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 FIG2A go 3 26) FIG2B ini 43 m3 FIGZC éu:5 : WFTWAIII$I|1$||aIII4LIIIbIIaIIIQéIIIh'Isl?lllalllsbll mans) mam-u) ‘limalmsl mums) Tmlw) mars) Patent Application Publication Apr. 26, 2007 Sheet 2 0f 15 US 2007/0092444 A1 F I Q 3 A G:52....55625.6 M55.“1%.....mw wm039M”, mvémm mi l2.” “$2M?2 Fig. 4A Fig. 4B WA) m -o- Basal // -¢- lMuMAmllux-Me a @mmmm ‘1/ / m I00 (PA) Fig. 4C “10 -o- llifluun Can-ml (mV) 4 400 Patent Application Publication Apr. 26, 2007 Sheet 3 0f 15 US 2007/0092444 A1 Breast Carcinoma MeIanOma ZR-75-1 SKMEL-Z 2“ im FIG. 5A 52 g _. E E4» 0 400 ‘W a ‘an "a r “me (m8) Time (ms) E :5“ FIG. 5B 3 5 £400“ 5 E E o ‘l'im4:°(ms) “a a; 400" g FIG. sc E s‘ o Tlm‘ams) a.“ FIG. 6A IASIC1:482 bp ASIC1:482 bp FIG. 6B Q <2-c9§c¥é°6c§°c§¢0¢ ‘ ASIC1=482 bp ii?iié-iiba uni era-ea - - ‘ ASIC12482 bp Patent Application Publication Apr. 26, 2007 Sheet 4 0f 15 US 2007/0092444 A1 SK-MG SK-MG ASICZ Patent Application Publication Apr. 26, 2007 Sheet 5 0f 15 US 2007/0092444 A1 FIG. 8A 15 n- 9“ “'° 3 g \ 7Z0 'm' 400 pM amiloride 8 o m” ASICZa a; ‘P :5 Cd) 1020 < 4010 . I - | - I 0 5 in ‘IR Time, seconds Dd pH 4.0 FIG. 8B .Jg -2O0D ; a; m- . ,f’ v m; 400 p.M amllorlde g E _ ASICla B Q m" +1 (O I (U 1 40000 _-§’@ M: < 44000: 0 ' 5' ' 1B ' 1% Time, seconds pH 4.0 0- 2 4000-: a 2:‘ -4ooo- 400 uM amiloride *2 g mi ":3 O m" -o-I (D ‘ ' g‘ | 40000: 35 @ 4m 5 . < 440m ‘1mm- ; o ' 5 ' 1'0 I 15 Time, seconds Patent Application Publication Apr. 26, 2007 Sheet 6 0f 15 US 2007/0092444 A1 FIG. 10A FIG. 10B FIG. 10C ASICl ASICZa FIG. 9 ASICI ASICZ ASICI ASICZ 207 - IP: ASIC2a IgG FIG. 11A LP. ASICl ASICI ASICl ASlCl FIG. 11B FIG. 11C Patent Application Publication Apr. 26, 2007 Sheet 7 0f 15 US 2007/0092444 A1 FIG.'12A FIG. 12B FIG. 120 Fig. 13A Fig. 13B \\ “6” 208 — 124 - lOl _ 36— Patent Application Publication Apr. 26, 2007 Sheet 8 0f 15 US 2007/0092444 A1 FIG. 14A FIG. 14;; = 4 n 5 As|c1 ASIC2 AS; C 112 Patent Application Publication Apr. 26, 2007 Sheet 9 0f 15 US 2007/0092444 A1 FIG16A ‘?an-MildredM-Cmltrbl‘if.cummm FIG16B uh“*6,“’astuna-summoncuminm - FIG16C a: an: a1: 1;» m'm 1mm uubwu Patent Application Publication Apr. 26, 2007 Sheet 10 0f 15 US 2007/0092444 A1 FIG. 17 '12 - 76 U a“" 0.3 l E _ ‘ .- ' o ,5 5 06- ~v E0 0.4 .._ O s E --I--N-amIdino-3,5-diemino E 0'2‘ pyrazlnecarhoxamlde . _ é‘ - 0 Benzamll '§ 0.0 II If Y I I I I l I a y | I ° 1° 100 Drug concentration (I09. uM) 10 nM PcTX1 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 Fig. 190 (pA)Amplitude TIME “me Fig. 20 ASIC1a-l-pgbd ASlC1a+ctl pep I. l_- 1000M 1000M 200s - Patent Application Publication Apr. 26, 2007 Sheet 12 0f 15 US 2007/0092444 A1 Fig. 21 Fig. 22A Fig. 223 1';,=205 an “>1 - " JJJFAL 'l'l “>4 4 5 '14?“'1 I WW“1-11 ' I a; Hi1 l > O 1 2 3 4 v :0 Illl?-MIEWII “ll?ih ‘ Log Dwell-"ma (ms) Log Dwell-"me (ms) Patent Application Publication Apr. 26, 2007 Sheet 13 0f 15 US 2007/0092444 A1 Fig. 23A Fig. 233 Ceu-Attachem Patch Outside-Out Fatah mum‘!!! Mimi-l ' 4?! E13? .urnlnm mmulai -& in? *“m‘ "-W'" "' ' ' ‘ dm'nwmwww" “Wm ?lm-4m 'l‘" mull-Am- ink-11h! - '81 UN “a.” I V V I‘ I In I - r.- -.-‘ -Q :mnlnnr [malls] --la1 mi’ :Iiml Wm“. Multan! llulullll I-I'Alln't' own Call-Attached Patch Current-Voltage Relation 1 m; hL M as i Us“, - I MD iii) an 46 +3.1 :15 oil) Patent Application Publication Apr. 26, 2007 Sheet 14 0f 15 US 2007/0092444 A1 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 1.0 _ ' 1.0 A 0.9 A 0.9 as ma ‘ag 0.3 E 0.1 E 0.1 _z‘: 0.6 § 0.6: 20 0.5 g 0.5 2 0.4 2 0'4 3 M —I— Scrambled Peptide n _.__ pcTx o v a 0-3- . n --0— POTX n - -O— Scrambled Peptlde 5 oz 5 02 2 OJ z 04 0" 1'.’ "'l I i g", _ " , H m , "I", . .. m, 0 0,1 1 1o 10D 0 H1 1‘ 1a 100 Concemm?o" (11M) Concentration (nM) 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 0.1 '- o" 0.Ou-f,'.-...| . ..-.-| FH-r-rr|——~|—\—r—y-1-rrr|-—|-—r-—|—|—|-m 0 1 10 on 0 1 10 Concentration (nM) Conoanhation (nM) Fig. 25 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.