US 20070092444Al (19) United States (12) Patent Application Publication (10) Pub. No.: US 2007/0092444 A1 Benos et al. (43) Pub. Date: Apr. 26, 2007

(54) INHIBITION OF INWARD SODIUM Related U.S. Application Data CURRENTS IN CANCER (60) Provisional application No. 60/502,034, ?led on Sep. (75) Inventors: Dale J. Benos, Birmingham, AL (US); 11, 2003. James K. Bubien, Pelham, AL (US); G. Yancey Gillespie, Birmingham, AL Publication Classi?cation (Us) (51) Int. Cl. Correspondence Address: A61K 51/00 (2006.01) BRADLEY ARANT ROSE & WHITE, LLP A61K 38/16 (2006.01) INTELLECTUAL PROPERTY A61K 38/1 7 (2007.01) DEPARTMENT-NWJ (52) U.S. Cl...... 424/1.69; 514/12; 514/8 1819 FIFTH AVENUE NORTH BIRMINGHAM, AL 35203-2104 (US) (57) ABSTRACT (73) Assignee: THE UAB RESEARCH FOUNDA Described is a constitutive inWard Na+ currents found in a variety of human cancers. The constitutive inWard Na+ TION, Birmingham, AL (US) current plays a role in increased cellular proliferation, cel (21) Appl. No.: 10/571,302 lular migration and volume regulation. The inWard current is mediated, at least in part, by AlSC-containing Na+ channels. (22) PCT Filed: Sep. 13, 2004 In addition, an inhibitor of the inWard current, the PcTXl peptide, is described. Also provided are methods for screen (86) PCT No.: PCT/US04/29970 ing compounds to inhibit the inWard Na+ current, methods for screening for tumors expressing the inWard Na+ current § 371(c)(1), and methods for treating tumors expressing the inWard Na+ (2), (4) Date: Aug. 17, 2006 current. Patent Application Publication Apr. 26, 2007 Sheet 1 0f 15 US 2007/0092444 A1

NORMAL GRADE I GRADE ll GRADE Ill GRADE IV GRADE IV freshly resectad freshly resected

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MW Difference Current W W 250pk

400 ms. Patent Application Publication Apr. 26, 2007 Sheet 11 0f 15 US 2007/0092444 A1

Breast Carcinoma MelanOma ZR-75-1 SKMEL-Z

Fig. 19A Ampllluda(pA)

Fig. 19B (pA)Amplitude

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Fig. 24A Fig. 24B The Effect ofFsalmoWxln (PXTX) 0" The Effect of Psalmotoxln (PcTX) on ‘ ‘ Transwell Mlgrellon of UB‘I-MG Cells 1 ‘ Transwell Mlgrallon of D54-MG Celts

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Fig. 24D * Fig- 24C The Effect of Psalmoloxln (PcTX) on The Effect of Pulmutoxm (PcTX) on _ Transwell Migration of primary human astrocytes Trangwell Mlgm?on of primary Gaul 1.:4 1.2: I 1.5 ,._ 1.1- A 1.4 1,3 to‘. 1.3 iE 0.9-me: i %E m E 0.1- 5 on 2 0.6: =1” 0'9 8 l — — ‘Scrambled Pe tide 8 °'7 ‘ ~5 " PCTX P 3 u -|-sc:amb|ed Peptide a . _ —.."' a 0-5 ‘ +Pc'rx 1: 0.3: D 0.4 I

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uB7-MG Scrambled Peptide NormalizedMeanCellVolume Patent Application Publication Apr. 26, 2007 Sheet 15 0f 15 US 2007/0092444 A1 Fig. 26 US 2007/0092444 A1 Apr. 26, 2007

