(12) United States Patent (10) Patent No.: US 9.468,603 B2 Mcchesney Et Al
USOO9468603B2
(12) United States Patent (10) Patent No.: US 9.468,603 B2 McChesney et al. (45) Date of Patent: Oct. 18, 2016
(54) NANOPARTICULATE COMPOSITIONS FOR A61 K3I/7076 (2013.01); A61K 45/06 TARGETED DELIVERY OF ACID LABILE, (2013.01); A61K 47/48061 (2013.01) LIPOPHILIC PRODRUGS OF CANCER (58) Field of Classification Search CHEMOTHERAPEUTICS AND THEIR CPC ...... A61K 47/48061; A61K 45/06; A61 K PREPARATION 9/0019; A61K 9/5123; C07D 305/14: CO7D 314/24: CO7D 407/12: C07D 487/04; C07D (71) Applicant: Arbor Therapeutics, LLC, Etta, MS 491/14: CO7D 493/06; C07D 493/08: C07D (US) 519700 See application file for complete search history. (72) Inventors: James D. McChesney, Etta, MS (US); Igor Nikoulin, San Diego, CA (US); (56) References Cited Steve J. Bannister, Tampa, FL (US); Douglas L. Rodenburg, Thaxton, MS U.S. PATENT DOCUMENTS (US) 4,762,720 A * 8/1988 Jizomoto ...... A61K9/1277 264/4.1 (73) Assignee: ARBOR THERAPEUTICS, LLC, 8.440,714 B2 * 5/2013 McChesney ...... CO7D 305,14 Etta, MS (US) 514,449 2004/0213837 A1* 10/2004 Mantripragada ...... A61K 9,127 424/450 (*) Notice: Subject to any disclaimer, the term of this 2011 0077291 A1* 3, 2011 Chen ...... A61K9/0019 patent is extended or adjusted under 35 514,449 U.S.C. 154(b) by 0 days. 2012/030861.6 A1* 12/2012 Liu ...... A61K9/0019 424/400 (21) Appl. No.: 14/485,713 2012/0309819 A1 12/2012 McChesney 2015,0297749 A1* 10, 2015 Hahn ...... A61K 49, 1839 (22) Filed: Sep. 13, 2014 424, 1.37 (65) Prior Publication Data FOREIGN PATENT DOCUMENTS US 2015/OO79189 A1 Mar. 19, 2015 WO 2014, 159851 A1 10, 1994 WO WOOOf 45791 * 8/2OOO WO WO 2011050739 * 5/2011 Related U.S. Application Data OTHER PUBLICATIONS (60) Provisional application No. 61/877,521, filed on Sep. 13, 2013. Brown, Sarah, et al., Journal of Drug Delivery, vol. 2012 (Jan. 2012) pp. 1-7.* (51) Int. Cl. Arasto, Docetaxel, pp. 1-3, accessed Feb. 23, 2016.* A 6LX 9/27 (2006.01) Nikanjam et al., “Synthetic nano-LDL witith paclitaxel oleate as a A6 IK3I/337 (2006.01) targeted drug delivery vehicle for glioblastoma multiforme”, Jour A6 IK3I/365 (2006.01) nal of Controlled Release, Elsevier, Amsterdam, NL, vol. 124, No. 3, Nov. 16, 2007 pp. 163-171. A6 IK3I/366 (2006.01) Hackett Michael J. et al., “Fatty acids as therapeutic auxiliaries for A6 IK 3/427 (2006.01) oral and parenteral formulations'. Advanced Drug Delivery A6 IK 3/4745 (2006.01) Reviews, vol. 65, No. 10, Aug. 17, 2012, pp. 1331-1339. A6 IK 3/475 (2006.01) A6 IK3I/5685 (2006.01) (Continued) A 6LX 3/575 (2006.01) A6 IK3I/704 (2006.01) Primary Examiner — Brian-Yong Kwon A6 IK3I/7056 (2006.01) Assistant Examiner — Mark V Stevens A6 IK3I/7068 (2006.01) (74) Attorney, Agent, or Firm — Sam L. Nguyen; A 6LX 3L/7076 (2006.01) Hamilton, DeSanctis & Cha, LLP A6 IK 45/06 (2006.01) A6 IK 47/48 (2006.01) (57) ABSTRACT A6 IK 9/00 (2006.01) In one embodiment, the present application discloses Syn A 6LX 9/5 (2006.01) thetic LDL nanoparticles comprising mixtures of compo A 6LX 3L/357 (2006.01) nents selected from the group consisting of phospholipids, (52) U.S. Cl. triglycerides, cholesterol ester and free cholesterol: option CPC ...... A6IK 9/1275 (2013.01); A61K 9/0019 ally further comprising an agent selected from the group (2013.01); A61 K9/5123 (2013.01); A61 K consisting of natural antioxidants, ubiquinol and vitamin E, 31/337 (2013.01); A61K3I/357 (2013.01); and methods for preparing the synthetic nanoparticles. The A6 IK3I/365 (2013.01); A61K 3 1/366 disclosed synthetic LDL nanoparticles are capable of selec (2013.01); A61 K3I/427 (2013.01); A61 K tively delivering lipophilic drugs and prodrugs to cellular 31/475 (2013.01); A61K 31/4745 (2013.01); targets expressing LDL receptors after intra venous injec A6 IK3I/5685 (2013.01); A61K 3 1/575 tion. (2013.01); A61 K3I/704 (2013.01); A61 K 3 1/7056 (2013.01); A61 K3I/7068 (2013.01); 10 Claims, 45 Drawing Sheets US 9,468,603 B2 Page 2
(56) References Cited Lundberg, B., "Preparation of Drug-Carrier Emulsion Stabilized with Phosphatidylcholine-Surfactant Mixtures”. Journal of Pharma ceutical Sciences, vol. 83, No. 1, 1994, 72-75. OTHER PUBLICATIONS Norvaisas P. et al. The Role of Payload Hydrophobicity in Nanotherapeutic Pharmacokinetics. Journal of Pharmaceutical Sci Nikanjam et al., “Synthetic nano-low density lipoprotein as targeted ences 2014; 103(7): 2147-56. Fayad W. et al., Identification of Agents that Induce Apoptosis of drug delivery vehicle for glioblastoma multiforme”. International Multicellular Tumour Spheroids: Enrichment for Mitotic Inhibitors Journal of Pharmaceutics 328 (2007) 86-94. with Hydrophobic Properties. Chemical Biology & Drug Design Lundberg et al., “A lipophilic paclitaxel derivative incorporated in 2011; 78(4):547-57. a lipid emulsion for parenteral administration'. Journal of Con trolled Release 86 (2003) 93-100. * cited by examiner U.S. Patent Oct. 18, 2016 Sheet 1 of 45 US 9,468,603 B2
Figure 1: Particle size reaches the 55-60 nm plateau after 40 discrete passes.
160
140
120 Filtration 100 s 8O wintensity
60 xx Number 40
s - - - C s Y 10 s 20 i. s s S s 0 or&s O O.O 20.0 40.O 6O.O 80.0 100.0 Fig.1. Number of passes
Figure 2: Particle size and stability of drug free formulation.
Particle size and stability of drug-free formulation.
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Figure 3: Cooling conditions allowed further decreasing of the particle size to 43 nm
Filtration
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Particle size and stability of drug-free firrituatics,
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Figure 5: Representative graph showing particle size reaches the plateau or resistance.
Lt C.S. &
15, d 1. 500 100,0 Filtrati 40. (mama Intensity s 80.0 * , alume
& O * Flumber O.
Figure 6: Representative graph showing different particle size and Stability over time.
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Time, Days U.S. Patent Oct. 18, 2016 Sheet 4 of 45 US 9,468,603 B2
Figure 7: Representative graph showing particle size at different temperatures.
OOO2.108.1
10 15 Fig. 8. Time, Days U.S. Patent Oct. 18, 2016 Sheet S of 45 US 9,468,603 B2
Figure 9: Representative graph showing change in particle size over time and temperature.
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Figure T 1: Representative graph showing relative particle size over processing time and temperatures
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Fig. 12. Time, Days U.S. Patent Oct. 18, 2016 Sheet 7 of 45 US 9,468,603 B2
Figure 13: Representative graph showing particle size over time and temperature
12O
1OO Filtration
8O
&N Interns ity 6 O x Volume Nsaw Number 4O
Fig.13. Processing time, min
w
Figure 14: Representative graph showing particle size and stability of ART-207 over time. Particle size and stability of ART-2O7 containing formulation. 80 70 60 50 x 40 30 nue -- OO2.111.2 20 10 on US 9,468,603 B2
Figure 16: Representative graph showing particle size and Stability of ART-207 formulation
&rlsrurrarvarvars rurrivers ravarvervs rurrivervs rurrive U.S. Patent Oct. 18, 2016 Sheet 9 of 45 US 9,468,603 B2
Figure 17: Representative graph showing particle size and processing times
6O Filtration
\ssa Inten sity 40 xxx Volume asa Number C
O 2O 40 Fig.17. Processing time, min
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Figure 18: Representative graph showing particle size and stability of drug-free formulations Particle size and stability of drug-free formulations.
ar.002.118.0 was OO2.118.00
Fig. 18. Time, Days
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Figure 19: Representative graph showing particle size over processing times and temperatures
Filtration
30
&N Intensit y 6 O 6O wVolume asa Number 4O 40
2 O
Fig.19. Processing time, min
Figure 20: Representative graph showing particle size and stability over time Particle size and stability of ART-207 containing formulation.
2OO
150
100 -O-OO2.119.4
Fig. 20. Time, Days U.S. Patent Oct. 18, 2016 Sheet 11 of 45 US 9,468,603 B2
Figure 21: Representative graph showing stability of drug-free and ART-207 containing formulations Stability of drug-free and ART- containing Tormulations shipped to/from MTD site (- - -) and retained by Arbor Therapeutics
O OO2-119-4
O02-119-3 -902-119-2 OO2-119-1
50 iss------OQ2-118-00
O 5 10 15 2O 25 Fig. 21. Days
Figure 22: Representative graph of effect of paclitaxel and formulated ART-207 on non-tumored
mouse weight Effect of Paclitaxel and formulated ART-207 on non-tumored mouse weight.
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Fig. 22. Days U.S. Patent Oct. 18, 2016 Sheet 12 of 45 US 9,468,603 B2
Figure 23: Representative graph of effect of paclitaxel and formulated ART-207 on tumored mouse weight
Effect of Paclitaxel and Formulated ART-207 on Tumored Mouse Weight.
12 15 18 21 24 27 30 33 36 39 DayS
Figure 24: Representative graph of paclitaxel and formulated ART-207 on tumor weight
Effect of Paclitaxel and Formulated ART 207 on Tumor Weight.
A. a. 9 -
2 l e - O e Y O e H U.S. Patent Oct. 18, 2016 Sheet 13 of 45 US 9,468,603 B2
Figure 25: Representative graph of mouse death rate in control and RX treated groups
Mouse death rate in control and Rx treated groups.
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Figure 26: Representative graph showing MF processing time
Filtration
1. OO s & Inters ity & Volume w Number - - - C
W
W U.S. Patent Oct. 18, 2016 Sheet 14 of 45 US 9,468,603 B2
Figure 27: Representative graph showing particle size and stability of ART-207 with formulation Particle size and stability of ART-207 containing formulation.
8 d N v -0-002122.0
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Figure 28: Representative graph showing particle size and processing times
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Figure 29: Representative graph showing particle size and stability of ART-207 containing formulation Particle size and stability of ART-207 containing formulation.
10-0- - - -
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Fig. 29. Time, Days & Figure 30: Representative graph showing particle size and processing time
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Figure 31: Representative graph showing particle size and stability of ART-207 containing formulation
Particle size and stability of ART-207 containing formulation.
5 O
4.O
3 O
2 O
1. O
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Figure 33: Representative graph showing particle size and stability of drug free formulation Particle size and stability of drug-free
formulation.
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O 2O AO 6O Fig. 33. Time, Days Figure 34: Representative graph showing particle size and processing times
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Figure 35: Representative graph showing particle size and stability of ART-207 containing formulation
U.S. Patent Oct. 18, 2016 Sheet 19 Of 45 US 9,468,603 B2
Figure 37: Representative graph showing particle size and stability of ART-207 containing formulation Particle size and stability of ART-207 containing formulation.
