US 20120264810A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2012/0264810 A1 Lin et al. (43) Pub. Date: Oct. 18, 2012 (54) COMPOSITIONS AND METHODS FOR 61/400,763, filed on Jul. 30, 2010, provisional appli ENHANCING CELLULARUPTAKE AND cation No. 61/400,758, filed on Jul. 30, 2010. INTRACELLULAR DELVERY OF LIPID PARTICLES Publication Classification (75) Inventors: Paulo J.C. Lin, Vancouver (CA); (51) Int. Cl. Yuen Yic. Tam, Vancouver (CA); C07. 4I/00 (2006.01) Srinivasulu Masuna, Edmonton A6II 47/24 (2006.01) (CA); Marco A. Ciufolini, CI2N 5/071 (2010.01) Vancouver (CA); Michel Roberge, A63L/73 (2006.01) Vancouver (CA); Pieter R. Cullis, A6II 47/22 (2006.01) Vancouver (CA) C07D 215/46 (2006.01) A 6LX 3L/705 (2006.01) (73) Assignee: The University of British (52) U.S. Cl. ............ 514/44A: 540/5: 546/163; 514/788: Columbia, Vancouver (CA) 514/44 R; 435/375 (21) Appl. No.: 13/497,395 (57) ABSTRACT (22) PCT Filed: Sep. 22, 2010 Compositions, methods and compounds useful for enhancing the uptake of a lipid particle b\ a cell are describedIn particu (86). PCT No.: PCT/B2O1O/OO2518 lar embodiments, the methods of the invention include con tacting a cell with a lipid particle and a compound that binds S371 (c)(1), a Na+/K+ ATPase to enhance uptake of the lipid particle b\the (2), (4) Date: Jul. 3, 2012 cell Related compositions useful in practicing methods include lipid particles comprising a conjugated compound Related U.S. Application Data that enhances uptake of the lipid particles b\ the cell The (60) Provisional application No. 61/277.306, filed on Sep. methods and compositions are useful in delivering a thera 22, 2009, provisional application No. 61/277.307, peutic agent to a cell, e g for the treatment of a disease or filed on Sep. 22, 2009, provisional application No. disorder in a Subject Patent Application Publication Oct. 18, 2012 Sheet 1 of 18 US 2012/026481.0 A1 Figure 1 A. 6OO 500 S. 5 e 4OO 1 ug/mL 3OO E35 ug/mL E S 10 ug/mL 2OO 15ug/mL g a 100 Time (Hr) siRNA Uptake 1.60 1.40 1.2O 1.OO O.80 O. 60 O. 40 O.2O O.OO O 2O 40 60 8O Compound Patent Application Publication Oct. 18, 2012 Sheet 2 of 18 US 2012/026481.0 A1 Punctate Distribution 18O 160 1. 40 1.20 1. OO O.8O O. 60 O. 40 O.2O O. OO O 2O 40 60 8O Compound Patent Application Publication Oct. 18, 2012 Sheet 3 of 18 US 2012/026481.0 A1 & Diprophylline & Isoxicam s r I l O 5 1O 15 2O 25 3O Concentration (uM) mano Chloroquine & Diphemani methylsulfate ...:... Trimethobenzamide hydrochloride O 1O 2O 3O Concentration (uM) Patent Application Publication Oct. 18, 2012 Sheet 4 of 18 US 2012/026481.0 A1 Intracellular Distribution of siRNACy3 120 100 Cytosolic Cy3 Signal Punctate Cy3. Signal 24.68 OOOO O SS (-) LN-siRNA (+) LN-siRNA + 0 (+) LN-siRNA + 10 (+) LN-siRNA + 30 uM chloroquine uM chloroquine uM chloroquine Treatment Patent Application Publication Oct. 18, 2012 Sheet 5 of 18 US 2012/026481.0 A1 Figure 3. 11-1-N/-N-11N1a1n O-SOMe C 3 n linoleyl-1-methanesulfonate NH HN1N1\- 2. C N1 OH 1 2 Ho-N-o-r 4,7-Dichloroquinoline 1,4-Diaminobutane 4 3-allyloxy-1,2-propane diol HN1a1a-NH2 2, 80 °C, 1 h 1 -> n 120 °C, 6 h 85% C N 5 N-(7-chloroquinolin-4-yl)butane-1,4-diamine 1) 5, THF NaH, Ph.H 1n 11 N1 n-1-1-1-1aO 2) NaBH 4 -- l -- 3, reflux o m O MeOH 60% R 23% from 7 6 R = CHO-CH-CH=CH ZnC,2, Pd(PPh3). Bu sSnH D. 7 R = CH-OH2 (75%)2 2 Swern oxidation 8 R. CHO C 11-1N1-FN-1N1-1-1- O 2 m m O lu NS-1-1-1N N N Patent Application Publication Oct. 18, 2012 Sheet 6 of 18 US 2012/026481.0 A1 Figure 4 350 -\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\ 3OO 2 25O DDLinDMA 200 BDLinDMA + CO 10 uM 15O SDLinDMA + CG 3OuM e OVO 8CG-DLinDMA O 100 ŠS s 5O s Š O S. 2O 1O 5 2.5 siRNACy5 (ug/mL) B. 60% 50% S 40% so-$o DLinDMA is 30% w8x DLinDMA + COR 1 OuM g \say DLinDMA + COR 30 uM 20% -- CG-DLin DMA 10% O% s is O 5 1O 15 2O siRNACy5 (ug/mL) Patent Application Publication Oct. 18, 2012 Sheet 7 of 18 US 2012/026481.0 A1 C. 60% -a- 50% S 40% --DLinDMA 9s 30% & DLinDMA + CG 10 uM 2 asa DLinDMA + COR 30 uM 20% -e-CG-DLinDMA 10% O% O 5 1O 15 2O siRNACy5 (ug/mL) D. DLinDMA:CQ-lipid DLinDMA:CQ-lipid (40:0) (35:5) E. DLinDMA.co-lipid T DLinDMA.co.ipid (40:0) (35:5) Patent Application Publication Oct. 18, 2012 Sheet 8 of 18 US 2012/026481.0 A1 Figures 5A and 5B A. 23 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 3. SSR 8. -- 43% ixiti:8& 48% Q-lipii:. as .38 flies. 