(12) Patent Application Publication (10) Pub. No.: US 2007/0053845 A1 Sengupta Et Al
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US 2007.0053845A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2007/0053845 A1 Sengupta et al. (43) Pub. Date: Mar. 8, 2007 (54) NANOCELL DRUG DELIVERY SYSTEM Publication Classification (76) Inventors: Shiladitya Sengupta, Waltham, MA (51) Int. Cl. (US); Ganlin Zhao, Arlington, MA A6IR 9/14 (2006.01) (US); Ishan Capila, Ashland, MA (US); A6IL 9/04 (2006.01) David Eavarone, North Quincy, MA (52) U.S. Cl. ................................................................ 424/46 (US); Ram Sasisekharan, Bedford, MA (57) ABSTRACT (US) Nanocells allow the sequential delivery of two different Correspondence Address: therapeutic agents with different modes of action or different CHOATE, HALL & STEWART LLP pharmacokinetics. A nanocell is formed by encapsulating a TWO INTERNATIONAL PLACE nanocore with a first agent inside a lipid vesicle containing BOSTON, MA 02110 (US) a second agent. The agent in the outer lipid compartment is released first and may exert its effect before the agent in the (21) Appl. No.: 11/495,947 nanocore is released. The nanocell delivery system may be formulated in pharmaceutical composition for delivery to (22) Filed: Jul. 28, 2006 patients Suffering from diseases such as cancer, inflamma tory diseases Such as asthma, autoimmune diseases such as Related U.S. Application Data rheumatoid arthritis, infectious diseases, and neurological diseases Such as epilepsy. In treating cancer, a traditional (63) Continuation-in-part of application No. 11/070,731, antineoplastic agent is contained in the outer lipid vesicle of filed on Mar. 2, 2005. the nanocell, and an antiangiogenic agent is loaded into the nanocore. This arrangement allows the antineoplastic agent (60) Provisional application No. 60/549,280, filed on Mar. to be released first and delivered to the tumor before the 2, 2004. tumors blood Supply is cut off by the antianiogenic agent. Patent Application Publication Mar. 8, 2007 Sheet 1 of 18 US 2007/00538.45 A1 ||30OueueJo??poup?sododd Patent Application Publication Mar. 8, 2007 Sheet 2 of 18 US 2007/00538.45 A1 Patent Application Publication Mar. 8, 2007 Sheet 3 of 18 US 2007/00538.45 A1 A O Chs O Cls (A) ------O o PLGA 5050 DL4A (C) ..pN.C. pyridine, CHCI HCO -S 2. Doxorubicin, DMF, TEA HCO O Oh O OH OCH Oh Oh Hac1 O O oh O O is O O CH O hC -- NH ouls O O O Oh O 200 nm Doxorubicin - Pl-GA conjugate emulsion-Solvent evaporation (B) Ultracentrifugation, sizing and phospholipid membrane coating. Combretastatin encapsulated in lipid layer. tumo --- Doxorubicin (ug) 2 (E) (F) Combretastatin (x10kg) 200 O t 9 2 2 8 5 150 is 7 will 6 k r 5 5 a 100 4 f 5 3 if s 2 # 50 S. S. O H -- &S c O 6 12 S 24 25 49 73 97 21 SS S d SSQ KS Time (hours) FIG. 3 Patent Application Publication Mar. 8, 2007 Sheet 4 of 18 US 2007/00538.45 A1 Effect of VEGF and HGF on tumor angiogenesis in vitro, specificity of a VEGF antagonist Tumor Cells es > C y Blood vessels forming CD Endothelial g Cells es > o y we na. OO NyN H d is . up d : vrr WY na OO n Y A as S t O N na o n Y H Only tumor cells express green fluorescent proteins, and all nuclei are stained with PI. In a merge image, the tumor cells look yellow as a result of green and red merging, while endothelial cells look only red. FIG. 4 Patent Application Publication Mar. 8, 2007 Sheet 5 of 18 US 2007/00538.45 A1 Effect of Doxorubicin, thalidomide, and COmbretastatin On VEGF-induced tumor angiogenesis in vitro. Tumor Cells s Blood vesses 5 forming O Endothelia Cells s ; This model allows the dissection of chemotherapeutics and anti-angiogenics. Doxorubicin kills only the yellow tumor cell, while the anti-angiogenics kill only the red endothelial cells. FIG. 5 Patent Application Publication Mar. 8, 2007 Sheet 6 of 18 US 2007/00538.45 A1 Effect of Doxorubicin, thalidomide, and COmbretastatin on HGF-induced tumor angiogenesis in vitro. Propidium iodide Tumor cells era C Blood vessels forming t Endothelia Cells SEsess This model allows the identification of exact drugs that would Work in defined clinicopathological cases. Doxorubicin works like it did in the case of VEGF-induced response, but unlike Fig. 5, both thalidomide and Combretastatin failed to inhibit the HGF-induced effect, This shows that the assay can detect unique effects of growth factors, FIG. 6 Patent Application Publication Mar. 8, 2007 Sheet 7 of 18 Effect of Doxorubicin, thalidomide, and COmbretastatin On VEGF-induced tumor angiogenes IS In VI trO When plated on Collagen Patent Application Publication Mar. 8, 2007 Sheet 8 of 18 US 2007/00538.45 A1 Effect of Doxorubicin, thalidomide, and COmbretastatin On HGF-induced tumor angiogenesis in - s > E. t e s forming h Endothelial Cells CD S Tumor Cels as is 2 --: th a 15 u?