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Patent Application Publication Oo) Pub. No.: US 2014/0336159 Al Clarke Et Al

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Patent Application Publication Oo) Pub. No.: US 2014/0336159 Al Clarke Et Al US 20140336159A1 (19) United States (12) Patent Application Publication oo) Pub. No.: US 2014/0336159 Al Clarke et al. (43) Pub. Date: Nov. 13,2014 (54) METHODS FOR TREATING AND 61/605,013, filed on Feb. 29, 2012, provisional appli­ DIAGNOSING RESPIRATORY TRACT cation No. 61/607,936, filed on Mar. 7, 2012, provi­ INFECTIONS sional application No. 61/648,960, filed on May 18, 2012, provisional application No. 61/648,822, filed on (71) Applicant: PULMATRIX, INC., Lexington, MA May 18, 2012. (US) Publication Classification (72) Inventors: Robert William Clarke, Medfield, MA (US); David L. Hava, Natick, MA (US); (51) Int. Cl. John P. Hanrahan, West Roxbury, MA A61K31/19 (2006.01) (US); Wesley Hugh Dehaan, A61K9/00 (2006.01) Chelmsford, MA (US); Paulette Wright A61K31/56 (2006.01) Andreotta, Charlton, MA (US); Stephen A61K4S/06 (2006.01) P. Arold, Billerica, MA (US); Jennifer (52) U.S. Cl. Kenyon Saunders, Boston, MA (US) CPC ................. A 61K 31/19 (2013.01); A 61K 45/06 (2013.01); A 61K 9/0078 (2013.01); A61K (21) Appl. No.: 14/350,235 9/0075 (2013.01); A 61K 31/56 (2013.01) USPC ............................................. 514/171; 514/557 (22) PCT Filed: Oct. 5, 2012 (57) ABSTRACT (86) PCT No.: PCT/US2012/059022 Described are methods of preventing, treating and diagnosing § 371 (c)(1), of a subject having a condition, such as, an inflammation or (2), (4) Date: Apr. 7, 2014 infection of the respiratory tract. Methods of treatment and prevention include administration of effective amounts of Related U.S. Application Data calcium salt formulations to a subject. Methods of diagnosing (60) Provisional application No. 61/544,400, filed on Oct. include the use of biomarkers and optionally the use of kits 7, 2011, provisional application No. 61/550,081, filed that can detect biomarkers. Further described are methods for on Oct. 21, 2011, provisional application No. 61/584, modulating an immune response that include the modulation 001, filed on Jan. 6, 2012, provisional application No. of Toll-like receptors. Patent Application Publication Nov. 13, 2014 Sheet I of 24 US 2014/0336159 Al FIGURE IA Calcium ion dose Lung Dose mg/kg matching Predinicai Model MCC infection Inil ammat Idri Patent Application Publication Nov. 13, 2014 Sheet 2 of 24 US 2014/0336159 Al FIGURE IB Lung Dose, Lung Mass Matching Precfinical Model Patent Application Publication Nov. 13, 2014 Sheet 3 of 24 US 2014/0336159 Al FIGURE IC 1I as £ S- '"iRS O CS Lung Dose E Lung Area hatching Predsnica! Model Patent Application Publication Nov. 13, 2014 Sheet 4 of 24 US 2014/0336159 Al A 7% HS FIGURE 2 Form. I (30) Form. I (30): 