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Figure 38: Representative graph showing particle size and processing time U.S. Patent Oct. 18, 2016 Sheet 20 of 45 US 9,468,603 B2
U.S. Patent Oct. 18, 2016 Sheet 21 of 45 US 9,468,603 B2
Figure 41: Representative graph showing particle size and stability of ART-207 containing formulation Particle size and stability of ART-207 containing formulation.
OO2.137.41 as as a OO2.137.42
Figure 42: Representative graph showing particle size and processing time
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Figure 43: Representative graph showing particle size and stability of drug free formulation
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Figure 44: Representative graph showing particle size and processing time U.S. Patent Oct. 18, 2016 Sheet 23 of 45 US 9,468,603 B2
Figure 45: Representative graph showing particle size and stability of ART-207 containing formulation
Particle size and stability of ART-207 containing formulation.
W. OO2.140.22 -HOO2.140.23 ---- OO2.140.24
1. O 2 O 3 O
Fig. 45. Time, Days
Figure 46: Representative graph showing particle size and processing time
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8O
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saxSas Intensity Ssssssssssss SS saw Volume N Number
XXY SS crysawstorysws
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Figure 47: Representative graph showing particle size and stability of drug-free formulation Particle size and stability of drug-free formulation.
× --OO2.151.5
40 60 Fig. 47. Time, Days W.*\YYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYY-\YYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYY-\YYYYYYY Figure 48: Representative graph showing particle size and processing time
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Figure 49: Representative graph showing particle size and stability of ART-207 containing formulation Particle size and stability of ART-207 containing formulation.
--OO2.151.8
10 30 40 50
Fig. 49. Time Days Figure 50: Representative graph showing particle size and processing time %>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>*
U.S. Patent Oct. 18, 2016 Sheet 26 of 45 US 9,468,603 B2
Figure 51: Representative graph showing particle size and stability of ART-207 containing formulation Particle size and stability of ART-207 containing w formulation. S S
w S S
w
w S S
w S S S
w S S
w 20 AO 60 S S
Fig. 51. Time, Days S
Figure 52: Representative graph of particle size and processing time
15 45 75 90 Fig.52. Processing time, min U.S. Patent Oct. 18, 2016 Sheet 27 Of 45 US 9,468,603 B2
Figure 53: Representative graph showing particle size and stability of ART-207 containing formulation sy Particle size and stability of ART-207 containing w formulation. S S S
w
S S
w
S S - OO2.153.2 S
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S S
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Figure 54: Representative graph showing particle size and stability of drug-free and ART-207
containing formulatio ºzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzzº Particle size and stability of drug-free and ART-207 containing formulations.
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AO 60 Fig. 54. Time, Days U.S. Patent Oct. 18, 2016 Sheet 28 of 45 US 9,468,603 B2
Figure 55: Representative graph showing effect of paclitaxel and formulated ART-207 on tumor growth Effect of Paclitaxel and Formulated ART 207 on Tumor
Figure 56: Representative graph showing effect of paclitaxel and formulated ART-207 on tumored mouse weight
Effect of Paclitaxel and Formulated ART-207 on Tumored Mouse Weight.
d
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Figure 57: Representative graph of mouse death rate in control and Rx treated groups Mouse death rate in control and Rx treated groups.
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Effect of Paclitaxel and Formulated ART-207 on
Tumored Mouse Weight U.S. Patent Oct. 18, 2016 Sheet 30 of 45 US 9,468,603 B2
Figure 59: Effect of formulated ART-207 on tumor weight Advanced Tumor. Effect of Formulated ART-207 on Tumor Weight. 6909 ally rarely rarely rarely rarely rarely st
Figure 60a; Representative graph showing effect of paclitaxel and formulated ART-207 on tumor
weight Effect of Paclitaxel and Formulated ART-207 on Tumor Weight.
Z. m
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Figure 60b: Representative graphs showing effect of paclitaxel and formulated ART-207 on tumored mouse weight Effect of Paclitaxel and Formulated ART-207 on Tumored Mouse Weight.
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Filtration
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Fig.61. Processing time, min U.S. Patent Oct. 18, 2016 Sheet 32 of 45 US 9,468,603 B2
Figure 62: Representative graph showing particle size and Stability of ART-207 containing formulation Particle size and stability of ART-207 containing formulation.
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Figure 64: Representative graph showing particle size and stability of ART-207 containing formulation.
Particle size and stability of ART-207 containing formulation.
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Figure 65: Representative graphs showing results of treatment of tumored and non-tumored mice
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Figure 66: Representative graph of plasma levels of paclitaxel
Plasma levels of Paclitaxel after Paclitaxel administration into non-tumored and tumored mice. 100000 as 5. 90OOO 80000 E 70000 e m 6OOOO O 50000 4. 4OOOO $ Non-Tumored ss 3OOOO & Tumored 20000 E 10000 A. O 8 Time, hrs
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S Non-Tumored 8; Tumored
Time, hrs U.S. Patent Oct. 18, 2016 Sheet 35 of 45 US 9,468,603 B2
Figure 68: Concentration of paclitaxel in tissues of non-tumored mice injected with paclitaxel or formulated ART-207 Concentrations of Paclitaxel in Tissues of Non-Tumored Mice Injected with Paclitaxelor Formulated ART-207.
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Paclitaxel
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Figure 70: Representative graph showing tumor concentrations of paclitaxel in tumored mice injected with palcitaxel or formulated ART-207. Tumor Concentrations of Paclitaxel in Tumored Mice Injected with Paclitaxel or Formulated ART-207, respectively.
& Paclitaxel SART-2O7
Tumor
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& Volume N Number C U.S. Patent Oct. 18, 2016 Sheet 37 Of 45 US 9,468,603 B2
Figure 72: Representative graph showing particle size and stability of ART-207 containing formulation Particle size and stability of ART-207 containing formulation.
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Figure 74: Representative graphs showing particle size and stability of ART-207 containing formulation Particle size and stability of ART-207 containing formulation.
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Figure 75: Representative graphs showing article size and processing time in minutes
120 Filtration
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Figure 76: Representative graph showing particle size and stability with time in days Particle size and stability of ART-207 containing s8 formulation. 3
O 10 2O 30 40 S. Figure 77: Representative graph showing Effect of TG/ART-207 ratio on ART-207 incorporation and particle stability
Effect of TG/ART-207 ratio on ART-207 incorporation ( ) and particle stability (- - -) 120 c 300
O.O O.5 1.0 1.5 2.0 2.5 3.0 Fig. 77. TG/ART-207 Ratio U.S. Patent Oct. 18, 2016 Sheet 40 of 45 US 9,468,603 B2
Figure 78: Dependence of particle size and stability on processing temperatures Dependence of the particle size and stability on processing temperature. 80 s. TGART-207 ratioSl-Os-l-6s 25% 75 2 O 70 65 15 60 in-u in 10 55 -- ParticleSize 50 5 45 y-axy... O - - ParticlePartiro Szci s 40 X increase, % A. 5 35 ry 30 seasyayaxaraxacaxa&x -10 O 2O 40 60 3O
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8
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U.S. Patent Oct. 18, 2016 Sheet 42 of 45 US 9,468,603 B2
Figure 82: Representative graph showing particle size and stability of ART-287 containing formulation with time in days. Particle size and stability of ART-287 containing formulation.
40 as as as as as as as as a salts as as as a d N
30 t --005.13.1 a 20 assays...a...a...a...a...assassassyaya.
10 N------w------Y------wx------w------wx------wx------w-x-x-x-xx-x-x-x-x-x-x-x-x-x-xx-x-x-x-x-x-x-x-x-x-x-x-x---
O Ray U.S. Patent Oct. 18, 2016 Sheet 43 of 45 US 9,468,603 B2
Acid Labile Lipophilic Molecular Conjugates
ar “Y”
COCC -ALL OALL'O OALLO, O O
CCNS O l, O-ALL ALLMC of aclarubicin O-ALL
C
ALL2-O F
ALLMC of camptothecin ALLMC of gemcitabine -ALL NH2 "\ My N y s N N C l 2NN
O OALL3 O ALL" "O ALL2 w 'i O-ALL OALL O-ALL
ALLMC of pentostatin ALLMC of amrubicin ALLMC of cladribine NH2
O-ALL o-s, O -ALL
O-ALL
ALLMC of cytarabine ALLMC of docetaxel ALLMC of elliptinium acetate
ART 449; ART 448; ART473; ART 471; ART472; ART 470; ART 489; ART 488, ART 467, ART 332 and ART 441. wherein-ALL', -ALL, -ALL and -ALL" are each independently hydrogen or an acid labile lipophilic group, provided that at least one of -ALL', -ALL, -ALL and -ALL" is an acid labile lipophilic group. Figure 83 U.S. Patent Oct. 18, 2016 Sheet 44 of 45 US 9,468,603 B2
Acid Labile Lipophilic Molecular Conjugates O
A-l All YuO
ÖAc ALLMC of a 10-alpha taxane analog (19C)
ALLMC of a 10-beta abeo-taxane analog (19q) ALLMC of a 10-beta abeo-taxane analog (19h) wherein-ALL, -ALL, -ALL and -ALL are each independently hydrogen or an acid labile lipophilic group, provided that at least one of -ALL', -ALL, -ALL and -ALL" is an acid labile lipophilic group. Figure 84 U.S. Patent Oct. 18, 2016 Sheet 45 of 45 US 9,468,603 B2
Acid Labile Lipophilic Molecular Conjugates
O-AI
ALLMC of epirubicin
O O-ALL ALL-O
H ALL-O CG)YCH)-SO-CH-)-cif. HC O AL4-o12 ALLMC of formestane ALLMC of fulvestrant
V ALL"-O CF ALL2 O ALLMC of pirarubicin ALLMC of topotecan ALLMC of walrubicin
wherein-ALL', -ALL, -ALL and -ALL" are each independently hydrogen or an acid labile lipophilic group, provided that at least one of -ALL". -ALL, -ALL and -ALL" is an acid labile lipophilic group.
