediated disorders (eng., autoimmune disorders, graft vs, host disease, prevention of rejection of organ transplants etc.), other inflammatory disorders, obesity and Type 2 diabetes, in human or animal subjects by administering to the subject a therapeutically effective (e.g., preventative or effective to reduce or eliminate symptoms or 15 disease progression) amount of a pharmaceutlcally acceptable preparation consisting or comprising an ShK analog of the present invention (e.g., including but not limited to those listed in Table 1 hereabove). Any suitable route of administration (e.g., oral. rectal, intravenous intramuscular subcutaneous, intradermal, intranasal, topcal, transmucosa. transdermal, by 20 drug delivery implant, etc.) may be used. When used to prevent or treat a I cell mediated disorder, the dosage(s) will be sufficient to inhibit Kv1.3 channels on T cell membranes in this regard, the ShK analogs of the present invention have the potential to be used to prevent or treat a wide variety of a T cell mediated autoimmune disorders. The following are some examples of 25 some T cell mediated autoimmune diseases that may be prevented or treated by the methods of the present invention, categorized with respect to the target organ that is principally affected by each such disease: Nervous System: Gastfrointestin! T react: Multiple sclerosis Crohn's Disease Myasthenia gravis Ulcerative colitis Autoimmune neuropathies Primary biliary cirrhosis such as Gullain-Barr6 Autoimmune hepatitis uveitis Bone resorption associated Autoimmune with periodontal disease S'thP r!a WO2006n042151 CT2USZSt/036234 Endocrine: Autoimmune hemolytic anemia Type I diabetes meiltus Pernicious anemia Addison's Disease Autoimmune Thrombocytopenia Grave's Disease Hashimoto's thyroidtis Vascular: Autoimmune cophoritis and Temporal arterits orchitis Anti-phospholipid syndrome Vasculltides such as Wegener's granulomatosis Behoet's disease Multiele gCmans and/or Musculoskeletat System: Rheumatoid arthritis (RA) Osteoarthritis (OA) 1-1 Systemic upus erythematosus Psoriasis Sceroderma Dermatitis herpetiformis Polymyositis.dermatomyosti Pemphigus vulgaris Spcndyioarthropathes such as V/itiiigo ankylosing spondylitis Sjogren's syndrome Irrespective of the particular organs) affected, T-iymphocytes are believed to contribute to the development of autoirmune diseases. The currently available therapies for these diseases are largely unsatisfactory and 5 typically involve the use of glucocorticoids (e~g, methylprednisolone, prednisone), non-steroidal anti-inflammatory agents, gold salts, methotrexate: antimalarials, and other immunosuppressants such as cyclosporin and FK 506. Also, another T cell mediated disorder that may be prevented or treated by the methods of the present invention is graft vs. host disease andior 0 rejection of transplanted organs. indeed, the outcomes of organ transplant procedures have progressively improved with the development of refinements in tissue typing, surgical techniques, and more effective inmunosuppressive treatments. However; rejection of transplanted organs remains a major problem. T-ymphocytes play a central role in the immune response and they 15 are responsible, in large measure, for the rejection of many transplanted organs. They are also responsible for the so-called graftersus host disease 19 WO 2006/042151 PCTiUS20050 364 in which transplanted bone arrow cells recognize and destroy MHC mismatched host tissues. Accordingly, drugs such as cyclosporin and FK50S that suppress T-cell immunity are used to preventtansplant rejection and graft-versusrhost disease, Unfortunately, these T cll inhibiting drugs are 5 toxic, with liver and renal toxicities limiting their use Thus, he methods of the present invention may provide les toxic alternatives for the treatment or preventIon of graft vs host disease or transplant rejection. Also, inhibitors of the voltage gated Kv1 3 potassium channel have been shown to be especially effective in suppressing effector memory T cells and, thus, the methods of 10 present invention may be particularly effective in preventing or treating diseases that are associated with effector memory T cells, such as; bone resorption and periodontal disease, psoriasis, rheumatoid arthritis, diabetes mellitus and multiple sclerosis, in addition to T cell mediated diseases, the Kv1.3 channel has been determined to regulate energy homeostasis, body 15 weight and peripheral insulin sensitivity, Thus, the methods of the present invention may be used to treat other diseases and disorders that involve abnormal homeostasis, body weight and peripheral insulin sensitivity by inhibiting Kvl.3 channels on cell membranes, such other diseases and disorders include but are not necessarily limited to bone resorpion in 20 periodontal disease, Type 2 diabetes, metabolic syndrome and obesity. Use of ShK Analogs of the Present Invention in Flow Cvytmetv Further in accordance with the present invention there are provided methods for diagnosing T cell mediated disorders or otherwise sorting or distinguishing between various cell types in vitro using fluorophore tagged 25 versions of ShK(LS) for use in flow cytometry alone, or in conjunction With class |l tetramers that can detect autoreactive cells. Flow Qytimeiy is a flexible method for characterizing cells in suspension wherein fluorescence activated cell sorting is used to select living cells on the basis of characteristics measured by flow cytometry, The types of cellular features and 30 functions that may be detected by flow cytometry include the expression of proteins outside and within cells, type of ONA content, viability and apoptosis, multiple drug resistance pump activity, enzyme activity, T-cell activation, -cell receptor specificity, cytcline expression, phagoncytosis and oxidative burst 20 