58 E8 : siR&A&3 girl. Patent Application Publication Oct. 18, 2012 Sheet 9 of 18 US 2012/026481.0 A1 Figures 5C-5E C. iA as is t-dipiti ta. ce-lipid 5% . a -ts. is Sir SX. f Sir R. S 8-it SS&clipsis Scripts Sassic 2ns w gira. S to 28 S 3 2 S is 23 St. 20 S s y 8 Atia scies s S. E. 8 tars y 2 cars 48% is a 48:3pths f saintain spinosa. f &K-Li Stairs s scalisir it. s s: s & calci Patent Application Publication Oct. 18, 2012 Sheet 10 of 18 US 2012/026481.0 A1 Figure 6 Increased siRNA Cy3 Accumulation Normalized siRNACy3 Normalized siRNACy3 Small Molecule Cytosolic Distribution Accumulation Levodopa 0.72 1.28 Naphazoline hydrochloride O.68 1.32 ACetohexamide O.68 1.32 Nicosamide O8O 1.35 Diprophylline O.64 1.38 ISOXicam O.65 142 Increased siRNA Cy3 Cytosolic Distribution Normalized siRNACy3 Normalized siRNACy3 Small Molecule Cytosolic Distribution Accumulation Diphemanil methylsulfate 1.43 0.70 lsoxSuprine hydrochloride 1.44 0.51 Trimethobenzamide hydrochloride 1.45 0.70 Chloroquine 160 O.34 Azaguanine-8 1.61 0.54 Isoflupredone acetate 1.61 0.50 Patent Application Publication Oct. 18, 2012 Sheet 11 of 18 US 2012/026481.0 A1 Figure 7A Incubation of LN compound 96-well optical plate Figure 7B CD c 9 o SPDO O D siRNA-Cy3 s s O Z Time (hrs) Patent Application Publication Oct. 18, 2012 Sheet 12 of 18 US 2012/026481.0 A1 Figure 8A 12 1525 O. 5 O 1 51 101 151 201 251 301 351 401 451 501 551 601 651 701 751 Small molecules Figure 8B 39 3.5 c 3 9. S 2.5 2 15 uM 2s 1.5 M a 1.5 D 0.15 uM O 1 N 0.5 E d O - t W S& s p. ŠP s S &Cu SS P s &S S s SS S Sy cse s S KS&S OS Q S o s ss isO o ' KSs Cardiac glycosides Patent Application Publication Oct. 18, 2012 Sheet 13 of 18 US 2012/026481.0 A1 Figure 9A Figure 9B O M 30 nM C. O. E 9 9 r 5 3 - 5 3 LN-SiRNA: GAPDH B-Actin Patent Application Publication Oct. 18, 2012 Sheet 14 of 18 US 2012/026481.0 A1 Figure 10 DSSE-REG. -H, settsrier excessarissed s: 83 is remit is Et pro f is siastics Patent Application Publication Oct. 18, 2012 Sheet 15 of 18 US 2012/026481.0 A1 Figure 11A & DSPE-PEG KSTR-PEG NP uptake in NCaP cells 140 :------------------------------------- SDSPF-PEG KSTR-PEG 2. ENP conic agfind Patent Application Publication Oct. 18, 2012 Sheet 16 of 18 US 2012/026481.0 A1 Figure 12A a - 350 250 150 & SPE-PEG 100 3: STRPFG e.g. ShATP. A. sh Scramble Ceiries Figure 12B O E CS al CD O -C C ATP1A1 {-actin Patent Application Publication Oct. 18, 2012 Sheet 17 of 18 US 2012/026481.0 A1 Figure 13A DSPE-PEG STR-PEG t '? c \r tr. C. r. 3 c - r a c - r an Figure 13B SEG : STR-PEG LNP concentiations (ug find Patent Application Publication Oct. 18, 2012 Sheet 18 of 18 US 2012/026481.0 A1 Figure 14 GAPDH mRNA relative to 18S rRNA i O.8 2.5 mg/kg Oa - i- - - - - - - - - - OSaling Kirray issues US 2012/026481.0 A1 Oct. 18, 2012 COMPOSITIONS AND METHODS FOR branes. Liposomal nanoparticle (LN) encapsulation of ENHANCING CELLULARUPTAKE AND siRNA has demonstrated significant potential for overcoming INTRACELLULAR DELVERY OF LIPID these problems for delivery of siRNA to hepatocytes in vivo PARTICLES and thus enabling siRNA to be used as therapeutics ((Zim mermann et al., 2006)). However, the design of LN formula CLAIM OF PRIORITY tions of siRNA (LN-siRNA) for other in vivo applications is 0001. This application claims priority to provisional U.S. far from optimized. In particular, effective targeting to spe Application Nos. 61/277.306, filed 22 Sep. 2009: 61/277.307, cific cells is lacking as the majority of systemic administered filed 22 Sep. 2009: 61/400,758, filed 30 Jul. 2010; and LN-siRNA is taken up by the reticular endothelial system in 61/400,763, filed 30 Jul. 2010; each of which is incorporated the spleen and liver (Fenske et al., 2008).