Figure 85 US 9,468,603 B2 1. 2 NANOPARTICULATE COMPOSITIONS FOR Requisite Particle Characteristics: TARGETED DELIVERY OF ACID LABILE, In one aspect, the advantageous disposition of these LIPOPHILIC PRODRUGS OF CANCER particles may be attributed to the particle's size, shape, CHEMOTHERAPEUTICS AND THEIR composition and charge. In one aspect, the particles may be PREPARATION Substantially spherical to move Smoothly through the cap illaries and may have a narrow size distribution with a mean RELATED APPLICATION of 60 nm. In one aspect, the size distribution range is about 40 to 80 nm. The composition may include cholesterol, other This application claims the benefit of U.S. Provisional lipids and Surface-active agents with or without the addition 10 of polymers used to define particle structure. In one embodi Application No. 61/877,521 filed Sep. 13, 2013. ment, a positive surface charge is achieved with the use of cationic Surface active agents. A fundamental characteristic SUMMARY of small particles creates inherent instability. As particle size The present invention describes compositions and pro goes down, the interfacial area per unit mass of the dispersed 15 system increases, and so does interfacial energy. This cesses used to create structured Submicron particles (nano increased energy will tend to drive the particles to coalesce, particles) suitable for drug delivery. The structure of the forming larger particles with lower total energy. particles prepared according to the methods disclosed herein Extreme particle size reduction can result in significant results in desirable biological and physical performance. In increases in drug solubility. Materials in a nanoparticle have one aspect, the structure of the particles is determined by the a much higher tendency to leave the particle and go into the selection of the formulation components and of the process Surrounding solution than those in a larger particle of the ing steps used to create the particles. Structural elements that same composition. This phenomenon can increase the avail determine particle performance include particle size (and ability of drug for transport across a biological membrane, size distribution in the population), particle shape, particle but it can also create physical instability of the nanoparticle charge and the distribution of individual components in the 25 itself. This instability is seen in Ostwald ripening in which particle, especially those at the particle Surface. Small particles disappear as material is transferred to large Particle Performance particles. The physical stability of nanoparticles may be Nanoparticles have particular advantages in parenteral improved by the use of appropriate Surface active agents and drug delivery. Nanoparticles are smaller than blood cells and excipients at the right levels to reduce the interfacial energy, can be suspended and transported by the blood to various 30 controlling the Surface charge of the particles to maintain the tissues of the body. Because they are smaller than microbes, dispersion, and manufacturing the particles in a narrow size they will pass through filters used to terminally sterilize distribution to reduce Ostwald ripening. parenteral products. Since the rapidly expanding vasculature In one embodiment, the high drug load in the particles of of tumors is inherently leaky, nanoparticles leave the cap the present application is achieved by creating a particle core illaries and are trapped in the poorly flushed extracellular 35 of lipophilic inactive components which will dissolve the space of tumors. drug or its lipophilic pro-drug and reduce its tendency to The nanoparticles included in the present application have leave the nanoparticle until it is released intracellularly at the unique biological performance. In one aspect, the nanopar target site. ticles of the present application are referred to as synthetic Particle Production: low density lipoprotein (LDL) nanoparticle, LDL-like nano 40 Very significant challenges are presented in the creation of particles or lipid emulsion (LDE), in part because the particles with the proper size, structure, charge and stability. nanoparticles are prepared without any protein. Upon Homogeneous nanoparticles can be created by either pre administration, these particles are not recognized as foreign, cipitation of nanoparticle material from Solution; or reducing i.e., they are not coated with proteins which trigger clearance the size of larger particles. Heterogeneous structured nano processes in the tissues of the reticuloendothelial system. 45 particles are not easily made by precipitation techniques Moreover, these particles were coated with a component that because of the improbability of a single physical system inhibits opSonization and phagocytosis. In fact, these par precipitating all of the components into the requisite struc ticles are recognized as endogenous constituents and receive ture. a coat of apo proteins that are recognized by receptors Particle size reduction requires energy; this energy is expressed on the surface of cells in need of cholesterol. After 50 necessary to disrupt the forces holding the molecules of bulk coating, these particles are preferentially taken up by lipo components together and to increase the interfacial contact protein receptor mediated endocytosis by cells with high area between the particle and the Surrounding medium. The cholesterol need. Such cells include those of rapidly divid energy must come from the process used to create the ing tissues, especially solid and liquid tumor tissue. nanoparticles. To be useful, any process for the production In one embodiment, the structured nanoparticles of the 55 of nanoparticles by size reduction, i.e., the system for present application are designed to carry a useful drug load imparting energy to the bulk formulation, must be control in a parenterally administered drug product. Drugs of par lable and scalable. Techniques demonstrated to be useful in ticular interest with respect to this delivery system are those nanoparticle production include ultrasonication and high drugs which have low or extremely low solubility in water pressure homogenization. Ultrasonic energy, in the form of due to high lipophilicity. In one embodiment, drugs to be 60 mechanical oscillations at greater than 20,000 Hz, has been delivered which are not sufficiently lipophilic to be suitable used to reduce particle size in fluids. The high-frequency for delivery in the nanoparticles of the present application mechanical oscillations in the fluid cause the rapid formation may be made suitable by formation of highly lipophilic and collapse of microscopic vacuum bubbles (cavitation). derivatives which can serve as pro-drugs of the drugs to be The high-velocity local mass transfer in this process imparts delivered. These particles are designed to be sufficiently 65 extremely high shear on the fluid and on Suspended particles. chemically and physically stable in a manufactured drug High shear can also be generated in flowing systems by product to allow a commercially adequate shelf life. forcing fluids at very high-pressure through an orifice or into US 9,468,603 B2 3 4 a very narrow passage. Different processing equipment are delivery to cancer cells has had limited success due to the known in the art and are available which uses either ultra large size of antibodies (MW=125-150 kilodaltons or KD) Sonication and or high-pressure homogenization working on and thus their relative inability to penetrate solid tumors. An a flowing stream of process fluid with sufficient control alternative strategy comprises the use of Smaller targeting parameters to optimize the process. ligands and peptides, which recognize specific receptors Synthetic LDL Nanoparticles (sLDL): unique to or overexpressed on tumor cells, as the targeting In one embodiment, the invention provides for synthetic vector. Such constructs have molecular weights of 2-6 KD, LDL nanoparticles comprising a lipid composition Sufi which allow ready penetration throughout solid tumors. ciently similar to the normal human LDL particle compo sition to be recognized by the body as “natural’. By being 10 Increased cell proliferation and growth is a trademark of recognized by the body as “natural these synthetic LDL cancer. The increase in cellular proliferation is associated nanoparticles become effective for selective delivery of with high turnover of cell cholesterol. Cells requiring cho lipophilic drugs or prodrugs to LDL receptor expressing lesterol for membrane synthesis and growth may acquire tissues, especially tumor tissues which overexpess LDL cholesterol by receptor mediated endocytosis of plasma low receptors. The synthetic LDL nanoparticles are produced as density lipoproteins (LDL), the major transporter of choles described herein or may be made by methods known to those terol in the blood, or by de novo synthesis. LDL is taken up of skill in the art. As used herein, “synthetic' means made into cells by a receptor known as the LDL receptor (LDLR): by chemical synthesis. the LDL along with the receptor is endocytosed and trans In one embodiment, the mean LDL nanoparticle size is 60 ported into the cells in endosomes. The endoSomes become nm, but can be 40 nm to 100 nm. In some embodiments, the acidified and this releases the LDL receptor from the LDL: LDL nanoparticle is between 50 nm to 60 nm in size. The the LDL receptor recycles to the surface where it can drugs or pro-drugs to be delivered can be complexed with participate in additional uptake of LDL particles. There is a microemulsions of a particular weight ratio of phospholipids body of evidence that suggests that tumors in a variety of (PL), triglyceride (TG) and cholesteryl ester (CE). In one tissues have a high requirement for LDL to the extent that embodiment the PL:TG:CE ratio is 36:5:1. In some embodi 25 ments, the phospholipid is egg yolk phosphatidylcholine plasma LDLs are depleted. The increased import of LDL (PC) or 1,2-dimyristoyl-sn-glycero-3-phosphorylcholine into cancerous cells is thought to be due to elevated LDL (DMPC). Suitable triglycerides that can be used include but receptors (LDLR) in these tumors. Some tumors known to are not limited to, soybean oil (SO), triolein (TO) and express high numbers of LDLRs include some forms of tripalmitate, and mixtures thereof. Suitable cholesteryl 30 leukemia, lung tumors, colorectal tumors and ovarian can esters that can be used include but are not limited to, cer. In vivo studies showed that LDLRs do appear in brain cholesteryl oleate (CO) or cholesteryl palmitate (CP), or malignancies. Leppala et al used PET imaging, and demon mixtures thereof. In another embodiment, the composition strated that 99mTc LDL localizes in human brain tumors in of the core lipids, CE and TG are altered in order to alter the vivo but not in normal brain. size of the emulsion. In another embodiment, the ratio of 35 This suggests that the LDL receptor is a potential unique lipids is 30:6:1 weight ratio of PL:TG:CE. Other ratios that molecular target in GBM and other malignancies for the can be used include PL:TG:CE of 37:7:1 and PL:TG:CE of delivery of anti-tumor drugs via LDL particles. A test of this 20:3:0.5. In some embodiments, the cholesteryl ester is possibility was undertaken by Maranhao and coworkers. A omitted from the microemulsion. In some embodiments the protein-free microemulsion (LDE) with a lipid composition drugs or pro-drugs can be complexed with microemulsions 40 resembling that of low-density lipoprotein (LDL) was used of PL and TG in the ratio of 7:1. in metabolic studies in rats to compare LDE with the native The microemulsions can be made by extrusion of the lipoprotein. Incubation studies also showed that LDE incor lipids through a nanometer filter, such as a 30 nm filter. For porates a variety of apolipoproteins, including apo E, a example, the lipids are sonicated at 40°C. in the presence of ligand for recognition of lipoproteins by specific receptors. 