WO 20461042151 PCT/US2005/036234 activity. ThUS, in this method of the present invenion, the amino acid residue attached to the ShK may incorporate a fluorophore tag for use in flow cytometry alone, or in conjunction with class II tetramers loaded with specific autoantigens that can detect autoreactive cells. Specific descriptions of the 5 methods by which such flow cytometry may be carried out are described in Beeton, C, et a[, A Novel Fluorescent Toxin to Detect and Investigate Kv13 Channel Up-Regulation in Chrcnicaly Activated T Lymhocytes J.Biol.Chem. Vol. 278, No. 1 !, 99289937 (March 2003). In general, a flow cytometer uses focused laser light to Illuminate cells as they pass the laser beam in a fluid 10 stream. Ought scattered by the cells and light emitted by fluorescent dyes attached to cells of interest are analyzed by several detectors and processed by a computer, Cells may be distinguished and selected on the basis of size and shape as well as by the presence of many different molecules inside and on the surface of the cells. 15 Examples of Potassium Channel ini bliing Effects and Therapeputic Utility of SMK Analogs of the Present Invention ShK blocks the neuronat Kvin I channel and the KvI.3 channel with roughly equivalent potency. Neurotoxicity is therefore a concern under 20 circumstances that compromise the blood-brain-barrier and allow the entry of sufficient amounts of ShK to block Kv1.1 channels. Our strategy to design a Kvl.3-specific inhibitor was guided by our finding that ShK-F6CA containing fluorescein-carboxylate {(FCA) attached through a 20 A-long Aeea linker to the N-terminus of ShK exhibited 80-fold selectivity for Kvl.3 over Kvi 25 (Beeton et al, 2003). Since FSCA can exist as a restricted carboxylate or also as a cyclized lactone, it was not clear whether ShK-FSQAs Kv1.3 specificty was clue to the negative charge of FSCA, the hydrophobicity created by this large bulky fluorescein nucleus, potential planar -p electronic stacking or a combination of all of these potential contributions, To distinguish between 30 these Possibilities and with the intention of developing a nocfuorescent Kv.3-selective inhibitor, we generated a series of 12 nove N-termnally substituted ShK analogs to probe some of These interactions. By attaching tyrosine, phenylalanine or their derivatives (varying in charge, size and hydrophoblcity) through an Aeea linker to the N-terminus of ShK, we could 21 WO 2006/0421$I PCT5US2005/036234 probe the effects of charge and hydrophobicity to gain insight into our selectivity enhancement seen with FOCA substitution, Selective KVWi.3 nhibitton over Kv1.1 Inhibition: In the example shown in Figures 2A-2D, L-phosphotyrosine (L-pTyr) a 5 negatively charged (net charge 2) post-translationally modified aromatic amino acid, was attached via the AEEA linker to ShK-Arg to generate the novel analog ShK(LS). The ShK toxin and ShK(L5) were testd on Kv1 3 and Kvl 1 channels stably expressed in L929 cells, Figure 21 shows the effects of ShK and ShK(LS) on Kv1.3 and Kv1.1 currents elicited by 200 ms 10 depolariing pulses from a holding potential of 80 mV to 40 mV, Both peptides reversibly blocked Kv1.3 and Kv1.1 in a dose-dependent manner with Hill coefficients of 1. Kjs were determined from the dose-response curves shown using Microcal Origin software. ShK blocked Kv, (Kd = 10 i 1 pM) and kvi. 1 (Kd= 28 ± 6 pM) with roughly equivalent potency as expected (Fig 1C 15 In contrast, ShK(LS) was 100fold selective for Kv1 3 (Kd 69 ±5 pM) over Kv1 (Kd = 74 0.8 nM) (Figs. 11, 10) The time course of Kv1.3 current block by ShK(L5) and its washout is shown in Figure 1D The time constant

(

T

o) of ShK(15) wash-in was 131 ± 21 sec (n =7) while the time constant (TOFF) for peptide vash-out was 150 ± 28 sec (n 4). The Kd (57 ± 7 pM) 20 calculated from the Kon (15 x 10 ± 0.5 x 106 Msec-) and KorF (0,0059 ± 0.0013 sec') values is consistent with the K, (69 ± 5 pM) determined with Microcai Origin software. Other ShK analogs were also tested on Kv1.3 and KvlA channels. ShK(D5) containing D-phosphotyrosine (D-pTyr) was .35-fold selective for 25 Kv1.3 over Kvf.l but was an order of magnitude less potent than ShK(LS), ShK(L6) containing L-pTyr-monomethyl showed modest (11-fold) KV3 specificity, while ShK analogs containing L-pTyr-dimeihy or L-Tyr were not selective for Kvl 3 over Kv1.1- Analogs that contained phenylalanine or its derivatives (varfng in bulk, p electron density and charge) were modestly 30 specific or not specific for KvI3 over Kv1.1 ShK(L5)'s 100-fold specificity for Kv1 3 over Kv11 is greater than that of ShK-F6CA (80-fokd ShK(D) (35 fold), ShK~Dap 2 2 (33-fold) or any other ShK analog tested 22 WO 2006/042151 PC/US20O5036234 Applcants also assessed ShK(L5)'s specificiy on a panel of 20 ion channels and these data are summarized in the following Table 2: * Channel K 1 otShK(LS}fM) K Er2 40,00&±&7,000 X~VI.3 (clotted! 6 ztS 0 t<,tL3 (native) 76 B lv1 41037000 &3000 KvtM 100,000 no effect 3tA M D3.0063,000 SVL7 105,000 go efect R*31100,000 at effect 43 100,000 no effect C-r13 10400 ne fet G1 100,000 no effect K , 100,000 no effect Ce 21i10,0060 effect 04,2. 