INHIBITION OF INWARD SODIUM CURRENTS IN shoWs the Whole-cell patch clamp recordings in the presence CANCER of 100 uM amiloride; and FIG. 2C shoWs the amiloride sensitive difference current. FIELD OF THE DISCLOSURE [0007] FIGS. 3A and 3B shoW a summary of absolute [0001] The present disclosure relates generally to inward outWard (+40 mV; FIG. 3A) and inWard (—60 mV; FIG. 3B) constitutive Na+ currents and the Na+ channels mediating currents obtained from a variety of gliomas and normal cells such currents, and to the identi?cation, characterization and in the absence and presence of 100 uM amiloride, using treatment of tumors expressing said Na+ currents. Whole-cell patch clamp. [0008] FIGS. 4A and B shoW summary I-V curves of BACKGROUND freshly resected normal astrocytes (FIG. 4A) and GBM cells [0002] The ever-expanding Degenerin/ENaC (Deg/ENaC; (FIG. 4B). InWard currents (—60 mV) Were —7.5:1.2 pA ENaC=Epithelial Na Channel) superfamily contains over 60 (normal) and —43.8:14.5 pA (GBM). OutWard currents (+40 proteins having a similar topology. As shoWn in FIG. 1, each mV) averaged 42.2:2.4 pA and 47.21125 pA for normal family member has a short intracellularly located Ni and and GBMs, respectively. FIGS. 4C and D shoW summary C-ter'mini, tWo predicted transmembrane spanning domains amiloride-sensitive (difference) currents of freshly resected (M1 and M2), and a large extracellular loop (1,2). All family normal astrocytes (FIG. 4C) and GBM cells (FIG. 4D). members are cation selective and blocked by the diuretic [0009] FIGS. 5A-5C shoW representative Whole-cell patch amiloride (1-3). Recently, another branch of this superfam clamp recordings. FIG. 5A shoWs Whole-cell patch clamp ily, the human BNaC (Brain Na Channel, also knoWn as recordings from ZR-75-1 and SKMEL-2 cells; FIG. 5B ASIC, Acid Sensing ) family has been identi?ed shoWs the Whole-cell patch clamp recordings in the presence (4,5). The six members of this family so far identi?ed in of 100 uM amiloride; FIG. 5C shoWs the amiloride-sensitive mammals are primarily expressed in the brain and in sensory difference current. organs. [0010] FIGS. 6A and B shoW RT-PCR detection of ASIC1 [0003] Individual members of the ASIC family co-as and ASIC2 in normal tissues, GBM tissues and cell culture semble to form heteromeric channels With differing proper samples. FIGS. 6A and B are the results of tWo separate ties, and are postulated to be involved in a Wide variety of experiments With partial overlap of tissues and cell lines cellular responses ranging from nociception to mechanosen tested. Primers for ASIC1 spanned bp 1091-1537 and bp sation (6,7). To date, six members of the BNaC/ASIC 1109-1587+3' UTR for ASIC2. N-nor'mal control cells; subfamily of the Deg/ENaC family have been cloned in G-freshly excised GBM; P-primary (1St passage) GBM cells; mammals (5,39-42). Table 1 gives a summary of these astrocyte-primary (1St passage) culture of normal human channels and their pseudonyms. Each of these channels, astrocytes. except for ASIC2b, share the common characteristic