20 uM BHT and N for sufficient time (e.g., about 1 hour), 45 Lipophilic Derivatives of Cancer Chemotherapeutic Agents: then extruded through a filter or a series of filters to obtain lipid particles having a suitable size. In one embodiment, the Arbor Therapeutics has developed unique lipophilic microemulsion is extruded through a 0.1 um (100 nm) filter, derivatives of the cancer chemotherapeutic agent which then a 0.03 um (30 nm) filter and isolated. In another have high Stability in normal systemic circulation and reten embodiment, the composition can be made using a micro 50 tion in the lipid core of the LDL particles but readily release fluidizer apparatus. the active chemotherapeutic agent in the acidic environment of the endosome. See U.S. Pat. No. 8,440,714, the disclosure BACKGROUND of which is incorporated herein in its entirety. In another embodiment, there is provided a active che Targeted cancer therapies that can selectively kill cancer 55 motherapeutic compounds of the formula 3a or 3b: cells without harming other cells in the body would repre sent a major improvement in the clinical treatment of cancer. It would be highly desirable to develop a strategy to directly 3a target cancer cells with chemotherapeutic agents in cancer treatment regimens. This could lead to reduction or elimi 60 nation of toxic side effects, more efficient delivery of the drug to the targeted site, and reduction in dosage of the administered drug and a resulting decrease in toxicity to healthy cells and in the cost of the chemotherapeutic regi men. Reports of targeting chemotherapeutic drugs using 65 antibodies have appeared in the literature since 1958. Tar ON geting drugs by conjugation to antibodies for selective US 9,468,603 B2 5 6 -continued from the group consisting of mesylates, Sulfonates and 3b halogen (C1, Br and I); and their isolated enantiomers, diastereoisomers or mixtures thereof, or a pharmaceutically acceptable salt thereof. The compound 1.1 includes the pure syn isomer, the pure anti isomer and mixtures of Syn- and anti-isomers, and their diastereomers. In another embodiment, there is provided the above acid labile lipophilic molecular conjugate of the formula 1 or 1.1 10 wherein: R is a hydroxyl bearing cancer chemotherapeutic ON agent; R' is hydrogen, C-C alkyl or Cs-C alkyl; R is Cs-C alkyl: Y is O or S. Z is O: Q is O; and T is O. In one wherein: R' is hydrogen, C-C alkyl or Cs-C2 alkyl; R is aspect of the acid labile lipophilic molecular conjugate of Cs-C alkyl: Y is selected from O, NR' or S wherein R' is 15 the formula 2 wherein: R is Cs-C alkyl: T is O; and X is hydrogen or selected from the group consisting of Cl, Brand hydrogen or C-C alkyl; Z is selected from O or S.; Q is O I. In another variation, R is Co-C. In another aspect of the or S; and T is O or S. In one aspect of the compound, R' is above acid labile lipophilic molecular conjugate comprising hydrogen or C-C alkyl; R is Cs-C alkyl: Y is O or S. Z. the formula 1a, 1b or formula 2a: is O: Q is O; and T is O. The activated compound of the formula 3a or 3b may be used to prepare the acid labile lipophilic conjugate when the activated compound is con la densed with a hydroxyl bearing cancer chemotherapeutic R1 R2 agent (HBCCA). As defined herein, the HBCCA is repre sented generically with the residue or group “R” in the 25 X, formulae 1, 1a, 1b, 1.1, 2 and 2a, for example, and where the HBCCA is not coupled to form the acid labile, lipophilic R1 os-N-- molecular conjugates, then the HBCCA may also be generi O cally represented as having the formula “R-OH' since the 30 1b HBCCA may be functionalized by one or more hydroxyl RI (—OH) groups. In one embodiment, there is provided an acid labile O lipophilic molecular conjugate (ALLMC) of the formula 1, O O 1.1 or formula 2: 35 R1 r O 2a X X, 40
wherein: R is a hydroxyl bearing cancer chemotherapeutic --- 45 agent (HBCCA); for formula 1a or 1b R' is hydrogen, C-C, 1.1 alkyl or Cs-C2 alkyl; and R is Cs-C2 alkyl; and for formula 2a: R is C-C2 alkyl; and X is hydrogen or is selected from the group consisting of Cl, Brand I. In one 50 variation of the compound that is the carbonate (i.e., —OC (O)O—) of the formula 1a or 1b the compound is the corresponding sulfonate (i.e., —OS(O)O ) of the formula 1a wherein the carbonate group is replaced by a Sulfonate group. The compound 1b includes the pure Syn isomer, the 55 pure anti isomer and mixtures of syn and anti isomers, and their diastereomers. In another variation of the compound of the formula 1, 2, 1a and 2a, R is hydrogen or C-C alkylor Cs-C2 alkyl, and 60 R’ is the carbon residue of an unsaturated fatty acid, such as the carbon residue selected from the group consisting of the wherein: R is a hydroxyl bearing cancer chemotherapeutic C, residue of eicosenoic acid (including the cis isomer, agent; for formula 1 or 1.1 R' is hydrogen, C-C alkyl or trans isomer and mixtures of isomers), C, residue of oleic Cs-C alkyl; R is Cs-C alkyl: Y is selected from O, NR acid and the C7 residue of elaidic acid. As used herein, the or S wherein R is hydrogen or C-C alkyl; Z is O or S.; Q 65 “carbon residue” (e.g., C, residue, Co residue etc . . . ) of is O or S; and T is O or S; for formula 2: R is a C-C alkyl: the fatty acid means the carbon chain of the fatty acids T is O or S; and X is hydrogen or a leaving group selected excluding the carboxyl carbon. US 9,468,603 B2 7 In another aspect of the above acid labile lipophilic molecular conjugate, the hydroxyl bearing cancer chemo la therapeutic agent is selected from the group consisting of R1 R2 taxanes, abeo-taxanes, camptothecins, epothilones, cucurbi tacins, quassinoids, anthracyclines, and their analogs and 5 -X, derivatives. In another aspect of the above acid labile lipophilic molecular conjugate, the hydroxyl bearing cancer R1 os-N-- chemotherapeutic agent is selected from the group consist ing of aclarubicin, camptothecin, masoprocol, paclitaxel, O 10 1b pentostatin, amrubicin, cladribine, cytarabine, docetaxel, RI gemcitabine, elliptinium acetate, epirubicin, etoposide, formestane, fulvestrant, idarubicin, pirarubicin, topotecan, O- R2 valrubicin and vinblastine. In one embodiment, there is provided an acid labile 15 O O S. lipophilic molecular conjugate (ALLMC) of the formula 1, 1.1 or formula 2: R1 O 2a X
R2 X, R No 1. O1 25 wherein: R is a hydroxyl bearing cancer chemotherapeutic agent (HBCCA); for formula 1a or 1b R' is hydrogen, C-C, --- alkyl or Cs-C2 alkyl; and R is Cs-C2 alkyl; and for 1.1 formula 2a: R is C-C alkyl; and X is hydrogen or is 30 selected from the group consisting of Cl, Brand I. In one variation of the compound that is the carbonate (i.e., —OC (O)O—) of the formula 1a or 1b the compound is the corresponding sulfonate (i.e., —OS(O)O ) of the formula 1a wherein the carbonate group is replaced by a Sulfonate 35 group. The compound 1b includes the pure Syn isomer, the pure anti isomer and mixtures of syn and anti isomers, and their diastereomers. In another variation of the compound of the formula 1, 2, 1a and 2a, R is hydrogen or C-C alkylor Cs-C2 alkyl, and 40 R’ is the carbon residue of an unsaturated fatty acid, such as the carbon residue selected from the group consisting of the C, residue of eicosenoic acid (including the cis isomer, trans isomer and mixtures of isomers), C, residue of oleic wherein: R is a hydroxyl bearing cancer chemotherapeutic acid and the C7 residue of elaidic acid. As used herein, the agent; for formula 1 or 1.1 R' is hydrogen, C-C alkyl or 45 'carbon residue” (e.g., C, residue, Co residue etc . . . ) of Cs-C alkyl; R is C-C alkyl: Y is selected from O, NR' the fatty acid means the carbon chain of the fatty acids or S wherein R is hydrogen or C-C alkyl; Z is O or S.; Q excluding the carboxyl carbon. In another aspect of the is O or S; and T is O or S; for formula 2: R is a C-C alkyl: above acid labile lipophilic molecular conjugate, the T is O or S; and X is hydrogen or a leaving group selected hydroxyl bearing cancer chemotherapeutic agent is selected from the group consisting of mesylates, Sulfonates and 50 from the group consisting of taxanes, abeo-taxanes, camp halogen (C1, Br and I); and their isolated enantiomers, tothecins, epothilones, cucurbitacins, quassinoids, anthracy diastereoisomers or mixtures thereof, or a pharmaceutically clines, and their analogs and derivatives. In another aspect of acceptable salt thereof. The compound 1.1 includes the pure the above acid labile lipophilic molecular conjugate, the syn isomer, the pure anti isomer and mixtures of Syn- and hydroxyl bearing cancer chemotherapeutic agent is selected anti-isomers, and their diastereomers. 55 from the group consisting of aclarubicin, camptothecin, masoprocol, paclitaxel, pentostatin, amrubicin, cladribine, In another embodiment, there is provided the above acid cytarabine, docetaxel, gemcitabine, elliptinium acetate, epi labile lipophilic molecular conjugate of the formula 1 or 1.1 rubicin, etoposide, formestane, fulvestrant, idarubicin, pira wherein: R is a hydroxyl bearing cancer chemotherapeutic rubicin, topotecan, valrubicin and vinblastine. In another agent; R' is hydrogen, C-C alkyl or Cs-C alkyl; R is 60 aspect of the above acid labile lipophilic molecular conju Cs-C alkyl: Y is O or S. Z is O; Q is O; and T is O. In one gate, the conjugate is selected from the compounds in FIGS. aspect of the acid labile lipophilic molecular conjugate of 18, 19 and 20. In one variation, only one of the groups the formula 2 wherein: R is C-C alkyl: T is O; and X is -ALL". -ALL. -ALL . . . to -ALL" is an -ALL group and hydrogen or selected from the group consisting of Cl, Brand the others are hydrogens. In another variation, two of the I. In another variation, R is Co-C2. In another aspect of the 65 groups -ALL". -ALL. -ALL... to -ALL" are -ALL groups. above acid labile lipophilic molecular conjugate comprising In another embodiment, there is provided a pharmaceu the formula 1a, 1b or formula 2a: tical composition comprising: a) a therapeutically effective US 9,468,603 B2 10 amount of a compound of the above, in the form of a single includes the pure Syn isomer, the pure anti isomer and diastereoisomer, and b) a pharmaceutically acceptable mixtures of syn and anti isomers, and their diastereomers. In excipient. In another aspect, the pharmaceutical composition one variation of the above, R is Co-C alkyl. In one aspect, is adapted for oral administration; or as a liquid formulation the method provides a higher concentration of the cancer adapted for parenteral administration. In another aspect, the 5 chemotherapeutic agent in a cancer cell of the patient. In composition is adapted for administration by a route selected another aspect, the method delivers a higher concentration of from the group consisting of orally, parenterally, intraperi the cancer chemotherapeutic agent in the cancer cell, when toneally, intravenously, intraarterial, transdermally, intra compared to the administration of a non-conjugated cancer muscularly, rectally, intranasally, liposomally, Subcutane chemotherapeutic agent to the patient, by at least 5%, 10%, ously and intrathecally. In another embodiment, there is 1 O 20%, 30%, 40% or at least 50%. provided a method for the treatment of cancer in a patient In another embodiment, there is provided a compound of comprising administering to the patient a therapeutically the formula 3a or 3b: effective amount of a compound or composition of any of the above compound or composition, to a patient in need of 5 Such treatment. In one aspect of the method, the cancer is 3a selected from the group consisting of leukemia, neuroblas R1 R2 toma, glioblastoma, cervical, colorectal, pancreatic, renal and melanoma. In another aspect of the method, the cancer is selected from the group consisting of lung, breast, pros QX, tate, ovarian and head and neck. In another aspect of the method, the method provides at least a 10%, 20%, 30%, 40%, or at least a 50% diminished degree of resistance ----Y expressed by the cancer cells when compared with the ON non-conjugated hydroxyl bearing cancer chemotherapeutic 25 3b agent. R1 In another embodiment, there is provided a method for reducing or Substantially eliminating the side effects of s R2 chemotherapy associated with the administration of a cancer T chemotherapeutic agent to a patient, the method comprising administering to the patient a therapeutically effective O C amount of an acid labile lipophilic molecular conjugate of the formula 1, 1.1 or formula 2: Orr 35 wherein: R' is hydrogen, C-C alkyl or Cs-C2 alkyl; R is C-C alkyl: Y is selected from O, NR' or S wherein R' X, is hydrogen or C-C alkyl; Z is selected from O or S.; Q is 40 O or S; and T is O or S. In one aspect of the compound, R' is hydrogen or C-C alkyl; R is Cs-C alkyl: Y is O or S; Z is O: Q is O; and T is O. The activated compound of the --- formula 3a or 3b may be used to prepare the acid labile 1.1 lipophilic conjugate when the activated compound is con 45 densed with a hydroxyl bearing cancer chemotherapeutic agent (HBCCA). As defined herein, the HBCCA is repre sented generically with the residue or group “R” in the formulae 1, 1a, 1b, 1.1, 2 and 2a, for example, and where the HBCCA is not coupled to form the acid labile, lipophilic 50 molecular conjugates, then the HBCCA may also be repre sented as having the formula "R-OH since the HBCCA may be functionalized by one or more hydroxyl (—OH) groups. 55 Similarly, the acid labile lipophilic group (i.e., the “-ALL group of the activated compound) that may be condensed with a HBCCA to form the acid labile, lipophilic molecular conjugate generically represented as "R-O-ALL. Accord wherein: R is a hydroxyl bearing cancer chemotherapeutic ingly, where more than one -ALL group is condensed or agent; for formula 1 or 1.1: R' is hydrogen, C-C alkyl or 60 conjugated with a HBCCA group, then each -ALL Cs-C alkyl; R is C-C alkyl: Y is selected from O, NR' group may be independently designated as -ALL". -ALL, or S wherein R is hydrogen or C-C alkyl; Z is O or S.; Q -ALL . . . to -ALL" where n is the number of available is O or S; and T is O or S; for formula 2: R is C-C alkyl; hydroxyl groups on the cancer chemotherapeutic agent that T is O or S; and X is hydrogen or a leaving group selected may be conjugated or couple with an -ALL group. As from the group consisting of mesylates, Sulfonates and 65 exemplified for the compound of formulae 1 and 2, for halogen (C1, Br and I); and their isolated enantiomers, example, the HBCCA and the -ALL groups as designated, diastereoisomers or mixtures thereof. The compound 1.1 are shown below. US 9,468,603 B2