100,100 a: effect 33l 500e l,00 N' 2 10 g,000neffect NyA4 100,00 o effect Swd4g-eetltoed T <dt M0000 uo e a chane) As may be appreciated from the data of Table 2 above $hK(LS) blocked the Kv1.3 channel in T cells with a KX (76 pM) equivalent to its K on the cloned channel (69 pM). It was 100-fold selective for Kvl.3 over KvA, 260-fold 15 selective over Kv1.6, 260-fold selective over Kv3,2, 680-fold selective over Kv.2 and >1000-told selective over all other channels tested. importantly, it was 1600-fold Kv1,3-selectlve over KM31, the calciurn-ativated K channel that regulates activation of human naive and T 00 cells (Wulff et al, 2003). Native ShK was less selective than ShK(LS). ShK was 2.8-fold selective for 20 Kvi.3 (K 4 10 * 1 pM) over KvI.1 (KS 23 ± 6 pM) 20-fold selective over Kv .6 (200 t 20 pM), 500-fold selective over Kv32 1( 5 000 ± I,000 pM), and >1000-tid selective-over Kvi 2 (10 &1 nM) and KCa3 1 (K, =28 t 3 nM). Margatoxin, a peptide from scorpion venom that has been touted as a specific Kv1.3 inhibitor (Koo et al,, 1997; Un et al., 1993; Middleton et al., 23 2003) was also not specific. It was 5-fold selective for Kv1.3 (10 ± 12 pM) over Kv1.2 (Kl =520 ± I pM), 9-fold selective over Kvi. (10 ± 1 nM) and > 1000-fold selective over Kvi.6 and Kv3,2 (K 0 d 100 M). Luteolin, a nutriceutical sold for autoimmune diseases (v lma>.cm) on the bass of It being a Kv1 .3 inhibitor (Lahey and Rajadhyaksha, 20041, blocked Kv11.3 3 WO 2006/042151 PCTiUS0 6234 weakly (.Kd 65 t 5 mM) and exhibited no selectivigt over Kv.1 (K77± 5mM), Kv12 (K =63 i 14 mM) or Kv.5 (K = 41 ± 3 mM). ShK(L-5s exquisite specificity for Kv1.3 together with its picomolar affinity for the channel makes it a potentially attracted immnosuppressant 5 Preferential Suppression of Human Tem cell Prolifferation With reference to Figures 3A-3D, in order to assess ShK(LSYs in vitro immunosuppressive activity, Applicants compared its ability to suppress anti 003 antibody-stimulated proliferation of human Thu cell lines versus human PBMCs that contain a mixture of naive and Tc cells. Flow cytometry 10 confirmed the cell surface phenotypes of the two populations studied, As seen in Figure 3A, the Tsu lines were -90% OCRTCD45RA, while as shown in Figure 3B the PBMCs contained 65 % CCR7*C[45RA* (naive) and 13% CCR7*CD45RK (

T

cw) cells, Figure 3C shows that ShK(L5) and ShK were 60-fold more effective in suppressing the proliferation of TEM cells (1C50 =-0 i5 pM) compared with PBMCs (Cj = 5 nM, p < 0,05) The lower sensitty of PBMCs might be explained by a rapid up-regulation of K~a3 channels in naive and Tcm cells upon stimulation as has been reported previously (Ghanshani et a, 2000: Wulff et al, 2003). In keeping with this interpretation, PBMCs activated for 48 hours to up-regulate KCa3.1 expression, then rested 20 for 12 hours, and re-activated Vith anti-OD3 antibody were completely resistant to ShK(L5) block, as shown in the upper row of Figure 3D. PBM~s that had been suppressed by ShK(L5) during the first round of stimuation exhiitied identical resistance to ShN(.5) when the ceA were washed, rested and re-challenged with anti-CD3 antibody. These results carroborate earlier 25 studies Indicating that naive and TcM cells escape Kv1.3 inhibitors by up regulating K~a31 channels. Thus, ShK(L5) preferentially and persistently suppresses the proliferation of Thu cells. Preferential Suppression of Rat T 5 m Cells Proliferation As a prearnble to evaluating ShK(LS)Js therapeulic effectiveness we 30 examined its ability to suppress proliferation of a memory T cell ine, PAS, that causes an MS-like disease in rats, As a control, Applicants used rat splenic T cells To confirm the differentiation status of the two cel population we 24 WO 206/04251 PCTaiS2005/03234 assessed the expression of CD45RC, a marker of naive T cells (Bunce and Bell, 1997), Rat splenic T cells were 76% CD46RC' (i e, mainly naive cells) whereas PAS cells were CD45RV suggesting that they are memory cells, as shown in Figure 4A. To determine whether PAS cells are in the 1 E- or rne 5 TOM-state we examined Kvl .3 expression before and 46 hous after activation Tac but not Toe cells are expected to significantly up-regulate Kv13 levels upon stimulation. With reference to Figure 43, patch-clamp experiments revealed a striking increase in Kv.3 current amplitude after MBP-stimulation of FAS cells consistent with their being Te celis, As an independent measure 10 of the number of Kvl.3 channels on PAS cells, we used ShK-FSCA, a fluorescently labeled ShK analog that has previously been reported to bind specifically to K13, The intensity of ShK-F6CA staining determined by flow cytometry reflects the number of Kv1.3 tetrarriers expressed on the cell surface, As seen in Figure 40, SI-hK-F6CA (10nM) staring Intensity increased 15 with MBP-activation of PAS celis and an excess of undabeled ShK(L5; (100 nM) competitively inhibited ShK-R6CA staining. As a final test, Applicants performed confocal microscopy on quiescent and MBP-stimulated PAS cells that had been fixed and stained with a Kv1.3-specific antibody. in keeping with data in Figures 48 and 4C, resting PAS T cells had a Kv1.3 staining 20 intensity of 4.4 ± 0.3 and this value increased to 10.5 ± 2.3 (p < 0.005) after antigen-Induced activation (See Figure 4D) showing augmentation in Kv13 protein expression following activation. Thus, MBP-activated PAS ceils are CD45RC- Kv1 3 h4" TaE cells whereas rat splenic T cels used in our experiments are predominantly in the naive state 2 MBP-triggered proliferation of PAS cells was suppressed ~4000-fld more effectively by ShK(L5) and ShK (IC0 = -80 pM) than mitogen-induced proliferation of rat splenic Tcells (See Figure 4E, 10cs '100 nM p < 0.05) These results corroborate the findings with human T ces described above As seen in Figure 4GShK(L5) inhibited MBP-induced 1L2 production by PAS 30 cells (Figure 4F): and exogenous U2 partially over-rode ShK(L) suppression of PAS cell proliferation (Figure 4G). Earlier studies reported similar findings with less specific K1,3 inhibitors on human, rat and mini-plo T cis In sumnmarv, ShK(L5) is a powerful and selective inhibitor of human and rat ThE 25 WO 2006/042151 P/S05343 cells, and may therefore have therapeutic use in autoimmune diseases by preferentially targeting T 5 o celis that contrtute to the pathogenesis of these disorders. Circulatin Half-Life and Stability S A patch-clamp bioassay was used to ascertain whether circulating levels of ShK(LS) following subcutaneous