of generating excitatory currents in response to acidic pH When [0011] FIGS. 7A-7C shoW representative Whole-cell patch studied in heterologous expression systems. ASIC2b, at least clamp recordings. FIG. 7A shoWs Whole-cell patch clamp in its homomeric form, does not appear to respond to loW recordings from U87-MG, SK-MG, and D54-MG glioma pH. Although the subunit composition of these brain sodium cells in the basal state; FIG. 7B shoWs the Whole-cell patch channels in native tissues is unknown, evidence for hetero clamp recordings in the presence of 100 uM amiloride; FIG. multimeric channel formation With distinctive functional 7C shoWs the amiloride-sensitive difference current. characteristics has been obtained (6,43,44). A role in chemi Amiloride (100 uW inhibited inWard currents in all three cell cal pain sensation, especially that associated With increased types, regardless of the absence or presence of ASIC2 acidi?cation, has been proposed for these channels in sen mRNA (FIG. 7D). sory neurons (45,46). [0012] FIGS. 8A-C shoW acid-activated ASIC currents in [0004] Like the degenerins and ENaCs, ASICs are gener Xenopus oocyles. ASIC 2 (FIG. 8A), ASIC1 (FIG. 8B) and ally thought to form mechanically gated ion channels and to the combination of ASIC2 and ASIC1 (FIG. 8C) Were be involved in cell volume regulation (32,33). ASICs may examined. InWard Na+ currents versus time Were measured also be involved in the small sodium in?ux that occurs in in voltage-clamped oocyles (—60 mV) in the absence and cells and thus contribute to the cell’s resting potential. presence of 400 uM amiloride folloWing activation by Alterations in membrane potential, either by activating or reduction of extracellular pH to 4.0 (solid bars). Each oocyle inhibiting these channels, may have deleterious effects on served as its oWn control. Each experiment Was repeated cell survival (34). Isolation of an inhibitor of these channels three times With similar results. may be useful as a therapeutic agent as Well as a diagnostic agent [0013] FIG. 9 shoWs analysis of the interaction betWeen ASIC1 and ASIC2 in proteoliposomes. In vitro transcription and translation of ASIC1 and ASIC2 Were performed using BREIF DESCRIPTION OF THE FIGURES either radioactive or non-radioactive methionine. Translated [0005] FIG. 1 shoWs the structure of the Deg/ENaC super proteins Were reconstituted into liposomes as per standard family of amiloride-sensitive Na+ channels procedures knoWn in the art. To test for co-precipitation, antibodies directed against non-labeled ASIC Were used, and [0006] FIGS. 2A-C shoW representative Whole-cell patch the presence of co-precipitated radioactively labeled ASIC clamp recordings. FIG. 2A shoWs the Whole-cell patch Was detected. clamp recordings from freshly isolated normal human astro cytes and GBM (WHO Grade IV), and primary cultures of [0014] FIGS. 10A-C shoW co-immuno-precipitation of different grades of glial tumors (astrocytomas); FIG. 2B ASIC1, ASIC 2 and y-hENaC from SK-MG cells. Whole US 2007/0092444 A1 Apr. 26, 2007