10
15 ART-2O7
HBCCA -- Capturing the great potential of selective and specific 1 R-O T delivery of chemotherapeutic compounds to cancer tissues via their over expression of LDL receptors and consequent high uptake of LDL particles from the systemic circulation, An example of an acid labile, lipophilic molecular con requires that the cancer chemotherapeutic agent have high jugate (ALLMC), where the HBCCA group is paclitaxel lipophilicity So as to remain entrapped in the lipid core of the having two -ALL groups, is depicted below: LDL particle and not diffuse into the plasma to lead to toxic 25 side effects from exposure of normal tissues to the agent. Further, once the LDL particle with its chemotherapeutic payload has entered the cancer cell via LDL receptor medi AcO ated uptake into the acidic environment of the endosome, the 30 LDL receptor is disassociated from the LDL particle and is recycled to the cell surface and the LDL particle releases its lipid contents and its lipophilic chemotherapeutic agent to the enzymes and acidic environment of the endosome. Further validity of this expectation was shown by Mara 35 nhao and coworkers who demonstrated that a cholesterol rich microemulsion or nanoparticle preparation (LDE) con centrates in cancer tissues after injection into the bloodstream. The cytotoxicity, pharmacokinetics, toxicity to Acid Labile Lipophilic Molecular Conjugate of animals and therapeutic action of a paclitaxel lipophilic Paclitaxel 40 derivative associated to LDE were compared with those of commercial paclitaxel. Results showed that LDE-paclitaxel In the above representative example of the acid labile oleate was stable. The cytostatic activity of the drug in the molecular conjugate of paclitaxel, each of the -ALL" and complex was diminished compared with the commercial -ALL is independently hydrogen or an -ALL group as paclitaxel due to the cytotoxicity of the vehicle Cremophor defined herein. For HBCCA groups having more than one 45 EL used in the commercial formulation. Competition experi hydroxyl groups, the inaccessible hydroxyl group or groups ments in neoplastic cultured cells showed that paclitaxel where the acid labile lipophilic group cannot be formed, then oleate and LDE are internalized together by the LDL recep the group that is designated as an -ALL group(s) is hydro tor pathway. Tolerability to mice was remarkable, such that gen. the lethal dose (LDs) was nine fold greater than that of the 50 commercial formulation (LD50–326 uM and 37 uM, respec In another aspect of the above acid labile lipophilic tively). LDE concentrates paclitaxel oleate in the tumor molecular conjugate, the hydroxyl bearing cancer chemo roughly fourfold relative to the normal adjacent tissues. At therapeutic agent is selected from the group consisting of equimolar doses, the association of paclitaxel oleate with taxanes, abeo-taxanes, camptothecins, epothilones, cucurbi LDE resulted in remarkable changes in the drug pharma tacins, quassinoids, anthracyclines, and their analogs and 55 cokinetic parameters when compared to commercial pacli derivatives. In another aspect of the above acid labile taxel (t=218 min and 184 min, AUC=1,334 ug-h/mL and lipophilic molecular conjugate, the hydroxyl bearing cancer 707 ug-h/mL and CL=0.125 mL/min and 0.236 mL/min, chemotherapeutic agent is selected from the group consist respectively). The therapeutic efficacy of the complex was ing of aclarubicin, camptothecin, masoprocol, paclitaxel, pronouncedly greater than that of the commercial paclitaxel, pentostatin, amrubicin, cladribine, cytarabine, docetaxel, 60 as indicated by the reduction in tumor growth, increase in gemcitabine, elliptinium acetate, epirubicin, etoposide, survival rates and % cure of treated mice. Maranhao et al formestane, fulvestrant, idarubicin, pirarubicin, topotecan, showed LDE-paclitaxel oleate is a stable complex and valrubicin and vinblastine. compared with paclitaxel, toxicity is considerably reduced Representative chemotherapeutic agents that may be and activity is enhanced which may lead to improved employed in the present composition or formulations are 65 therapeutic index in clinical use. Maranhao and coworkers disclosed in Figures A, B and C. In one aspect of the above, followed up their preliminary animal studies with a pilot the chemotherapeutic agent is ART-207. clinical study in breast cancer patients. The clinical study US 9,468,603 B2 13 14 was performed in breast cancer patients to evaluate the dimyristoyl-sn-glycero-3-phosphorylethanolamine, egg tumoral uptake, pharmacokinetics and toxicity of paclitaxel phospholipids, egg phosphatidylglycerol, dipalmitoylphos associated to LDE nanoemulsions. Twenty-four hours phatidylglycerol, egg lecithin, soy lecithin, lecithin (NOS) before mastectomy H-paclitaxel oleate associated with 'C- and mixtures thereof. cholesteryl oleate-nanoemulsion or H-paclitaxel in Cremo 5 In another aspect, the LDL nanoparticle further comprises phor EL were injected into five patients for collection of cholesterol ester (CE) or cholesterol (C), or mixtures of blood samples and fragments of tumor and normal breast cholesterol ester and cholesterol. In another aspect, the tissue. A pilot clinical study of paclitaxel-nanoemulsion cholesterol ester is selected from the group consisting of administered at 3-week intervals was performed in four C-2 esters of cholesterol, cholesterol and mixtures breast cancer patients with refractory advanced disease at 10 175 and 220 mg/m dose levels. The half-life (t) of thereof, and the triglycerides is selected from the group paclitaxel oleate associated to the nanoemulsion was longer consisting of Soybean oil, triolein, glyceryl tripalmitate and than that of paclitaxel (t=15.4+4.7 and 3.5+0.80h, respec mixtures thereof. In one aspect of the above, the esters of tively). Uptake of the ''C-cholesteryl ester nanoemulsion cholesterol is selected from the group consisting of choles and H-paclitaxel oleate by breast malignant tissue was 15 teryl oleate, cholesteryl palmatate, cholesteryl Stearate and threefold greater than the normal breast tissue and toxicity cholesteryl lenolenate. In another aspect, the LDL nanopar was minimal at the two dose levels. Their results suggest that ticle further comprises an agent selected from the group the paclitaxel-nanoemulsion preparation can be advanta consisting of triolein, natural antioxidants, BHT, ubiquinol, geous for use in the treatment of breast cancer because the ubiquinol 10, Vitamin E, alpha-tocopherol, gamma-tocoph pharmacokinetic parameters are improved, the drug is con erol, lycopene, retinyl derivative and betacarotene, or mix centrated in the neoplastic tissue and the toxicity of pacli tures thereof. In another aspect, the lipophilic anti-cancer taxel is reduced. Additional reports from the Maranhão agent is an anti-cancer agent or a prodrug of the anti-cancer laboratory of small human trials with the LDL-like lipid agent. In one aspect of the above, the ratio of PL:TG may emulsion show that lipophilic drugs incorporated into the range from 8:1 to 3:1. In another aspect, the ratio of core of the emulsion are targeted to tumor tissue and side 25 PL:TG:CE may range from 8:1:0.5 to 3:1:0.1. In another effects are significantly reduced. The difficulty of prepara aspect, the ratio of the lipophilic anti-cancer agent:PL:TG tion of the emulsion, manufacture by long term Sonication may range from 1:10:3 to 1:3:0.5. In another aspect, the ratio and extended centrifugation for particle size selection pre of the lipophilic anti-cancer agent:PL:TG:CE may range cluded them from further clinical exploration and develop from 1:10:3:1 to 1:3:0.5:0.1 ment. 30 In another aspect, the anti-cancer agent is selected from We have discovered how to prepare a nanoparticulate the group consisting of a taxane, abeo-taxane, camptothecin, “pseudo LDL lipid microemulsion as a delivery formula epothilone, cucurbitacin, quassinoid and an anthracycline. In tion for Sufficiently lipophilic chemotherapeutics, including another aspect, the anticancer agent is selected from the our unique acid labile, lipophilic prodrug derivative of the group consisting of aclarubicin, camptothecin, masoprocol, cancer chemotherapeutic agent. In one embodiment, the 35 lipophilic chemotherapeutic agents have a measured or paclitaxel, pentostatin, amrubicin, cladribine, cytarabine, calculated Log P of greater than 4. We further demonstrate docetaxel, gemcitabine, elliptinium acetate, epirubicin, in animal tumor models that the acid labile, lipophilic etoposide, formestane, fulvestrant, idarubicin, pirarubicin, molecular conjugates of cancer chemotherapeutic agent topotecan, valrubicin and vinblastine. In yet another aspect, when dosed in a nanoparticulate, LDL-like lipid emulsion, is 40 the pro-drug of the anti-cancer agent is an acid labile more useful for tumor reduction due to reduced toxicity and lipophilic molecular conjugates is as disclosed herein, and in greater efficacy due to selective delivery to neoplastic/tumor Figures A, B and C. In one particular aspect, the acid labile tissue. lipophilic molecular conjugates is ART-207. In one embodiment, the application discloses a stable, synthetic low density lipoprotein (LDL) nanoparticle com 45 prising: a) a lipophilic anti-cancer agent; b) phospholipids
(PL); and c) triglycerides (TG); wherein the LDL nanopar AcO O ticle has a particle size less than 100 nm, less than 90 nm or less than 80 nm. As referred to herein, a stable synthetic low density lipoprotein (LDL) nanoparticle is a nanoparticle as 50 defined herein that has a shelf life at about 25°C. of greater than 90 days, greater than 120 days, greater than 180 days, or greater than 1 year when stored in a sealed container and away from exposure to light. In another aspect, the nano particle has a shelf life at about 25°C. that is more than 1 55 year, or about 2 years or more when stored in a sealed container and away from exposure to light. In one aspect if the LDL nanoparticle, the particle size distribution is between 40 to 80 nm. In another aspect, the particle size distribution is between 50 and 60 nm. In one aspect of the 60 above, the LDL nanoparticle has a mean size distribution of ART-2O7 60 nm. In another aspect, the LDL nanoparticle has a mean size distribution of about 50 nm. In another aspect, the phospholipids is selected from the group consisting of phosphotidylcholine, phosphotidylethanolamine, symmetric 65 In another aspect of the above, the lipophilic anti-cancer or asymmetric 1,2-diacyl-sn-glycero-3-phosphorylcholines, agent has a log P greater than 4.0, 6.0 or 8.0. In one aspect, 1.2-dimyristoyl-sn-glycero-3-phosphorylcholine, 1.2- the weight ratio of PL:TG:CE:C ranges from 73:12:2:1 to US 9,468,603 B2 15 16 78:12:2:1, optionally further comprising an additive selected In another embodiment, there is provided a method for the from the group consisting of triolein, natural antioxidants, treatment of cancer in a patient comprising administering to BHT, ubiquinol, ubiquinol 10, vitamin E, alpha-tocopherol, the patient a therapeutically effective amount of the stable, gamma-tocopherol, lycopene, retinyl derivative and betac synthetic low density lipoprotein (LDL) nanoparticle of any arotene, or mixtures thereof. In one variation, the weight one of the above embodiments, aspects and variations, to a ratio of PL:TG:CE:C is 77:10:2:1. In one aspect, the natural patient in need of Such treatment. In another aspect of the antioxidant is selected from Coenzyme Q10, resveratrol, method, the cancer is selected from the group consisting of pterostilbene and mixtures thereof. In another aspect, the leukemia, neuroblastoma, glioblastoma, cervical, colorectal, ratio of the lipophilic anti-cancer agent to the triglyceride is 10 pancreatic, renal melanoma, lung, breast, prostate, ovarian from 1:1 to 0.6:1. In another aspect, the LDL nanoparticle and head and neck. contains a total solids content of 6.0 to 8% wt/wt. In another aspect, the LDL nanoparticle contains a total lipid content of 5.0 to 7.0% wit/wt. In one variation, the LDL nanoparticle DETAILED DESCRIPTION OF THE further comprises a poloxamer selected from the group 15 INVENTION consisting of P188, P237, P338, P407, SYNPERONICS, PLURONICS and KOLLIPHOR, or mixtures thereof. In another embodiment, there is provided a process for Brief Description of the Figures preparing a stable, synthetic low density lipoprotein (LDL) nanoparticle comprising: a) a lipophilic anti-cancer agent; b) phospholipids (PL); and c) triglycerides (TG); the process FIG. 1 is a representative plot showing that particle size comprising: 1) combining the lipophilic anti-cancer agent, reaches the 55-60 nm plateau after 40 discrete passes that is phospholipids and triglycerides to form a mixture; 2) equal to 20 min of processing. 