injection were sufficient to inhibit Tsu cells. The results of these experiments are shown in Figures 5A-5F, Serum samples from ShK(L5)-treated and control rats were tested for blocking activity on Kv1.3 channels. Control serum did not exhibit detectable 10 blocking activity indicating an absence of endogenous channel blockers To standardize the. assay, known amounts of ShK(L5) were added to rat serum and these samples were tested on Kv1.3 channels. The spiked serum samples blocked Kv1.3 currents in a dose-dependent fashion (K 77 ± 9 pM) that was indistinguishable from ShK(L5)'s effect in the absence of serum (Fig. 15 4A). Levels of SIhK(L5) in treated animals were determined by comparison with the standard curve, ShK(LS) was detectable in serum 5 minutes after a single subcutaneous injection of 200 mglkg. Peak levels (12 nM) were reached in 30 minutes and the level then fell to a baseline of about 300 pM over 420 m minutes The disappearance of ShK(L5) from the blood could be 20 fitted by a single exponential. The circulating half-life was estimated to be -50 min, Since the peak serum level after 200 mg/kg (12 nM) significantly exceeds the requirement for selective blockade of K1.3 channels and TEM cell function, ve tested lower doses. After a singlinjection of 10 mg/kg the 25 peak serum concentration of ShK/L5) reached ^ 500 pM within 30 mi (data not shown), a concentration sufficient to block >90% Kv1.a but not affect Kv .t Repeated daily administration of this dose (10mg/kg/day) resulted in steady-state levels of -300 pM (measured 24 hours after injection, Figure SD), which is sufficient to cause 60-70% suppression of TEM cells with little effect 30 on niaIve/Tou celis, The "sieady-state' level is expected given the estimated circulating half-life of -50 min and indicates that ShK(LS) "accumulates on repeated administration To determine whether the "depot" was in the skin or WO 2006/0421s5 PCT/UTS20S/036234 elsewhere in the body, we measured blood levels of ShK(t5) 10 hours after rats received single intravenous or subcutaneous injections of 10 mg/kg ShK(Lo). The peptide disappeared witn the same time course following administration by either route (Figure 5E) indicsting that the skin is riot 5 responsible for the steady-state level of 300 pM ShK(LS) reacted after a single 10mg/kg daily injection (Figure 50), and the depot(s) resides elsewhere. The successful achievement of a steady-state level of 300 pM ShK(LB) following daily single injections of I 0mg/kg/day suggests that the peptide may 10 be stable in vivo. To directly examine its stability we Incubated ShK(L5) in rat plasma or in PBS containing 2 % rat plasma at 37 0 C for varying durations and then measured Kvi 3 blocking activity, in both sets of spiked samples (plasma and PBS) we observed a 50% reduction in Kv.3-blocking activity in about 5 hours, presumably due to peptide binding to the plastic surface of the 15 tube. and the level then remained steady for the next 2-days (gure 5F). As an added test of stability we compared the Kvl,3- versus KvIl-blocking acivtes of sera from ShK(LS)-treated rats. if ShK(L5) is modified in vivo, either by dephosphorylation of pTyr or cleavage of the Aeea-pTyr side-chain, would yield ShK(L4) and ShK respectively, neither of which is selective for 20 Kv 3 over Kvi,1. Serum samples from ShK(L5)-treated animals exhibited the same selectivity for Kvl.3 over Kvi as ShK(LS), indicating that the peptide does not undergo the modifications stated above Taken together, these results indicate that ShK(LS) is remarkably stable in plasma and attains pharmacologically relevant serum concentrations after single daily 25 subcutaneous injections of 10 mg/kg. Nontoxiclv Applicants conducted several n vitro and in vvo tests to determine if ShK(LS) exhibits any toxicity. The results of these studies are summarized in Appendix A. Human and rat lymphoid cells incubated for 48 hours with a 30 concentration (100 nM) of ShK(L5) >1200 times greater than the Kv1.3 half blocking dose or the iC 6 for TEM suppression (70-80 pM), exhibited minimal cytotoxicity. The same high concentration of ShK(L5) was negative in the 27 NVO 2006/04251 PCT/IUS2005/036234 Ames test on tester strain TA97A suggesting that it is not a mutagen. Both in vfo tests failed to detect any significant toxicity Drug-induced blockade of Kvl 1I1 (HERG) channels has contributed to major cardiac toxicity and the withdrawal of several medications from the 5 market ShK(LC) has no effect on Kvi 1 channels at 100 nM ('1430-fold the K for Kv1.3 and Applicants' chosen therapeutic regimen (10 mg/kg/day. 300 pM steady-state circulating level) should therefore not cause cardiotoxicity, As a further test Applicants performed heart rate variability analysis in conscious rats administered vehicle (PBS + 2% rat serum) on day-I, followed 10 by 10 mg/kg/day ShK(L5) on day-2. ShK(LS) had no effect on head rate or the standard HRV (heart rate variability) parameters in both time and frequency domains (Task force of the European Society of Cardiology and the North American Society of Pacing Electrophysiology, 1996), Encouraged by the acute toxicity experiments, Applicants performed a 15 sub-chronic toxicity study in which rats were administered daily subcutaneous injections of 10mg/kg ShK(LS) or vehicle for 2 weeks (n = 6 in each group). ShK(LS)-treated animals gained weight to the same degree as rias receiving vehicle (Appendix A). Hematological and blood chemistry analysis showed no difference between ShK(LS- and vehicle-treated rats, and flow cytometric 20 analysis revealed no differences in the proportions of thymocyte or lymphocyte subsets (Appendix A). Collectively, these studies suggest that ShK(LS) is safe. To determine the therapeutic safety index, we administered a 60-fold higher dose (600 mg/kg/day) of ShK(LS) to