30pg/cm2 Form. I (10): 10|ig/cm2 Form. I (10) 30 40 SO 60 Tim© (min) Form. If (30) Form. If (30): 30pg/cm Form. I! (10): iOpg/cm WaCI DP Form. CO 10 18 30 46 60 76 90 T im e (m in ) Patent Application Publication Nov. 13, 2014 Sheet 5 of 24 US 2014/0336159 Al FIGURE 3 2 § - - 7% HS 1§» A Vehide ■#> Formulation Il I mg/kg V Formulation I!. 0 5 mg/kg ■ Formulation II. 0.25 mg/kg §J T* 40 80 80 & Vehicle <#> Formulation li, 1 mg/kg. 2hr Dur V Formulation II. 0 5 mg/kg, 2hr Dur O 7% Saline (% of Baseline) (% of Baseline) Mucociliary Clearance Mucociliary Clearance -I 280 Tsme (m in) Patent Application Publication Nov. 13, 2014 Sheet 6 of 24 US 2014/0336159 Al FIGURE 4 Mean Central Lung MCC No Treatment Controf Form. !I 22mg Patent Application Publication Nov. 13, 2014 Sheet 7 of 24 US 2014/0336159 Al FIGURES ILB B ILS 2900© 260-j 18000 1800©I 200- j $Q m 150- £ "3s 4000- 0 . 100 2000 50- 0j 0- A o4' A o%' I-------------!!-------------S i______ il------- —S 5.5mg 11mg 5.5mg 110¾ GM-CSF ILIP 800- 50O-| 400I SO©-** I 120-r E OS Ss 80- a CL 40- ©■ 5.5mg 11 mg S.Smg 111¾¾ Patent Application Publication Nov. 13, 2014 Sheet 8 of 24 US 2014/0336159 Al FIGURE 6 A Total Cells/g Sputum I DG D2 DO D2 S.S mg 11 mg B Total Neutrophils/g Sputum «53 E 5 D 3 4' Q, CO as 3- W © 2 U O ^ 1 » 3 Z DO D2 DO D2 S.S mg 11 m g Patent Application Publication Nov. 13, 2014 Sheet 9 of 24 US 2014/0336159 Al FIGURE 40 I Mean Change in % Eosinophils fn=6) I 50 Mean 15.8 I OO 5? Mean 6,7 .£ 111 Baseline Form. V Placebo Patent Application Publication Nov. 13,2014 Sheet 10 of 24 US 2014/0336159 Al FIGURE 8 A. K, pneumoniae S. pneumoniae xrX£ 100- 25 80- LL O 40- -L W / O / $ 0 "> o—¢5—o~—~-O T- 0 m 120 ISO 240 0 60 120 180 240 Time (mini Time (min) c. D, P. aeruginosa S. aureus 0 60 1 20180 240 0 60 120 180 240 Time {mm} Time (mm} Patent Application Publication Nov. 13,2014 Sheet 11 of 24 US 2014/0336159 Al FIGURE 9 InfSuema ,A 3-» SC 2- o Na 1X 1QX Ca dose Rhinovirus -®- Sal:ne -O- 0.12M CaCb in saline -Si- 0 STofvI CaCi2 in saiine -Q- I 2M CaCI2 m saline M 120 180 Time (min) Patent Application Publication Nov. 13,2014 Sheet 12 of 24 US 2014/0336159 Al FIGURE 10 80' Saline 80' 2X 1:1.3, Form. V -J 70' 2X 8:1 E SO- CS C SO- 8x81, Form.! 40- Q. j.. 30' at "S3 20- 10- 0- 120 240 380 Time (min) Patent Application Publication Nov. 13, 2014 Sheet 13 of 24 US 2014/0336159 Al FIGURE 11 p<C-<JS S- 5" E S S u 3“ SS 2- O »1 I- T ~ T * ~ T ~ Mt Form. V !.0 0.01 Zartatrevir Zanamtvir * Form. V B PatentApplication Publication Nov. 13, 2014 Sheet 14 of 24 US 2014/0336159 Al FIGURE 12 A Infection model Rhinovirus P. aeruginosa: LPS I 0"» p-'0 05 7.5 «* p<0.01 t................ I I L I 0 8 - 5 .0 “ 0 6 - 0 4- 2 .5 “ 0 2 - 0 0- 0.CT & 4! E .£ ‘u E U S 3 3 o Q) o & u. w-! Inflammation model Tobacco smoke Ozone 2.5- I 1.5 ” 2.0 - 1.5- 1.0 - 1 , 0 ” 0.5 - 0.5- Neutrophils IxieSf IxieSf Neutrophils 03 (xlQ5) Neutrophils 0. 