cell lysate from SK-MG cells Was immunoprecipitated using cells/ml Were added to the upper chamber), in the presence ASIC2 antibodies and probed on Western blots With anti or absence of benZamil, and migration Was alloWed to bodies against ASlCl (FIG. 10A) ASIC2 (FIG. 10B) or proceed for 3 hours. Migration Was determined according to y-hENaC (FIG. 10C). Control immunoprecipitations Were standard procedures (120). N-amidino-3,5-diamino-pyraZi performed using IgG and probed on Western blots as indi necarboxamide Was used as a control. This pyraZine ring cated above. compound is an inactive analog of amiloride. [0015] FIGS. 11A-C shoW co-localiZation of syntaxin 1A [0022] FIGS. 18A and B shoW the effect of PcTXl (10 and ASlCl in SK-MG cells. All of the panels represent nM) and randomly scrambled control peptide (10 nM) on epi?uorescent images. FIG. 11A: ASlCl Was stained using inWard Na+ currents in a freshly resected GBM (FIG. 18A, commercially available polyclonal anti-ASlCl antibodies upper panel), SK-MG cell (FIG. 18A, loWer panel), or (Chemicon). FIG. 11B: Syntaxin 1A Was stained using normal human astrocyte (FIG. 18B). As a control, a highly speci?c monoclonal antibodies (no cross reactivity scrambled 40-mer peptide having the same amino acids as betWeen syntaxin 1A and syntaxin 1B). FIG. 11C: Double PcTXl Was used. staining With anti-syntaxin 1A and anti-ASlCl antibodies. Overlap is observed, as indicated by yelloW. [0023] FIGS. 19A-19C shoW representative Whole-cell patch clamp recordings. FIG. 19A shoWs Whole-cell patch [0016] FIGS. 12A-C shoW Co-localiZation of syntaxin 1A clamp recordings from ZR-75-1 and SKMEL-2 cells in the and y-hENaC in SK-MG cells. All of the panels represent basal state; FIG. 19B shoWs the Whole-cell patch clamp epi?uorescent images. FIG. 12A: y-hENaC Was stained recordings in the presence of 100 uM PcTXl; FIG. 19C using a commercially available antibody (source). FIG. 12B: shoWs the PcTXl-sensitive di?ference current. syntaxin 1A Was stained using highly speci?c monoclonal antibodies (no cross reactivity betWeen syntaxin 1A and [0024] FIG. 20 shoWs the effect of PcTXl (1 nM) and syntaxin 1B). FIG. 12C: Double staining With anti-syntaxin randomly scrambled control peptide (1 nM) on acid-induced 1A and anti-y-hENaC antibodies. Overlap is observed, as ASIC currents in voltage-clamped Xenzopus Oocytes. Mem indicated by yelloW. brane potential Was held at —60 mV, and the pHO Was step decreased to 4.0 for 10 s, and then returned to 7.4 for 30 s [0017] FIGS. 13A and B shoW expression and secretion of before repeating the sequence. Oocytes Were superfused MT-SPl in several glioma cell lines. FIG. 13A shoWs the With PcTXl solution (solid bars). PcTXl only inhibited presence of MT-SPl in glioma cells lines SK-MG, SNB19, inWard currents mediated by ASlCla and not the inWard U87-MG and U251. MT-SPl Was not detected in normal currents mediated by ASlC2 or the combination of ASlCl astrocytes or in a Grade II astrocytoma. FIG. 13B shoWs and ASIC2. The control scrambled peptide Was without gelatin Zymography of proteases excreted from SK-MG effect. cells. From left to right, lane 1 