healthy rats for 5 days and 25 observed no clinical signs of toxicity, and no toxicity was seen when healthy rats received a single injection of 1000 mg/kg ShK(LS). The'situation is less sanguine when the blood-brain-barrier is compromised as happens in EAE and MS. Rats with EAE that received ShK(LS) 10 mg/kg/day for 10 days showed no signs of toxicity. in contrast, forty percent of rats (5/12) 30 administered 600 ing/kg/day for five days died on the fifth day when they developed clinical signs of EAE (extrapolated L0 5 =750 mg/kg/day). Since the peak concentration of ShK(L5) in the serum (12 nM) after administration of 25 WO 2006/042151 PCT/US200536234 a single injection of 200 rng/kg is sufficient to block >50% of Kv.11 channels; toxicity observed in EAE rats administered 600 mg/kg/day ShK(LS) is likely due to the ingress into the brain of sufficient amounts of ShKL5) to block KvIA Thus, the effective therapeutic safety index of ShK(LS) is well in 5 excess of 100 in situations where the blood-brain barer is not compromised (as seen in autoimmune diseases that do NOT affect the central nervous system (CNS) whereas the therapeutic safety index is 75 when the blood brain barrier is breached. Prevention of DTH and Acute Adoptfive AE 10 With reference to Figures 6A-6C, ShK(LS) was evaluated for immunosuppressive activity in vivo in two animal models. Applicants tested its aity to prevent and treat acute EAE induced by the transfer of MBF activated PAS Tos cells into Lewis rats, as well as to suppress the 0TH reaction mediated by ThE cells, PAS cells were activated with MBP for 48 45 hours in vitro and then adoptively transferred (6-8 :< I 0'viable cells) into Lewis rats. For the prevention trial, rats then received subcutaneous injections of saline (controls) or ShK(L5) (10 pg/kg/day) for 5 days. in the first prevention trial control rats developed mild EAE (mean maximum clinical score 2.0 ± .2) with an average onset of 5,6 ± 0,6 days (not shown). ShK(L5) reduced 20 disease severity (mean maximum clinical score 0.7 ± 0.6, p < 0.05) In the second prevention trial, control rats developed more severe EAE (mean maximum clinical score 3.2 * 0.4) with a mean onset of 4.8 ± 0.4 days (Figure 6A) ShK(LS) significantly reduced disease severity (mean maximum clinical score 0.6 * 0.4 p < 0.007) but did not significantly delay disease onset (5.5 25 0. days: p =0.07. No signs of toxicity were noted in these studies. In the treatment trial (Figure 68) rats were injected with MBP..activated PAS cels, administered saline or 10 pg/kg/day ShK(LG) when they initially developed signs of EAE (limp tail, hunched posture and loss of 6% or more of their weight over 24 hours) and therapy was continued for three days, Oical 30 signs of EAE peaked on day 6 in the control group (score =39 ± 0.7) and on day 7in the treated group (score = 1,9 ± 0.9: p <0 05), 2- WO 2006/042151 PCIU S2005/036234 As an independent assessment of ShK(LS)'s immunosuppressive activity in vvo, Applicants also examined its effectiveness in inhibiting the DTH reaction that is mediated predominantly by skin-horning TM cells Lewis rats immunized with ovalbumin and adiuvant were challenged 7 days later 5 with ovalbumin in one ear and saline in the other ear. Rats then received injections of saline (controls) or ShK(L5) (10 pg/kg/day) and ear thickness was measured as an indication of DTH, All control rats developed ear swelling 24 and 48 hours after ovalbumin challenge while the DTH reaction wras substantially milder in ShK(LS)-treated animals (Figure 60 Thus, ShK(LS) 10 inhibits the TEe-mediated DTH response, and. prevents and ameliorates severe adoptive EAB induced by myelinactivated TrE cd KvI3 Clusters At The IS Durin Antigen Presentation But K* Efflux Through Kvt 3 Is Not Required For IS Formation Or Stability Referring to Figures 7A-7G, ShK(LS), a highly selective Kv1.3 inhibitor 15 (21, blocked Kv1 .3 currents in GAD65-speoific TEM cells with a KA of 72 ± 3 pMst We used ShK(LS) as a pharmacological probe to define those steps in Tem cell activation that require Kv1,3 function. Biochemical studies have shown that KvIt3 and Kvb2 belong to a signaling complex that includes SAP97 (Synapse-Associated-Protein-97), ZIP (PKC-zeta-nteracting-protein, 20 p5t7,ssociated-p52-protn AlTO) p56&c and 0D4 (Figure 7). The existence of this cornplex in human Ta cells is supported by Applicants' results showing co-capping of Kvl.3, Kvb2. SAP97, ZIP and p56ci with C04. Furthermore, FRET (fluorescence energy transfer) studies show Kv1 .3 in close proximity to CD3 in Kvl.3-transfected human T cells, and the channel 25 preferentially localizes at ite point of contact between Kvl3-transfected numan cytotoxic I cells and their targets. Since CD4 traffics to the IS, the zone of contact between T cells and antigen presenting cells (APO), it is possible that Kvl3 and other proteins in the signaling complex also localize at the iS during antigen-presentation, To test this idea, GADB5-specific Kvl 3 h 30 Tev clones from a TI DM patient were incubated with HLX+matched APOs that had been loaded with GAD65 5571 peptide and stained with DAP to aid visualization, After 20 min, ARCj-:t conjugates were immunostained for proteins in the signaling complex CD4 co-localized at the IS with KvI.3, 30U WO 2006/04215 PCT/US2005/034234 Kvb2, SAP97, ZIP and p5 kt In the absence of APC-Tsa contact, CD4 and Kvl .3 were distributed throughout the cell. Furthermore, CD4 and Kv1.3 failed to localize at points of contact when GAD65-specific T 5 & cells were exposed to APCs loaded with MBP (an irrelevant antigen), verifying that IS-clustering s 5 antigen-specific. Thus in GAD65-specific Tac cells, a KNl3-containing signaling complex traffics together with CD4 to the IS during antigen presentation, suggesting that Kvl.3 is an integral component of the machinery