25 FIG. 2 is a representative plot showing particle size homogenizing the mixture by dissolution in a volatile sol increased from 63 to 79 nm over 36 days. vent; 3) removing the solvent; 4) forming a coarse emulsion FIG. 3 is a representative graph showing cooling condi by blending of the mixture in a buffer to form an emulsion tions to allow further decreasing of the particle size to 43 mixture; 5) microfluidizing the emulsion mixture in a micro . fluidizer apparatus for a sufficient amount of time to produce 30 FIG. 4 is a representative graph showing a particle size a particle preparation of 100 nm or less; and 6) sterilizing the analysis of drug-free formulation where the resultant particle nanoparticle preparation through a 0.22 micron filter to size was significantly smaller after processing at <30° C. obtain the synthetic LDL nanoparticles with a range of 40 temperatures compare to processing at 60° C. nm to 80 nm. In onve variation, the synthetic low density FIG. 5 is a representative graph showing particle size lipoprotein (LDL) nanoparticle mixture wherein the phos 35 reaches the plateau or resistance (Resistance 1—R1) at ~130 pholipids is selected from the group consisting of phospho nm after 30 min of processing at 10-20° C. tidylcholine, phosphotidylethanolamine, symmetric or FIG. 6 is a representative graph showing different particle asymmetric 1,2-diacyl-sn-glycero-3-phosphorylcholines, size and stability over time. 1.2-dimyristoyl-sn-glycero-3-phosphorylcholine, 1.2- FIG. 7 is a representative graph showing particle size at dimyristoyl-sn-glycero-3-phosphorylethanolamine, egg 40 different temperatures. phospholipids, egg phosphatidylglycerol, dipalmitoylphos FIG. 8 is a representative graph showing particle size phatidylglycerol, egg lecithin, soy lecithin, lecithin (NOS) increase over time. FIG. 9 is a representative graph showing change in and mixtures thereof. In another aspect, the LDL nanopar particle size over time and temperature. ticle further comprises cholesterol (C) or cholesterol ester 45 (CE) selected from the group consisting of C-2 esters of FIG. 10: Representative graph showing stability of ART cholesterol, cholesterol and mixtures thereof, and the tri 207 formulation over time. glycerides is selected from the group consisting of soybean FIG. 11 is a representative graph showing relative particle oil, triolein, glyceryl tripalmitate and mixtures thereof; or size over processing time and temperatures. mixtures of cholesterol and cholesterol esters. In one aspect 50 FIG. 12 is a representative graph showing particle size of the above, the slow speed blending is performed at a and stability over time. speed of between 200 and 800 rpm, about 200 rpm, 400 rpm, FIG. 13 is a representative graph showing particle size 600 rpm or 800 rpm. In another aspect, the microfluidizing over time and temperature. of the warm coarse emulsion mixture is performed at a 55 FIG. 14 is a representative graph showing particle size processing temperature of about 45 to 65° C. In another and stability of ART-207 over time. aspect of the above process, the solvent is removed in FIG. 15 is a representative graph showing particle size vacuum. In another embodiment, there is provided a stable, over time and temperature. synthetic low density lipoprotein (LDL) nanoparticle com FIG. 16 is a representative graph showing particle size prising: a) a lipophilic anti-cancer agent; b) phospholipids 60 and stability of ART-207 formulation. (PL); and c) triglycerides (TG) prepared by the process as FIG. 17 is a representative graph showing particle size disclosed herein. In one embodiment of the above, the and processing times. synthetic LDL nanoparticle is prepared by any of the dis FIG. 18 is a representative graph showing particle size closed process, wherein the LDL nanoparticle becomes 65 and stability of drug-free formulations. coated with apolipoprotein upon intra venous injection and FIG. 19 is a representative graph showing particle size are recognized and internalized by cellular LDL receptors. over processing times and temperatures. US 9,468,603 B2 17 18 FIG. 20 is a representative graph showing particle size FIG. 53 is a representative graph showing particle size and stability over time. and stability of ART-207 containing formulation. FIG. 21 is a representative graph showing stability of FIG. 54 is a representative graph showing particle size drug-free and ART-207 containing formulations. and stability of drug-free and ART-207 containing formula FIG. 22 is a representative graph of effect of paclitaxel tion. and formulated ART-207 on non-tumored mouse weight. FIG. 55 is a representative graph showing effect of FIG. 23 is a representative graph of effect of paclitaxel paclitaxel and formulated ART-207 on tumor growth. and formulated ART-207 on tumored mouse weight. FIG. 56 is a representative graph showing effect of FIG. 24 is a representative graph of paclitaxel and for paclitaxel and formulated ART-207 on tumored mouse mulated ART-207 on tumor weight. 10 weight. FIG. 25 is a representative graph of mouse death rate in FIG. 57 is a representative graph of mouse death rate in control and RX treated groups. control and RX treated groups. FIG. 26 is a representative graph showing MF processing FIG. 58 is a representative graph showing effect of time. paclitaxel and formulated ART-207 on tumored mouse 15 weight. FIG. 27 is a representative graph showing particle size FIG. 59 is a representative graph showing the effect of and stability of ART-207 with formulation formulated ART-207 on tumor weight. FIG. 28 is a representative graph showing particle size FIG. 60a is a representative graph showing effect of and processing times. paclitaxel and formulated ART-207 on tumor weight. FIG. 29 is a representative graph showing particle size FIG. 60b is a representative graphs showing effect of and stability of ART-207 containing formulation. paclitaxel and formulated ART-207 on tumored mouse FIG. 30 is a representative graph showing particle size weight. and processing time. FIG. 61 is a representative graphs showing particle size FIG. 31 is a representative graph showing particle size and processing time. and stability of ART-207 containing formulation. 25 FIG. 62 is a representative graph showing particle size FIG. 32 is a representative graph showing particle size and stability of ART-207 containing formulation. over processing time. FIG. 63 is a representative graph showing particle size FIG. 33 is a representative graph showing particle size and processing time. and stability of drug free formulation. FIG. 64 is a representative graph showing particle size FIG. 34 is a representative graph showing particle size 30 and stability of ART-207 containing formulation. and processing times. FIG. 65 is a representative graphs showing results of FIG. 35 is a representative graph showing particle size treatment of tumored and non-tumored mice and stability of ART-207 containing formulation. FIG. 66 is a representative graph of plasma levels of FIG. 36 is a representative graph showing particle size paclitaxel. and processing time. 35 FIG. 67 is a representative graph showing plasma levels FIG. 37 is a representative graph showing particle size of ART-207 after administration. and stability of ART-207 containing formulation. FIG. 68 is a representative graph showing concentration FIG. 38 is a representative graph showing particle size of paclitaxel in tissues of non-tumored mice injected with and processing time. paclitaxel or formulated ART-207. FIG. 39 is a representative graph showing particle size 40 FIG. 69 is a representative graph showing concentration and stability of ART-207 containing formulation. of paclitaxel in tissues in tumored mice injected with pacli FIG. 40 is a representative graph showing particle size taxel or formulated ART-207. and processing time. FIG. 70 is a representative graph showing tumor concen FIG. 41: Representative graph showing particle size and trations of paclitaxel in tumored mice injected with palci stability of ART-207 containing formulation. 45 taxel or formulated ART-207. FIG. 42 is a representative graph showing particle size FIG. 71 is a representative graph showing particle size and processing time. and processing time. FIG. 43 is a representative graph showing particle size FIG. 72 is a representative graph showing particle size and stability of drug free formulation. and stability of ART-207 containing formulation. FIG. 44 is a representative graph showing particle size 50 FIG. 73 is a representative graph showing particle size and processing time. and processing time. FIG. 45 is a representative graph showing particle size FIG. 74 is a representative graphs showing particle size and stability of ART-207 containing formulation. and stability of ART-207 containing formulation. FIG. 46 is a representative graph showing particle size FIG. 75 is a representative graphs showing article size and and processing time. 55 processing time in minutes. FIG. 47 is a representative graph showing particle size FIG. 76 is a representative graph showing particle size and stability of drug-free formulation. and stability with time in days. FIG. 48 is a representative graph showing particle size FIG. 77 is a representative graph showing Effect of and processing time. TG/ART-207 ratio on ART-207 incorporation and particle FIG. 49 is a representative graph showing particle size 60 stability. and stability of ART-207 containing formulation. FIG. 78 is a representative graph showing dependence of FIG. 50 is a representative graph showing particle size particle size and stability on processing temperatures. and processing time. FIG. 79 is a representative graph showing particle size on FIG. 51 is a representative graph showing particle size processing time and temperature. and stability of ART-207 containing formulation. 65 FIG. 80 is a representative graph showing particle size FIG. 52 is a representative graph of particle size and and stability of ART-287 containing formulation with time in processing time. days. US 9,468,603 B2 19 20 FIG. 81 is a representative graph showing particle size All of the log P calculations for the anti-cancer agents and temperature with time in minutes. noted in the table below were done online at www.chemi FIG. 82 is a representative graph showing particle size calize.org which uses the log P predictor from ChemAxon. and stability of ART-287 containing formulation with time in The ChemAxon algorithm is based on: Vellarkad N. Viswa days. 5 nadhan et al., Journal of Chemical Information and Com FIG. 83 describes some embodiments of the Acid Labile puter Sciences 1989 29 (3), 163-172. Lipophilic Molecular Conjugates. FIG. 84 describes some embodiments of the Acid Labile Lipophilic Molecular Conjugates. Anti-Cancer Anti-Cancer FIG. 85 describes some embodiments of the Acid Labile 10 Agents ogP Approved Agents LogP Approved Lipophilic Molecular Conjugates. ART-153 24.61 na ART-137 6.22 DEVELOPMENT OF LIPID-BASED DRUG AND ART-164 2.91 na Mitotane 6.11 Adrenal PRO-DRUG FORMULATIONS ART-1S2 2.86 na Epothilone D 5.10 15 ART-209 2.56 na Epothillone C 4.85 Optimization of Drug/ProDrug incorporation capacity, ART-163 2.19 na Masoprocol 4.76 particle size and stability. General procedures for prepara ART-151 2.04 na Cabozantinib 4.66 Thyroid tion of nanoparticulate lipid based “pseudo LDL formula ART-2O7 1.68 na Lapatinib 4.64 Breast tions are found in Arbor Therapeutics, LLC Standard Oper ART-156 1.42 na ART-448 453 ating Procedures: ART 001 Coarse Emulsion Preparation ART-185 140 na Valirubicin 4.49 Bladder Rev. 1, ART 002 Microfluidics Model 110P Gen II (MF) Rev ART-161 1.31 na ART-287. 441 1, and ART 003 Nicomp 380 ZLS Particle Size Analyses ART-467 1.15 na Imatinib 4.38 Leukemias ART-2O8 1.OS na Sorafenib 4.34 Liver, Kidney, Rev. 1. Exceptions to these SOPs are noted. Thyroid Abbreviations: PC phosphatidylcholine, TG triglycer ART-162 O.69 na Epothillone 4.21 ides, TC total cholesterol, FC free cholesterol, CE— 25 ART-449 9.77 na Cabazitaxel 4.20 Prostate esterified cholesterol, U Ubiquinol, VitF. Vitamin E ART-441 9.32 na Carfilzomib 4.20 Multiple (mixed tocopherols), P188 Poloxamer 188, DMPC -1,2- myeloma Dimyristoyl-sn-glycero-3-phosphorylcholine, PS Phos Gossypol 8.02 Vinblastine 4.18 Breast, photidyl serine; MFP microfluidizer processing, IC in myeloma, teraction chamber, ICJ interaction chamber jacket; 30 testicular TSPM total solids premix: REM resultant emulsion; Fulvestrant 7.57 Breast Axitinib 4.15 Kidney % percent of total solids, TL. 96 percent of total lipids, Everolimus 7.40 Kidney Epothilone B 4.12 W/V weight to volume; Recovery, 96 percent of ART ART-332 7.19 Bosutinib 4.09 CML 207 recovered in formulation after microfluidizer processing Temsirolimus 7.13 Kidney and sterile 0.22 um filtration; mfg. manufacturing; Pre 35 mix pre-mixture; ND not determined; BDL below detection limit. R—Resistance. Materials Used:
Reagent Vendor Part Number Lot Number Phosphatidylcholine Lipoid, GMBH Lipoid E 80 O32718-121052 Mfg. Date July 2010 Retest Date July 2013 DMPS (1,2-Dimyristoyl-sn-Glycero Avanti Polar Lipids, Inc. 840033P 4OPS-83 3-Phospho-L-Serine) (Sodium Salt) Soybean Oil (triglycerides) Crisco Pure Vegetable Oil O9S420 Soybean oil Best Before 5 Apr. 2013 Cholesterol, 95% Alfa Aesar A11470 L23WO24 Cholesterol Oleate (cholesterol ester) Alfa Aesar A-11378 GO3YO33 Vitamin E, mixed tocopherols Swanson (AMD) SW152 legible Lot #, Mfg. Date uly 2012 Ubiquinol, co-enzyme Q Kaneka Corporation Kaneka QHTM FBO2-0104 Polaxamer P-188 (Pluronic F68; Spectrum Chemicals P 1169 2AKO895 Polyethylene-Polypropylene Glycol) CAS 90OO3-11-6 DMPC (1,2-dimyristoyl-sn-glycero Avanti Polar Lipids, Inc. 850345P 4OPC-261 3-phosphorylcholine) ART-2O7 Arbor Therapeutics, LLC ART-207 AWOO1 - 243 AWOO4-13 AWOO4-24 ART 287 Arbor Therapeutics, LLC ART 287 Sodium Chloride, ACS Alfa Aesar 12314 L12XO71 Distilled Water Kroger Grocery Distilled Water NA Methylene Chloride, ACS Stabilized Fisher Scientific L-14119 125544 Nitrogen Nex Air HP NI-3 3904713-10-4 Glacial Acetic Acid, ACS Reagents, Inc. S-1 OO60-3 1004NE Sterile Filters, 0.22 im, PES Fisher Scientific 50 mL 09-741-88 NA 150 mL 09-741-01 NA 500 mL 09-761-107 NA Cuvettes, polystyrene 4.5 mL Fisher Scientific 149SS 12S NA US 9,468,603 B2 22 Equipment Used: -continued
A = Acetonitrile B = % 0.01M HPO. Water Taxane Prodrug 7 Taxane Prodrug.M Taxane Prodrug Short.M min.M Description Manufacturer Model Number 5 Injection Volume Injection Volume Injection Volume 2 || 1 IL 20 IL 400 gram balance Denver Instrument SI-403 100 gram balance Denver Instrument APX-100 Min. 96 A 96 B Min. % A % B Min. 96. A 96 B Emersion Hand Blender Oster (R) 2605 Variable Voltage Control Glas-Col (R) 104APL12O2 11 50 50 7 50 50 8 50 50 MicroFluidizer (R) MicroFluidics, Inc. M11OP GEN II 13 50 50 9 50 50 10 50 50 Particle Sizer Particle Sizing Systems NicompTM 380 ZLS 10 HPLC Agilent HP 1100 Series Taxane Prodrug.M was used to determine linearity of response which was shown to be R=0.9997 for ART-207 Analytical Quantitation of ART-207: The Analytical Method Development for Quantitation of from 0.6 to 5.2 mg/mL. ART-207 concentration in LDL like lipid emulsion nano 15 Taxane Prodrug.M was also used to quantitate the emul particles was an evolutionary process over approximately 18 sion prepared for the In-Vitro Cytotoxicity Study. months. All Taxane Prodrug.M methods showed carryover of Summary of Methods Used and Method Changes: emulsion components from a previous injection to the Taxane Prodrug.M. Variable sized neat emulsion injec subsequent injections. Part of this carryover co-eluted with tions: 13 Aug. 2012 Calibration Curve/Response Linearity the ART-207 peak. Buffer blanks between samples helped a 17 Jan. 2013 Dilution of emulsion samples 1:10 with IPA little. A 3 minute column wash method using 100 uL Taxane Test.M. Dilution of emulsion samples 1:10 with injections of 50/50 chloroform/methanol reduced the carry IPA, 1 uL injections: over considerably. Analyses of drug free formulations for the 25 MTD Study showed no drug presence in the analyzed samples that was not possible to demonstrate with previous analytical procedures. Blank Subtractions were not appro Rev.O 20 Jan. 2013 Higher column temperature 55° C. vs. 40° C. Rev.1 9 Feb. 2013 External Standard Preparation change to priate since emulsions had a peak eluting as the same time 100 mg/100 mL. 30 as ART-207. Rev.2 11 Mar. 2013 Injection Volume increase from 1 IL to 3 IL Rev.3 10 May 2013 Use of bracketing external standards and A new method was developed which solved the emulsion ART duplicate sample analyses defined in draft component carryover problem. The higher column tempera Standard Operating Procedure ture 55° C. vs. 40°C. may assist in liquefying and dissolving 005, HPLC Analysis of Formulated ART-207 the emulsion particles better, allowing them to be washed off 35 the column in the column wash. The gradient starts at higher Methods used to Quantitate ART-207 in emulsions prepared acetonitrile concentration and the gradient is shallower to for the following studies: provide for resolution of any impurities. Analyses of drug free emulsions may be performed with confidence in a “Below Detection Limit” statement of result. Taxane Prodrug.M 9 Oct. 2012 In-Vitro Study 40 Taxane Test..M., Rev.0 20 & 21 Jan. 2013 MTD Study The HPLC analytical method used to quantitate ART-207 Taxane Test..M., Rev.1 6 Mar. 2013 MTD Study in lipid emulsions is TAXANE TEST.M., Rev.0, performed Taxane Test..M., Rev.2 13 Mar. 2013 Efficacy Study on an Agilent 1100 quaternary pump and single wavelength Taxane Test..M., Rev.3 13 May 2013 PKPD and Particle Size ComparisonToxicity. system. The column is a Phenomenex 4.6x50 mm Luna 5u Efficacy Study 45 C18(2) 100A, part number 00B-4252-E0. The method con ditions are: flow rate: 1.5 mL/minute, detection: 230 nm, column temperature: 55° C., and injection volume: 1 uL. The three HPLC Methods used were similar. Emulsion The gradient table is as follows: samples were injected neat during this time. All methods used a Phenomenex 4.6x50mm Luna 5L C18(2) 100A, part 50 number 00B-4252-E0 column, flow rate: 1.5 mL/minute, Time, minutes % Acetonitrile % 0.01M HPO. Water detection: 230 nm, column temperature: 40° C., and injec O 75 25 tion volume: variable. 7 100 O The gradient tables and injection volumes for each of these 9 100 O 55 10 75 25 three methods are as follows: 11 75 25
A = Acetonitrile B = % 0.01M HPO. Water Taxane Prodrug 7 The typical retention time of ART-207 is +5.8 minutes in Taxane Prodrug.M Taxane Prodrug Short.M min.M this method. Lipid emulsion sample preparation is 1 part Injection Volume Injection Volume Injection Volume 60 emulsion into 9 parts isopropanol (1:10). Linearity of 2 IL 1 IL 20 ! response was shown to be R=0.9997 for ART-207 from 0.6 Min. 96 A 96 B Min. % A % B Min. 96 A 96 B to 5.2 mg/mL in a similar acetonitrile/water C18 method. Quantitation is accomplished by using a response factor O 50 50 O 50 50 O 50 50 1 50 50 1 50 50 1 50 50 calculated from an external standard. The data are shown in 3 100 O 3 100 O 3 100 O 65 the table. During the preparations of the lipid emulsions for 10 100 O 6 100 O 7 100 O this MTD study, unexplained fluctuations in the concentra tion of ART-207 were observed and investigated. The exter US 9,468,603 B2 23 24 nal standards were prepared using approximately 1 mg of Bracketing external standards and duplicate analyses of ART-207 dissolved in 1 mL of solvent. Accuracy and samples were used in analytical quantitation of ART-207 and consistency were improved when the external standard are defined in Standard Operating Procedure—ART 005, preparation was changed to 100 mg of ART-207 dissolved in HPLC 100 mL of solvent (Taxane Test.M. Rev.1). Taxane Test.M, Analysis of Formulated ART-207, Taxane Test.M. Rev.3. Rev.1 method was used to re-determine and revise concen Analytical Quantitation of 287 (Loth ISI-30052013-1). trations of ART-207 in emulsions prepared for the MTD Taxane Test.M. Rev 3" Analytical Method was used for study. The values reported initially (Taxane Test.M. Rev.0) Quantitation of ART 287 concentration in LDL like lipid and the more accurate re-determined values (Taxane Test...M. emulsion nanoparticles. Sterile 0.22 um filtration of resultant Rev.1) are shown in the table below. 10 emulsions did not significantly affect the ART-207 content in experiments described below. The particle size for all pre pared coarse Suspensions was always in the range 400-800 Taxane Test...M., Rev.0 Taxane Test..M., Rev.1 nm and did not affect the rate of particle size decrease during Original Concentration Recalculated MF processing. MF processing volume was 100 ml unless Lot Number Reported, mg/mL Concentration, mg/mL 15 specified. For all examples described below, see also Master OO2-119-1 2.01 2.26 Tables 1, 2 and 3. OO2-119-2 3.02 340 OO2-119-3 3.98 4.49 Experiment 1 OO2-119-4 6.13 6.91 Preparation of Drug-Free Lipid Emulsion Using On 11 Mar. 2013 the injection volume was increased from Original Formulation 1 LL to 3 ul to reduce the impact of injection to injection variability on the standard area counts used to determine To investigate effect of discrete passes VS recycling mode response factor as well as sample area counts, Taxane (return of the material into the feed reservoir) and controlled Test...M., Rev.2. (só0° C.) ICJ temperature on particle size. TABLE 1 a Formulation composition. Components Weighed, ng (per 100 ml
Date Lotti Material PC TG FC CE U Vit E ART P188 DMPC PS
17 Dec. 2012 OO2.102.1 TSPM 233O 1239 206 1S3 10 10 O O O O
35 TABLE 1b. Ratios of major formulation components. TG.ART- PCART 40 2O7 2O7 PCTG FCCE NA NA 1.88 1.35
Coarse Suspension was Prepared and MF Processed (Loti 002. 103.1). TABLE 1c Particle size, ART-207 content, and particle stability of resultant emulsion. Particle size ART-2O7 Formulation Stability Manufacturing by intensity, Content, Days past Particle size Date Lotti ill mg/ml Recovery, % mfg ill 18 Dec. 2012 OO2.103.1 63 NA NA 36 79
MFP. FIG. 1 shows that particle size reaches the 55-60 nm plateau after 40 discrete passes that is equal to 20 min of processing (one discrete pass is ~30 sec). MFP was stopped 60 after 80 passes and material filtered. Particle size slightly increased after 0.22 um filtration from 55 to 63 nm Process ing via discrete passes at ~60-65° C. (temperature of IC jacket) did not result in improvement of particle size relative to lipid formulations generated in prior studies. Particle size 65 analysis and Stability. The resultant emulsion was unstable. In FIG. 2 and Table 1 c, particle size increased from 63 to 79 nm over 36 days. US 9,468,603 B2 25 26 Experiment 2 Preparation of Drug-Free Lipid Emulsion Using Original Formulation 5 Investigate the effect of lower (<30°C.) ICJ temperature (i.e. effect of “local cooling of IC jacket) on resultant particle size. TABLE 2a Formulation composition. Components Weighed, ng (per 100 ml
Date Lotti Material PC TG FC CE U Vit E ART P188 DMPC PS
17 Dec. 2012 OO2.102.2 TSPM 2338 1247 203 152 10 10 O O O O
TABLE 2b Ratios for major formulation components. TG.ART- PCART 2O7 2O7 PCTG FCCE NA NA 1.87 1.34 25 Coarse emulsion was prepared and MF processed (loti 002.104.1). MF processing was performed in recycling mode. TABLE 2c Particle size, ART-207 content, and particle stability of resultant emulsion. Particle size ART-2O7 Formulation Stability Manufacturing by intensity, Content, Days past Particle size Date Lotti ill mg/ml Recovery, % mfg ill
19 Dec. 2012 OO2.104.1 45 NA NA 35 82
40 2a. Ice cubes were placed around IC jacket. After 10 min Smaller after processing at <30°C. temperatures compare to of MF processing the particle size dropped from 439 nm processing at 60° C. Both processing conditions resulted in (coarse emulsion) to 66 nm, and in 20 min it reached a unstable particles. 63 to 79 and 45 to 82 nm particle size plateau at ~58 nm. Further processing to 30 min did not increase was observed in loti 002. 103.1 and 002. 104.1, decrease the particle size (FIG. 3). 45 respectively. 2b. To achieve better contact of cooling agent with IC and to further lower its temperature, ice was removed from lower Experiment 3 tray and glycol bath set to 10° C. was used to submerge interaction chamber (with Surrounding pipelines and back Preparation of ART-207 Containing Lipid Emulsion pressure chamber) in 10° C. glycol. These cooling condi 50 Using Original Formulation tions allowed further decreasing of the particle size to 43 nm (FIG. 3). Investigating ART-207 (Loti AW-001-243) incorporation Particle size analysis of drug-free formulation. In FIG. 4, capacity of original formulation and effect of ART-207 on Table 1 c, and 2c, the resultant particle size was significantly particle size and stability. TABLE 3a
Formulation composition.