- E 50CR -- Changing the extra-cellular matrix from matrigel to collagen results in the loss of protective function of HGF against the anti-angiogenic effects of Combretastatin and thalidomide. FIG. 8 Patent Application Publication Mar. 8, 2007 Sheet 9 of 18 US 2007/00538.45 A1 Combretastatin (30 h) Doxorubicin (30 h) a & Y - C Y S--s-St. f. ! G a. --- - N. C. " O Ef 2. se d . y Nanocell (12 h) Nanocell (30 h) FIG. 9A Vascular Tumor component component Veh NCVeh NCND Veh NCVeh NCND 12 h 3O h 12 h 3Oh FIG. 9B 2.0 1.5 A DOXOrubicin Dox-PLGA conjugate 1.0 DOX-PLGA O.5 5 10 15 20 DOX) ug FIG. 9C Patent Application Publication Mar. 8, 2007 Sheet 10 of 18 US 2007/00538.45 A1 NCD+LC NC (ld) lewis Illing carcinoma B 16F10 melanoma FIG. 10A FIG. 1 OB Lewis lung carcinoma B16/F10 melanoma Veh -- Veh 55OO - - VeNCD+LIC) 1OOOO -O- NCD+LCNE +LIC) 5OOO - A - LC) 7500 -0- NCD) 4500 0- NCD -E- NC -E- NC SOOO - V - LICD) E 3500 E 5C 3000 CU 2000 S 2500 9 5 5 1500 E 2OOO E D H 15OO 1000 1OOO 500 5OO O 9 tilt is is 17 O t 11 is 1st 17 Days Days Patent Application Publication Mar. 8, 2007 Sheet 11 of 18 US 2007/00538.45 A1 Tumor ck k 220 1 7 O Liver Spleen LungS Plasma 1. 2 O 7 O 2O O 5 10 24 5 10 24 5 10 24 5 10 24 5 10 24 hours FIG. 1 OF Patent Application Publication Mar. 8, 2007 Sheet 12 of 18 US 2007/00538.45 A1 × × × × × ***+ Cj]ON º o o o co o w y N. H Se3 epXOdAH 9/6 O O O O t r N y Aasuap IeSSew Patent Application Publication Mar. 8, 2007 Sheet 13 of 18 US 2007/00538.45 A1 1.O) O% 2, ©2. CD 5 (1. o v g 2% 6 | %,s 9. O ?yn n y O Z. (uen Oh emeel) Q Seme uOSseudx. d5 2U O Z CD m 1. R E& H 2 SE- 8 is E.1 - > t . H ?o th C % 1 2. S2 8, it ?y N O (ue w Oh emeeu) Seme uoSsedX Patent Application Publication Mar. 8, 2007 Sheet 14 of 18 US 2007/00538.45 A1 9000 -- Vehicle - A - LC) 7OOO - V - NCC) 5 O O O 3OOO 3OOO 25 OO 2OOO 15OO 1OOO 500 7 9 11 .." 15 17 D FIG. 12A 12OOO r 9 OOO -- Veh - A - NC long term 6OOO 3OOO 5 O 15 2O 25 3O t t t t t t Days FIG. 12B Patent Application Publication Mar. 8, 2007 Sheet 15 of 18 US 2007/00538.45 A1 Vehicle Control NCD) Ncid+lic. Vehicle Control NCD) LC NCD)+LIC) FIG 3B Patent Application Publication Mar. 8, 2007 Sheet 16 of 18 US 2007/00538.45 A1 6 4. 2 O Veh NCD LC) NCD) NC LCD) +LIC) FIG. 13C 5. O 2 .5 O. O Veh NCD LC) NCD) NC LCD +LIC) FIG. 13D Patent Application Publication Mar. 8, 2007 Sheet 17 of 18 US 2007/00538.45 A1 NO2 O CH3 O CH3 -- House O oh-oh osci O n O p-nitrophenyl PLGA 505 O DL 4A Chlorofromate (pNC) Pyridine, CH,Cl, O CH3 O CH3 o,N-(y-o-&-ou.O O O OH O O n Activated PLGA Doxorubicin, DMF, TEA O OH O CCCCfor OH Hc O OH O HC-2 O CH O CH OH O O n Doxorubicin - PLGA conjugate FIG. 14 Patent Application Publication Mar. 8, 2007 Sheet 18 of 18 US 2007/00538.45 A1 SunOH 0 (fu) peseee. 6nup eno 000Z (fr) peSeeleubnup eno US 2007/0053.845 A1 Mar. 8, 2007 NANOCELL DRUG DELIVERY SYSTEM 0005 Though effective in some kinds of cancers, the use of systemic chemotherapy has had to minor Successes in the RELATED APPLICATIONS treatment of cancers of the colon-rectum, esophagus, liver, 0001. The present application claims priority to U.S. pancreas, and kidney, and skin. A major problem with provisional application, U.S. Ser. No. 60/549,280, filed Mar. systemic chemotherapy for the treatment of these types of 2, 2004, entitled “Nanocell Drug Delivery System” and is a cancers is that the systemic doses required to achieve control continuation-in-part of U.S. Ser. No. 11/070,731 filed Mar. over tumor growth frequently result in unacceptable sys 2, 2005 and also entitled “Nanocell Drug Delivery System’, temic toxicity. Efforts to improve delivery of chemothera both of which are incorporated by reference herein.