that transdOces signals in Tau cells. Based on these studies, ShK(LS) at a concentration that blocks approximately 99% of Ku .3 channels (100 nM) did 10 not prevent IS-clustering and did not disrupt the IS once formed, indicating that K' efflux through KvI.3 channels is unnecessary for IS formation or stability. Suppression of Hurnermy Cells With reference to Figures &A-BE, ShK(L5) inhibited calcium signaling in 15 Tsm cells, an early and essential step in T cell activation. GAD65-specific T 2 k clones from TI M patients were loaded with the calcium indicator dye Fluo3, pre-incubated in medium alone or with increasing concerations of Sh LS) and imaged by flow cytometry before and after the addition of an activating anti-CD3 antibody and a cross-linking secondary antibody. Peak calium rise 20 occurred in 242t35 seconds after Stirmulation and was blocked by ShlK(L5) with an lCi of -200 pM (Figure 8A). ShK(L5) was 10-fold more effective in suppressing 3 H-lthymidine-incorporation by autoreactive Tew cells from T1DM and RA patients compared with naive/Tcu cells from these patents (Figure 8B, left). In a second set of experiments (Figure B1, right), PA-SF and RA-PB 25 T cells were activated with anti-C antibody for 48 hours to generate "Ts effectors" and 'naive/Tc-effectors respectively. Cells were rested overnight in medium, re stimulated with anti-CD3 antibody in the presence or absence of ShK(L5) for a further 48 hous arid 3 H-ithymidine incorporation was measured. RA-SF-Thu-effectors retained their sensitivity to ShK(LS) 30 inhibition, whereas RA-P8-nalve/Tcweffectors were resistant to Kvi1.3 blockade (Figure 8B, right), most likely because they up-reguiate the calcium activated KCa3,1KCa1 channel, which substitutes for KvI.3 in promoting calcium entry. ShK(LS) profoundly suppressed the production ofinterleukin 2 311 WO 2006/042151 PCTIUS2005/036234 1L2) and interferon-g (IFN-g) by TEM celis from TIDM and RA patients, whereas IL2 and JFN-g production by naive/Te 5 cells from these patients was ess affected (Figure 80). The production of tuior necrosis factor-a and interieukin 4 by both TaA celis and naivefT 05 cells was less sensitive to 5 ShK(L5) (Figure 80). Verification of Rat Model of Delaved Tyeo Hypersgnsitivity(DTW Caused by Effector Memory T Cells. As shown in Figure 9, rats were immunized with ovalbumin (OVA) in adjuvant. They were challenged in one ear 7 days later with OVA and in the 10 other ear with saline. Ear swelling was measured 24 1 later as a sign of delayed type hypersensitivity (DTH). The FACS histograms shown in Figure 9 indicate that T cels in the ears challenged with OVA are CD45R0-negative memory Cells while T cells in the blood and spleen of the same rats are mostly naive T cells. 15 Treatment Protocol For ShkIL5) In A Rat Model Of Delaved Type H ypesenstivity (DTH) Caused By Effee tar Memory T Cells As shown in Figure 10, rats received ShK(L5) 10 pg/kg/day as a subcutaneous injection either from day 0 to day 7 (during the priming phase) to prevent the differentiation of naiv cells to effector memory TEM cells, or 20 during the effector phase after challenge to the ear with ovalbumin to prevent the function of the TEM cells. Shk(LE) Specificalty Suppresses Effector Memory Responses In Vivo In Rats Without Impairing The Function Of Naive And Central Memry T Cels Or B CellA 25 As shown in Figure 11, control rats developed ear swelling i.e. a positive DTH response. ShK(LS) was NOT effective in suppressing DTH when administered during the priming phase, indicating that It did not suppress the differentiation of naive and central memory T cells into effector memory cells, ShK(L5) suppressed DTH when administered during the effector phase K0 indicating that it either prevented the ability of effector memory T cells to reach the ear and/or suppressed the activation of effector memory T cells The first possibility was excluded because the number of T celsin the ears of WO 2006/042151 PCV/US2005/036234 ShK(L5-treated rats was the same as in the ears of rats given the vehicle. ShK(LO) suppressed effector mermory T cell activation in the ear because these i ceis were Kvit3-negative, while the rnemory T cells in the ears of vehicle-treated animals were Kvi1.3 positive, IgM and igG -cell responses in S these animals was also not affected. Kvl.3 Exvression in T Cells Specific For GAD6555-567,inslin9 23. And Myelin Antigens From Patients With TIDM Or MS And earthy Controls Figure 12A shows Kv .3 currents (top) and channel numbercell 10 (bottom) from antigen-specific T cells from patients with new onset type-i diabetes mellitus, health controls and patients with mulitple sclerosis. Each data-point represents the mean ± SEM from 20-50 cells from 2-4 T cell lines from a single donor measured 48 hours after the third antigen stimulation. Due to the low frequency of T cells specific for insulin and GAD65 in the blood 15 of TIDM patients and controls, we amplified these populations by generating short-term autoantigen-specific CD4" T cell lines using the split-well method. As controls, we generated T cell lines specific for the irrelevant autoantigen myein basic protein (MBP) that is implicated in MS but not TIDM, Following the third antigenic stimulation, K1 .3 currents were measured by whole-cell 20 patch-clarmp in activated cells with a membrane capacitance greater than 4 pF (cell diameter > i gm). Representative Kvi .3 currents and Kv1.3 channel numbers/I cell are shown in Figure 12A, The currents displayed biophysical and pharmaciological properties characteristic of Kv.3. T cells specific for insulin (2-23) or GAD65 (555-567) from patients wih new onset T1DM 25 displayed large Kv1.3 currents and expressed high numbers of Kv1.3 channelswhereas disease-irrelevant MBP-specific T cells from these patients were Kv1 .3" (p - 0001). For