served as a control; lane 2, indicates treatment With 10 mM EDTA; lane 3 indicates [0025] FIGS. 21 shoWs single channel recordings of the treatment With 10 mM Aprotinin; and lane 4 indicates ASlCl reconstituted into planar lipid bilayers in the absence treatment With 10 mM of Galardin (Sigma-Aldrich), matrix (upper panel) and in the presence (loWer panel) of the metalloproteinase inhibitor. PcTXl. An expanded time scale is shoWn beloW each trace. [0018] FIGS. 14A and B shoW the effect of syntaxin 1A on [0026] FIGS. 22A-B shoW the effect of PcTXl on kinetic ASIC1+ASIC2 (FIG. 14A) and ASlC1+ASlC2+y-hENaC properties of the ASlCl in planar lipid bilayers. The number (FIG. 14B) in planar lipid bilayers. The holding potential of events used for construction of the closed and open time Was +100 mV and records Were ?ltered at 200 HZ. Addition histograms shoWn Were: 811 and 812 (FIG. 22A, in the of syntaxin 1A Was to the cis chamber; addition of syntaxin absence of the PcTXl) and 989 and 988 (FIG. 22B, in the 1a to the trans side Was without effect. presence of 10 nM PcTXl). [0019] FIG. 15 shoWs the effect of syntaxin 1A on ASlCl, [0027] FIGS. 23A and B shoW single channel records of ASIC2, ASIC1+ASIC2 and ASlC1+ASlC2+y-hENaC fol ASIC-containing channel activity in cell attached (FIG. loWing expression in Oocytes. Currents (lp) Were normaliZed 23A) and outside-out patches (FIG. 23B) from U87-MG to the values measured at —60 mV in the absence of syntaxin cells. 1A. Currents Were evoked by a step decrease in pHO to 4.0. Co-expression of syntaxin 1A With ASlC1+ASlC2+y [0028] FIGS. 24A-D shoW the effect of PcTXl or ran domly scrambled control peptide on cell migration in U87 hENaC resulted in signi?cantly (P<0.01) loWer mean cur MG cells (FIG. 24A), D54-MG cells (FIG. 24B), primary rents. GBM cultures (FIG. 24C) and primary human astrocytes [0020] FIGS. 16A-C shoW concentration dependent inhi (FIG. 24D) cells. bition of cell proliferation of SK-MG (FIG. 16A), U373 (FIG. 16B), and U251 (FIG. 16C) glioma cells by amiloride, [0029] FIGS. 25 shoWs the time course of regulatory phenamil, and/or benZamil. Cells Were plated in 96-Well volume increase (RVI) in U87-MG cells folloWing osmotic plates at 1000, 4000, and 2000 cells/Well for SK-MG, U373, shrinkage With no peptide added (control) or in the presence and U251 cells, respectively. Drug Was added at speci?ed of 80 nM PcTXl or randomly scrambled control peptide Was concentration on day 3 after plating (at the beginning of log added. U87-MG cells Were mechanically dispersed, Washed, phase of groWth). and resuspended in PBS. At t=2-3 min, the osmolality of the bathing medium Was increased to 450 mOsM/kg by the [0021] FIG. 17 shoWs inhibition of TransWell migration of addition of NaCl from a 3M stock solution. The time course D54MG cells by benZamil. 5-8 um polycarbonate TransWell of volume recovery Was continuously folloWed by Coulter ?lters Were coated on the loWer surface With or vitronectin counter analysis in the absence (control) or presence of 80 (10 mg/ml in PBS). 100 ml of D54MG cells (400,000 nM PcTXl or scrambled PcTXl peptide. US 2007/0092444 A1 Apr. 26, 2007