0 - 0.0 J — ^L™^iW,T."nnrr,rrj f I E 3 Li* E 3 S & O PatentApplication Publication Nov. 13, 2014 Sheet 15 of 24 US 2014/0336159 Al FIGURE 12C Ozone Exposed PatentApplicationPublication Nov. 13,2014 Sheet 16 of 24 US 2014/0336159 Al FIGURE 13 Upregulaled with Upregulated with Tobacco smoke Rhmovirus-Ib infection X"""" / ,/ / / s\reg / bsuU / CdOAKP- i SUgl CU "/SgCC x CdC Cdl ? Cxd (U T C /O d d a C d eV CM-SlfKC CxrUA C x d w O V d O u d OcSCdUWts b i d CxdS Him CxdlC5 Cf \ SLS \ IdxdS5 TUC - \ NV xV x V-""" '-SVsv^ vsW. -X x - .... Downregulated with Downregulated with Tobacco smoke Rhinovirus-Ib infection Adrbl Cxc r4 Proc Apinr CxcrS Pou2afl Bdnf Fasl Ppbp Gusb Bm p6 Hspbl Pri2c2 Hifla CSa IgfbpS Rgs3 Pmaipl Cd5 i!l6 T irl GprSl Serpinala Ccr6 KcnaS TIrS 5od2 CcrS Lefl TIrS Ccrll Lep Xcil CcrU N od CrntrnS Perl Crebl Pin PatentApplication Publication Nov. 13, 2014 Sheet 17 of 24 US 2014/0336159 Al FIGURE 14 Supplemental CaCI ifmM) 2000 JsP* Supplemental Cad ^fmIVS) Patent Application Publication Nov. 13,2014 Sheet 18 of 24 US 2014/0336159 Al FIGURE 15 A & SS KC E «3 JZ u 2 o u Se * ^ ' p <§> Supplemental CaCfg fmfV!} 60 H B IL-S 0! as E fS JZ U 4 0 -i TS O «5 Jj 20- Supplemental CaCtg fmtVf) TNFalpha Supplemental CaQg (raMj PatentApplication Publication Nov. 13, 2014 Sheet 19 of 24 US 2014/0336159 Al FIGURE 16 ENA78 GM-CSF 4.S- 4.0- 3 .S- 3.0- 2 5- 2. 0 - I 5- 1 C- 0.5- 0.0J ■ i c-* ■;>* .o* s?' #• 1 ngftnl LPS 1 ng.'mf LPS !VttP-2 (P-10 25-, Si 2 0 - f I SO- p-n* /Ct I u. S- Qj I ! I f .................I..................................... T * S ^ '-? ip I n^'nrsL LPS 1 ag/mi LPS NRfPt (R1P140) I ng/s«t LP S PatentApplication Publication Nov. 13, 2014 Sheet 20 of 24 US 2014/0336159 Al FIGURE 17 TNFa IL-8 {pgSrnl} IL-8 SuppSemental GaGi 2 JmMS B 2800- _ 2 0 0 0 - § , 1500- CL C9 1000- JtC ~ 500 - !----------- r 4? SuppismentaS CaC!? (miVI) PatentApplication Publication Nov. 13, 2014 Sheet 21 of 24 US 2014/0336159 Al FIGURE 18 A Calcium Chloride -I- Caicum Lactate 1500 - a Magnesmin Chloride --4- Sodium Chloride £5 1000 -< 500 ' SuppiementaS Salts (mM) # Caidum Chloride S Calcium Lactate a Magnesium Chloride -jS- Sodium Chloride Suppiementai Salts (mM) Patent Application Publication Nov. 13,2014 Sheet 22 of 24 US 2014/0336159 Al FIGURE 18 cont. C IL-S Calcium Chicnde ~m CaiQurr Lactate ■& MagnfsSiur Chlonde Sodiun Chionde a. 400 • Supplemental Salts (mM) D IL-6 CaiQLm Chionde ■m Ca!citm Lactate •4- Magnesium Chionde -*-■ sC'd'UTi Chlorde Supplemtntal Salts (mM) Patent Application Publication Nov. 13, 2014 Sheet 23 of 24 US 2014/0336159 Al FIGURE 19 KC TNFa KC (pgfml) KC Supplements* CaCI3 (mft*) Supplements! CaC3? t KC TNFa C 1000«, 300- v I 600- £ ^oo- 200- S A 0- HF- SS *5 NS> ❖ Supptewntsl CaCf2 {mNi) SuppteJiientat CaCij (snW) PatentApplication Publication Nov.
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