comparison we have plotted our published Kvl,3 data on MS patients in whom the opposite patten was observed, In MS patients, T cells specific for MBP or myelin oligodendrocyte glycoprotein 30 (peptide 35-55) or proteolipid protein (peptide 139-1:51) were Kvi3, while Insulin- and GAD65-specific T 6elis were Kv'L3tp 0,000) Autoreactive T cells isolated from healthy controls were Kv1 3" regardless of specificity. n one individual with both MS and T1DM, T cells specific for al three autoantigens were Kvit3 .s GAD65-specific and insulin-spefic T cells from 33 WO 2006/042151 PCOU820051036234 patients with longstanding T1DM were Kv1.3" reflecting the persistence of autoreactive TCm cells, whereas a Kvi, 3 1C" pattern was found in GAD65- arid insulin-specific T cells from patients with non-autoimmune type-2 DM. As seen in Figure 12B, Kv1.3 staining (top) and fluorescence intensities of 5 individual cells (bottom) Applicants confirmed the patch-clamp data by immunostaining for Kv13. Insulin- and GAD6-specific T cells from TDM patients and MBP-specific T cells from MS patients stained brightly whereas cells specific for irrelevant autoantigens stained dimly. Figure 12C shows CCR7 expression, Flow cytometry revealed that Kv1 3

",

9 " T cells were CCR7 10

T

Et cells, while Kv1,3 w cells were CCR naive or ToA cells. Figure 12D shows Kvi.3 number/cell in autoreactive 7 cells from a patient with both T1 M and MS, and from patients having TI DM or type-2 DM for greater than 5 years and 2 years, respectively, Figure 12E shows Kv1.3 numbers in CD4'GADS5-tetramer T cells from patient with new-onset T1DM. As a 15 further control, we used fluorescent MHC class fi tetramers containing the GAU65 5571 peptide, to isolate GAD65-specific CD4' T cells from a DR-040 positive patient with new onset TIDM. Tetramer-sorted GAD65-activated T cells displayed the same Kvl.3h9 pattern observed in GAD65-specific I cell lines from T1DM patients; in summary, disease-relevant, autoantigen 20 activated T cells in both T1DM and MS are Kv13'isCCRT Tse4effectors while disease-irrelevant autoreactive cells in these patients are Kvi,3"CCR7 naIvei cells. Kv1.3 expression in Rheumatoid Arthritis and Osteoarthritis in RA, disease-relevant T cells can be isolated from affected joints, 25 Applicants patch-clamped I cells from the synovial fluid (SF) of 7 RA patients 48 hours after stimulation with anti-CD3 antibody As seen in Figure IA, as controls Applicants analyzed SF-T cells from 7 patients with degenerative non-autoimmune osteoarthritis (OA) (which had been activated with the same protocol. RA-SF T cells were Kv1 3 sh whereas OA-SF T cells were KvI w3 p c 0001). Aplicants found the Kvif" pattern in anti-C3-activated f cells from the peripheral blood (PB) of RA patients (p a 0.0001) because autoreactiNe Kvi i cells are Infrequent in the blood immunostaining 34 WO 20061042151 PCT/UlSz2 J003624 for Kvl.3 and its associated Kvh2 subunit corroborated the patch-clamp data. Figure 133 shows confocal images of Kvl;3 (light grey as seen in the figure) and Kvp2 (darker grey as seen in the figure) staining. RA-SF T cells stained brightly for both Kv1t3 and Kvp2, while CA.SF and RA-PB T cells displayed 5 weak staining. Figure 130 lustrates CCR7 expression. Flow cytometry verified that Kv1,3 5 9 RA-SF T cells were CCRT T cells, while Kvi1".3 OA SF and RA-PB T cells were CCR7' naiveT 0

,

4 cells. Figure 130 (top) shows micrographs of synovium from RA and OA patients stained with anti-CD3 or anti-K1.3 antibodies and counter-stained with hematoxylin/eosin (4XL As a 10 further test, we immunostained paraffin-embedded synovial tissues (S) from 5 RA and 5 OA patients for 0D3, Kv1.3 and CCR7. We have previously shown that our staining method does not detect Kv13 in naiv/aTo 0 cells because of their low numbers of Kv13 channels In RA-ST, a preponderance of CD3KvI.3 GORT Tcells was seen, whereas CD3+ cells were sparse in 15 OA-synovium and these were mairly Kv.3CCR7* naive/TMcells Degree of infiltration by C3 Kvl.3* and CCRT t cells assessed by grading system in Figure S2A. CDtinlammatory-index RA O32±0 CA 102 pd01t Kvil3-infiammatory-index: RA = 2.8:0.3; CA = 060.3 (p<0, Thus, in tree different autoimmune disorders, our results are consistent with disease 20 associated autoreactive T cells being Kvi .3,*qCCRTiTeifectors It is to be appreciated that the invention has been described herein with reference to certain examples or embodiments of the invention but that various additions, deletions alterations and modifications may be made to those examples and embodimens Mthout departing from the Intended spirit 25 and scope of the invention. For example, any element or attribute of one embodiment or example may be incorporated into or used with another embodiment or example, unless to do so would render the embodiment or example unsuitable for its intended use, Also, where the steps of a method or procedure are listed or stated in a particular order, the order of those steps 30 nay be changed unless otherwise specified or unless such change in the order of the steps would render the invention unpatentable or unsuitable for its intended use, All reasonable additions, deletions, modifications and alterations are to be considered equivalents of the described examples and kaid-menw e\NR3P 4O-hCC\AA I7034 !.do\-6,A)3I20 1 embodiments and are to be included within the scope of the folowing claims. Throughout this specification, unless the context requires otherwise. the word "comprise" or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or method step or group of elements or integers or method steps but not the exclusion of any other element or integer or method steps or group of elements or integers or method steps. Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country. 36-

Claims (1)