[0030] FIG. 26 shows the effect of PcTXl on cell growth. formation can occur in all glial cell types, thereby producing a large range of pathological and morphological variants. [0031] FIGS. 27A-C show the effect of PcTXl on the Most primary brain tumors derived from glial cells that have growth of U25 1 -MG brain tumors is SCID mice. SCID mice lost growth control regulation, giving rise to astrocytomas, were implanted with U251-MG cells and treated with either glioblastomas, or oligodendrocytomas. High-grade gliomas saline (27A, upper panels), scrambled peptide (27B, middle account for 30% of primary brain tumors in adults, and are panels), or PcTXl (27C, lower panel). After sacri?ce, brain the second most common cause of cancer death in children tissue was removed, embedded with para?in and sectioned under 15 years of age (8,9). High-grade gliomas are divided (10 pm thick). Sections were stained using hemotoxylin and by grade into two categories: anaplastic astrocytomas (WHO eosin. Magni?cations are 1x, 4x and 20x as indicated. Grade III) and glioblastoma multiforme (GBM; WHO Grade IV) (10). There are also two other histopathologically clas DETAILED DESCRIPTION si?ed grades of brain tumors, namely, Grades I and II. [0032] It has been observed that ion channels may be Increasing grades represent increasing malignancy and intimately involved in the cellular pathophysiology of can decreasing di?‘erentiation, which is associated with cer. Several different laboratories have demonstrated that the increased mitotic activity and enhanced cell migration (11, expression of certain oncogenes directly affect sodium (13 12). Thus, glioma cells exhibit a remarkable degree of 15), potassium (16-19), and calcium (13,20,21) channel heterogeneity that includes not only histological and karyo function. For example, the ras oncogenes, known to be typic features, but changes in cell motility and selective involved in metastasis (22), in?uence nerve growth factor alterations and cellular oncogenes and tumor suppressor induced neuronal differentiation and voltage sensitive genes. expression and calcium currents (21 ,23,24). Moreover, cell adhesion (25), motility (26,27), interaction [0036] In spite of this high degree of heterogeneity of with extracellular matrix (28), and proliferation (13,19,29 gliomas, in all cells isolated from biopsy material obtained 31) are all intimately linked to ion channel activity. There from patients who were diagnosed with high-grade gliomas, fore, inhibition of ion channel activity serves as a point for the presence of a novel, constitutive, amiloride-sensitive, pharmacological inhibition of the cellular pathophysiology inward Na+ conductance was observed. This constitutive, amiloride-sensitive, inward Na+ conductance was not of cancers. present in normal glial cells or in WHO Grade I and II stage [0033] The present disclosure is directed to the description tumors. The presence of this amiloride-sensitive, inward of a constitutive amiloride-sensitive inward Na+ current that Na+ conductance persisted in primary cultures of cells is associated with various tumor types and carcinogenesis in derived from high-grade gliomas, as well as continuous cell a variety of mammalian cell types. The ion channel medi lines that were originally derived from GBMs. Molecular ating the inward Na+ current is also described. In one biological, immunocytochemical, and pharmacological data embodiment, the ion channel mediating the inward Na+ suggest that the ion channels mediating the inward Na+ current comprises an ASIC component, such as an ASIC1 current may be comprised of subunits of the Deg/ENaC component. In an alternate embodiment, the ion channel superfamily of ion channels, such as ASIC and ENaC mediating the inward Na+ current may lack a functional subunits, as wells as other subunits. This suggests that the ASIC2 component. The constitutive inward Na+ current is constitutive amiloride-sensitive, inward whole-cell Na+ cur associated with tumor cell invasion, tumor cell volume rents may be a selective property of high-grade glial-derived recovery after cell shrinkage and tumor cell proliferation. tumors and other tumor types, such as breast tumors and Therefore, inhibition of this constitutive inward Na+ current melanomas. serves as a point for pharmacological intervention in the treatment of carcinogenesis. [0037] As described in the present disclosure, all high grade glioma cells, derived either from freshly resected [0034] Described herein are methods of treating tumors tumors or from established cell lines, express a constitu characterized by the expression of a constitutive inward Na+ tively active, amiloride-sensitive inward Na+ current. This current mediated by a Na+ channel containing an ASIC inward Na+ current is important in the proliferation and component, such as an ASIC1 component. Methods for the invasiveness of tumor cells. In contrast, this constitutively diagnosis/identi?cation of tumors characterized by the active, amiloride-sensitive inward Na+ conductance can not expression of a constitutive inward Na+ current are be detected in astrocytes obtained from normal brain tissue described. Methods for visualization of such tumors are also or from glioma cells derived from low-grade or benign provided. In addition, methods for screening and identi?ca tumors. Constitutive, amiloride-sensitive inward Na+ cur tion of novel therapeutic agents useful in the treatment of rents have also been detected by Applicants in breast cancer disease states expressing a constitutive inward Na+ current and melanoma cells. are described. The present disclosure describes in detail the application of these teachings to glial-derived tumors, such Methods of Treatment as gliomas. However, the teachings of the present disclosure [0038] The present disclosure provides for methods of are applicable to any tumor characterized by the expression treating tumors characterized by the expression of a consti of a constitutive inward Na+ current mediated by a Na+ tutive inward Na+ current mediated by a Na+ channel channel having an ASIC component Such tumors include, containing an ASIC component, such as an ASIC1 compo but are not limited to, glioma, breast cancer and melanoma. nent The tumor may be derived from glial cells, epithelial [0035] Glial-derived tumors comprise a diverse group of cells, melanocytes or other cell types. The tumors derived neoplasms that dilfer in their morphology, their CNS loca from glial cells may be gliomas, such as, but not limited to, tion, their degree of invasiveness, their tendency for pro astrocytomas, glioblastomas and medulloblastomas. The gression, and their growth characteristics. Neoplastic trans tumors derived from epithelial cells may be breast carcino US 2007/0092444 A1 Apr. 26, 2007