114. A composition according to Claim I wherein the chemical entity is 2 AEEAc-L-Tyr I 156 A coroition according to daim 1 wherein the chemical entity is 2 AEEAc-L-Tyr(P0sH 2 ) $/0 206/042S p qCT d'200;/0362 1 1& A composition according to daim 1 wherein the chemica entt s 2 AE.Ac-L-Phe(p-N02H> 1 1. A composition according to claim I wherein the chemical entity is 2 AEEAC-L-Asp-ate 1 19. A composion according to claim 1 wherein the chemical entity is 2 AEEAcD-Aspartate, 1 20. A composition according to daim I wherein the chemical entity is 2 AEEAc-L-Glutamate, 21. A composition according to ian 1 wherein the chernical entity is 5 AEEAc-D-Glutamate, 22 A method for causing inhibition of Kv1 3 potassium channels n a human or animal subject, said metnod comprising thetep of: ,A) administering to the subject a composition tht comprises ShK toxin 10 atiadhed to an organic or inorganic chemical entity thathas an anionic charge, in a forn and amount that is effective to inhibit of Kvi 3 potassiumn changes. 21 A rnethod according to claim 22 wherein the metod is carried out to prevent or treat an autoimmune disorder. 1 24 A method according to claim 23 wherein te autoimmune disArder is 2 selected from the group consisting of: Multiple sclerosis Myasthenia graves Autoimmune neuropathies such as GuMain-Barre Autdirmune uns Crohnis Disease., 39 WO 2056/0421 I PCUS2005O/36234 Ulcerative clitis Prmary bMry crhossI Autoinimune hepatitis Autirmmune thrombocytopenia Type diabetes menlitus Addison's Disease Grave's Disease Hashimoto's thyroiditis Autoimnmune oophoriis and orchNs Behoets disease Rheumatoid arthrits bone resorpon associated with periodontal disease Systemic lupus erythematosus Scieroderma Polymyositis, dermatomyositis Pemphigus vulgaris Spondyloadthropathies such as ankylcsrng spondylitis Sjogien's syndrome 25 A method according to claim 22 wherein the method s carried owt to 2 prevent or treat graft vs. host disease. 26 A method according to cairn 22 wherein the method :s carried out to 5 treat or prevent rejection of a transplanted tissue or organ. 2T A method according to cisin 22 wherein the nethod is carried out to prevent or treat metabolic syndrome. 10 28 A method according to claim 22 wherein the method is carried out to treat or prevent Type 2 diabetes, 29 A method according to claim 22 wherein the method is earned out to reat or prevent obesity, 15 30. A method according to claim 22 wherein the methos carried out to 40 WO 2006/0421S1 PCUJS2005/036234 treat or prevent bone resorption associated with periodonta disease 31, A method according to claim 22 wherein the composition comprises $nK toxin attached to a chemical entity selected frornthe group cons;sung or: amino acids; poypeptides; amino acid residues; unnatural amino acid residues: threonire; threonine derivaves; phospho-tbrechine; serine; scine derivatives: phospho-serine: t5 glutamic acid; glutamic acid derivative; gammacarboxyyuamic acid aspartic acid; aspartic acid dervatives 20 inorganic compounds or groups: organs compounds or groups; succinic anhydride; and phthalic anhydrde. 25 22. A method according to claim 22 whnerein the chemical entity is AEEAW L-Pmp(OH2). 33. A method according to claim 22 wherein the chemical entiy is AEEAo 30 DnPmp(OH4 321 A method according to claim 22 whrein the chemical entity is AEEAc D-Pmp(OH. Et4 41 WO 20061042151 PCOYUS2005/036234 35. A method according to claim 22 wherein the chemical entity is AEFzAC LPmp(Et 2 ), 5 36. A method according to claim 22 wherein the chemica entit is AEEAc Pmp( i 37. A method according to dairn 22 wherein the chemical entity is AEEAc is 38. A method according to claim 22 wherein the chemical entity is AEEAc- L-Tyr(PO-AHr 39, A ernthod according to claim 22 wherein the chemical entity is AEEAc 15 L-Phe(p-H ). 40, A method according to claim 22 wvherein the chemical entity is AEEAc L-Phefp-00 2 H). 20 41. A method according to claim 22 wherein the chernical entity i AEEAc L-Aspartate, 42, A method according to claim 22 wherein the chemical entity is AEEAc D-Aspartate, 43. A method according to claim 22 wherein the chemical entity is AEEAc L-Gutamate, 44, A method according to claim 22 whereinhe chemical entity is AEEAc 30 0-Glutamate. 45 A method for performing fiw cytometry, said method comprising the steps of: (A) pividing a composition that comprises ShK attached to an /42 W< 204/02 15 .1 ?C T IS2 003O2csj organic or inorganic chemical entity that has an anionic charge and a fuorophore tag B ) combining the composition provided in Step A with cells and C) using a flow cytometer to count isolate, or distinguish cells thai have affinity for the composition provided in Step A 46, A method according to clalin 45 wherein Step C comprises using the flow ytiometerto count, isolate or distinguish T yrnphocytes 10 47. A composition according to claim 1 whereir the ShK is rnodified by substituton of the Met residue at position 211 46 A composition according to claim 47 whefeinthe sbstian at Met residue 21 deters oxidation. 15 49. A composition according to claim I wherein the 5iK s modified by substitution of the of the C-temrina acid function with an aide. 50. A composition according to claim 49 whereir the substitution of the of the C-termina acid function with an aide imparts stability to C-terminal 20 corboxypeptidase enzynes, 51. A method according to claim 22 or 45 wherein the SiK is modified by substittion of the Met residue at position 2I. 25 52. A method according to claim 51 wherein the substitution at Met residue 21 deters oxidation. 53. A method according to claim 22 or 45 wherein the ShK is modified by subsbtution of the of the C-tenrinal acid function with an amioe. 30 54. A rnethod accorciing to caim 53 whereinthe substation of the of the C-terminal acid function with an amide imparts sabt to CE-terminal corboxypeptidase enzymes, 4 3