mas. The tumors derived from melanocytes may be mela may be an antibody, such as a monoclonal antibody. The nomas. Given the teachings of the present disclosure, one of second compound may be fused to a cytotoxic agent. The ordinary skill in the art could identify other tumor types cytotoxic agent may be any agent that is capable of killing expressing such a constitutive inWard Na+ current. or inhibiting the groWth of said tumors, such as, but not [0039] In one embodiment, the method of treating limited to, a radiolabel, gelonin, , saponin, pseudomo involves administering to a subject in need of such treatment nas , pokeWeed antiviral protein, diphtheria a therapeutically effective amount of a pharmaceutical com and complement proteins. The radiolabel may be any radio position containing a compound that inhibits the activity of label, such as, but not limited to, 131I and 125I. In a speci?c the Na+ channel mediating a constitutive inWard Na+ cur example, the compound may be PcTXl and the tag protein rent. Such a compound may be identi?ed as described beloW may be glutathione-S-transferase; the second compound in this speci?cation. Alternatively, such a compound may be may be a monoclonal antibody recognizing said glutathione PcTXl, or a variant of PcTXl. The inhibition of the Na+ S-transferase that is fused to a cytotoxic agent. channel mediating a constitutive inWard Na+ current by the compound may be a direct inhibition or indirect inhibition. [0042] Psalmotoxin l (PcTXl) is a peptide isolated from Direct inhibition may occur by blocking the activity of a the venom of the South American tarantula Psalmopoeus component of the Na+ channel mediating the constitutive cambridgei. PcTXl is a 40 amino acid peptide possessing 6 inWard Na+ current. In one embodiment, the inhibition may cysteine residues linked by three disul?de bridges. The occur by blocking the activity of the ASIC component, such amino acid sequence of PcTXl is shoWn in SEQ ID NO: 1. as an ASICl component Indirect inhibition may occur by PcTXl has a limited homology With other blocking an activity required for the activity of the Na+ knoWn in the art HoWever, PcTXl does share a conserved channel mediating the constitutive inWard Na". In one cysteine distribution found in both spider and cone snail embodiment, such activity may be a protein required for the peptide toxins (64). As used in the present disclosure, activation of the Na+ channel mediating the constitutive PcTXl is de?ned as the peptide the amino acid composition inWard Na+ current or that is involved in the doWn-regula ofWhich is shoWn in SEQ ID NO: 1 or SEQ ID NO. 2. The tion of such Na+ channel mediating the constitutive inWard present disclosure is also directed to variants of PcTXl that Na+ current, such as a protease or a PKC family members. retain the activity of the peptide disclosed in SEQ ID NO: 1 A “therapeutically effective amount”, in reference to the or SEQ ID NO. 2. Generally, differences are limited so that treatment of a tumor or other disease or condition, refers to the sequences of the reference polypeptide and the variant an amount of a compound that is capable of having any are closely similar overall and, in many regions, identical. A detectable, positive effect on any symptom, aspect, or char variant and reference polypeptide may differ in amino acid acteristics of the tumor or other disease or condition. sequence by one or more substitutions, additions, deletions [0040] In an alternate embodiment, the method of treating in any combination. A variant may be a naturally occurring involves administering to a subject in need of such treatment or it may be a variant that is not knoWn to occur naturally. a therapeutically effective amount of a pharmaceutical com Non-naturally occurring variants of may be made by position containing a compound that binds to the Na+ mutagenesis techniques or by direct synthesis. Avariant may channel mediating the constitutive inWard Na+ current Such also include conservative amino acid substitutions. PcTXl a compound may be identi?ed as described beloW in this also includes fragments of the polypeptide shoWn in SEQ ID speci?cation. Alternatively, such a compound may be NO: 1 or SEQ ID NO. 2, Where said fragments are at least PcTXl, or a variant of PcTXl. Such compound may be ?ve amino acids in length. In one embodiment, the fragment linked to a cytotoxic agent The cytotoxic agent may be any of PcTXl contains all six cysteine residues. PcTXl or a agent that is capable of killing or inhibiting the groWth of variant of PcTXl may be puri?ed from natural sources, may said tumors, such as, but not limited to, a radiolabel, gelonin, be produced synthetically, or may be produced as a recom ricin, saponin, , pokeWeed antiviral binant protein from genetically engineered cells. In one protein, and complement proteins. The embodiment, PcTXl or a variant of PcTXl is used in a radiolabel may be any radialoabel, such as, but not limited puri?ed form. In an alternate embodiment, PcTXl r a variant to, 131I and 125I. Such binding of the compound to the Na+ of PcTXl is used in a partially puri?ed form. channel mediating the constitutive inWard Na+ current may, but is not required to, inhibit the activity of such Na+ [0043] Pharmaceutical compositions of the present disclo channel. sure containing the compounds discussed above, such as, but not limited to, PcTXl may be formulated in combination [0041] Furthermore, the compound may be conjugated to With a suitable pharmaceutical carrier for administration to a protein sequence that serves as a protein tag (the tag a subject in need of treatment. Such pharmaceutical com protein). As above, such compound may be identi?ed as positions comprise a therapeutically effective amount of the described beloW in this speci?cation or such compound may polypeptide or compound, and a pharmaceutically accept be PcTXl, or a variant of PcTXl. In the instance Where the able carrier or excipient Such carriers include but are not compound is PcTXl, or a variant of PcTXl, such PcTXl or limited to, saline, buffered saline, dextrose, Water, glycerol, variant of PcTXl may have a tyrosine residue or other ethanol, and combinations thereof. Formulation should suit residue at one end thereof to aid in the linking to the tag the mode of administration, and is Well Within the skill of the protein. Such as PcTXl molecule is shoWn in SEQ ID NO. art The invention further relates to pharmaceutical packs and 2 and has been shoWn to have activity identical to the kits comprising one or more containers ?lled With one or unmodi?ed PcTXl sequence. In this embodiment, the more of the ingredients of the aforementioned compositions method of treatment further includes administering to the of the invention. Compounds of the present invention may subject a therapeutically effective amount of a second com be employed alone or in conjunction With other compounds, pound Which binds to the tag protein. The second compound such as therapeutic compounds.