Components Weighed, mg (per 100 ml)
Date Lotti Material PC TG FC CE U VitE ART P188 DMPC PS
18 Dec. 2012 OO2.103.2 TSPM 2176 1145 184 141 10 10 32O O O O US 9,468,603 B2 27 28 TABLE 3b Ratio for maior formulation components. TG.ART- PCART 2O7 2O7 PCTG FCCE
3.58 6.8O 1.90 1.30
Coarse Suspension was prepared and MF processed (loth 002.105.1). TABLE 3c Particle size, ART-207 content, and particle stability of resultant emulsion. Particle size ART-2O7 Formulation Stability Manufacturing by intensity, Content, Days past Particle size Date Lotti ill mg/ml Recovery, % mfg ill
19 Dec. 2012 OO2.105.1 82 3.14 98.1 35 196
MFP. In FIG. 5, particle size reaches the plateau or Addition of ART-207 to formulation notably decreases resistance (Resistance 1—R1) at ~130 nm after 30 min of stability of resultant emulsion. processing at 10-20°C. Raising the temperature to 50-60° C. resulted in lowering particle size to 100 nm and reaching R2. Lowering the temperature back to 10-20° C. resulted in 25 further particle size decrease to 77 nm MFP was stopped and Experiment 4 material was filtered. Slight increase of particle size from 77 to 82 nm was observed after filtration. HPLC Analysis. ART-207 content in resultant emulsion Preparation of ART-207 (Loti AW-001-243) determined by HPLC (Taxane Prodrug.M) was 3.14 mg/ml. 30 Data indicate that 98% of the drug used for preparation of Containing Lipid Emulsion this formulation was incorporated into lipid particles (Table 3c). Investigating the effect of increased phospholipid content Particle size analysis of ART-207 containing formulation. The resultant ART-207 containing emulsion was unstable. and decreased FC/CE ratio while keeping the amount of TC FIG. 6 and Table 3c show that particle size increased from 35 the same (table 4b and 4a) on drug incorporation capacity 82 to 196 nm over 35 days. and stability of resultant nanoparticles. TABLE 4a Formulation composition. Components Weighed, ng (per 100 ml
Date Lotti Material PC TG FC CE U Vit E ART P188 DMPC PS
9 Jan 2013 OO2.107.2 TSPM 4157 1397 7O 347 10 10 648 O O O
TABLE 4b. Ratios for major formulation components. 50 TG.ART-2O7 PC.ART-2O7 PCTG FCfCE
2.16 6.42 2.98 O.2O
Coarse Suspension was prepared and MF processed (loth 002.108.1). TABLE 4c Particle size, ART-207 content, and particle stability of resultant emulsion. Particle size ART-2O7 Formulation Stability Manufacturing by intensity, Content, Recovery, Days past Particle size Date Lotti ill mg/ml % mfg ill
9 Jan 2013 OO2.108.1 129 4.81 74.2 14 184 US 9,468,603 B2 29 30 MFP MFP. In FIG. 9, particle size reaches R1 at ~105 nm after In FIG. 7, particle size reaches R1 at ~125 nm after 40 min 30 min of processing at ~60° C. Lowering the temperature of processing at ~60° C. Lowering the temperature to -20° to -20° C. resulted in lowering particle size to 73 nm and C. resulted in lowering particle size to 80 nm and reaching R2. Raising the temperature to 50° C. resulted in particle 5 reaching R2. MFP was stopped and filtered. No increase of size increase to ~108 nm MFP was stopped and filtered. particle size was observed after filtration. Increase of particle size from 108 to 129 nm was observed HPLC Analysis. ART-207 content in resultant emulsion after filtration. determined by HPLC (Taxane Prodrug.M) was 5.5 mg/ml. HPLC Analysis. ART-207 content in resultant emulsion Data indicate that 85% of the drug used for preparation of determined by HPLC (Taxane Prodrug.M) was 4.81 mg/ml. 10 Data indicate that 74% of the drug used for preparation of this formulation was incorporated into lipid particles (Table this formulation was incorporated into lipid particles (Table 5c). The resultant drug content of the particles was higher 4c). Particle size analysis of ART-207 containing formula than in previous experiment indicating that increase of tion. The resultant ART-207 containing emulsion was coating material (phospholipid) facilitates drug incorpora unstable. In FIG. 8 and Table 4c, particle size increased from 15 tion (see Tables 3a, 3c., 4a, and 4c). 129 to 184 nm over 14 days. Increased phospholipid content and decreased FC/CE Particle size analysis of ART-207 containing formulation. ratio resulted in higher ART-207 particle content but did not Although the resultant particle size was significantly smaller improve stability of resultant emulsion. (Table 5c) compared to previous experiment (Table 4c), the emulsion was extremely unstable. In FIG. 10 and Table 5c, Experiment 5 particle size increased from 74 to 149 nm over 14 days and from 74 to 169 nm over 33 days. Preparation of ART-207 (Loti AW-001-243) Further increase of phospholipid and decrease of CE Containing Lipid Emulsion 25 and Subsequently TC content resulted in Smaller particles Investigate effect of further increase of phospholipid and higher (relative to previous experiment #4) capacity concentration and decrease of CE on drug incorporation of the formulation to incorporate ART-207 but did not capacity and stability of resultant nanoparticles; and B. To improve stability of resultant emulsion. A repeat of this optimize the temperature control strategy: raising the tem experiment gave similar results which indicate the process perature from 20° C. to 60° C. in experiment #4 resulted in 30 ing parameters are reproducible and give reproducible out undesirable increase of the particle size. COCS. TABLE 5a
Formulation composition.
Components Weighed, mg (per 100 ml)
Date Lotfi Material PC TG FC CE U Vit E ART P188 DMPC PS
9 Jan. 2013 OO2.108.2 TSPM SO2S 1397 71 21 6 10 10 648 O O O
TABLE 5b 45 Experiment 6 Ratios for major formulation components. Preparation of ART-207 (Loti AW-001-243) TG ART-2O7 PC.ART-2O7 PCTG FCICE Containing Lipid Emulsion 2.16 7.75 3.60 O.33 50 Increased ART-207 content results in decreased stability of the resultant particles, experiment #6 was performed to Coarse Suspension was prepared and MF processed determine the effect of lowering ART-207 concentration on (loth 002. 109.1). particle size and stability. TABLE 5c
Particle size, ART-207 content, and particle stability of resultant emulsion.
Particle size ART-2O7 Formulation Stability
Manufacturing by intensity, Content, Days past Particle size Date Lotti ill mg/ml Recovery, % mfg ill
9 Jan 2013 OO2.109.1 74 5.50 84.9 33 169 US 9,468,603 B2 31 32 TABLE 6a Formulation composition. Components Weighed, ng (per 100 ml
Date Lotti Material PC TG FC CE U Vit E ART P188 DMPC PS
10 Jan. 2013 OO2.109.3 TSPM S17O 1392 7O 213 10 1 O 213 O O O
10 TABLE 6b Ratio for major formulation components. TG ART-2O7 PC.ART-2O7 PCTG FCICE 15 6.54 24.27 3.71 O.33
Coarse Suspension was prepared and MF processed (loti 002.111.1). TABLE 6c Particle size, ART-207 content, and particle stability of resultant emulsion. Particle size ART-2O7 Formulation Stability Manufacturing by intensity, Content, Recovery, Days past Particle Date Lotti ill mg/ml % mfg size, nm 11 Jan 2013 OO2.1111 47 186 87.3 24 66
30 MFP particle size compared to the size previously attained in In FIG. 11, particle size reaches R1 at ~66 nm after 20 min experiment #5 (see also tables 5a,b,c and 6a,b, c), and b) of processing at ~60° C. Lowering the temperature to -20° more stable particles (relative to emulsions obtained in C. resulted in lowering particle size to 42 nm MFP was previous experiments). stopped and was filtered. Increase of particle size from 42 to 35 47 nm was observed after filtration. HPLC Analysis. ART-207 content in resultant emulsion determined by HPLC (Taxane Prodrug.M) was 1.86 mg/ml. Experiment 7 Data indicate that 87% of the drug used for preparation of this formulation was incorporated into lipid particles (Table 6c). Particle size analysis of ART-207 containing formula 40 tion. The resultant ART-207 containing emulsion was more Preparation of ART-207 (Loti AW-001-243) stable relative to previous formulations. In FIG. 12 and Containing Lipid Emulsion Table 6c, particle size increased from 47 to 66 nm over 24 days. Lowering ART-207 and maintaining high phospholipid 45 Effect of lowering triglycerides content on ART-207 content (Table 6a and b) resulted in: a) significantly smaller incorporation, particle size and stability. TABLE 7a Formulation composition. Components Weighed, mg (per 100 ml
Date Lotti Material PC TG FC CE U Vit E ART P188 DMPC PS
11 Jan 2013 OO2.110.4 TSPM S900 652 69 150 1 0 1 0 648 O O O
TABLE 7b 60 Ratios for major formulation components. TG.ART-2O7 PC.ART-2O7 PCTG FCfCE
1.01 9.10 9.OS O46
65 Coarse Suspension was prepared and MF processed (loti 002.111.2). US 9,468,603 B2 33 34 TABLE 7c Particle size, ART-207 content, and particle stability of resultant emulsion. Particle size ART-2O7 Formulation Stability Manufacturing by intensity, Content, Recovery, Days past Particle size Date Lotti ill mg/ml % mfg ill
11 Jan 2013 OO2.111.2 66 3.88 59.9 38 75
MFP. In FIG. 13, particle size reached R1 at ~105 nm after Experiment 8 20 min of processing at ~60° C. Lowering the temperature to -20° C. and further to 10° C. resulted in further increase Preparation of ART-207 (Loti AW-001-243) of the particle size from 105 to 118 nm and reaching R2. Is Containing Emulsion Increasing temperature back to 50-60° C. resulted in particle size decrease to 90 nm and reaching next resistance point at Investigating the effect of Poloxamer P188 (1% V/W) 94 nm Additional 30 min processing at ~~25° C. decreased addition on ART-207 incorporation, particle size and stabil particle size from 94 to 84 nm MFP was stopped and filtered. ity. Poloxamers are non-ionic poly (ethylene oxide) (PEO)- Decrease of particle size from 84 to 66 nm was observed 20 poly(propylene oxide) (PPO) copolymers, Poloxamers are after filtration. broadly used in clinical applications (1). Ability of P188 to intercalate in lipid monolayers and to seal the membranes HPLC Analysis. ART-207 content in resultant emulsion (2) Suggests their usefulness in improving stability of the determined by HPLC (Taxane Prodrug.M) was 3.88 mg/ml. lipid particles by possibly relieving the Surface tension. Data indicate that 60% of the drug used for preparation of P188 coating of nanoparticles reduces their opsonization by this formulation was incorporated into lipid particles (7c). SU proteins and macrophage uptake (3) that is particu ART-207 incorporation capacity of the particles with low larly relevant for in vivo applications. One of the most triglycerides content was decreased. Particle size analysis of relevant features of P188 to this application is that P188 ART-207 containing formulation. The ART-207 containing coating of the lipid nanoparticles does not prevent binding of emulsion was more stable compared to formulations higher so Apollipoprotein E (4). in triglycerides processed in experiments #3-6. In FIG. 14 and Table 7c, particle size did not increase from 66 to 75 nm REFERENCES over 38 days. For the next 30 days (68 days total) particle size increased to 92 nm. 1. Hitesh R. Patel et al. “Poloxamers: A pharmaceutical excipient with therapeutic behaviors'. 2009. International Thus, lowering triglyceride content and maintaining high Journal of PharmTech Research, Vol. 1, No. 2, pp. 299-303. phospholipid content resulted in reasonably small and fairly 2. Guohui Wu et al. “Interaction between Lipid Monolayers stable particles. ART-207 incorporating capacity of low and Poloxamer 188: An X-Ray Reflectivity and Diffraction triglycerides emulsion was significantly decreased. Increase Study”. 2005. Biophysical Journal Volume 89 November of phospholipid results in smaller particles with increased 20053159-3173.3. Zhang, Wen-liet al. "Stealth tanshinone ART-207 content; Increase of phospholipid does not IIA-loaded solid lipid nanoparticles: effects of poloxamer improve the particle stability; Decrease of ART-207 content 188 coating on in vitro phagocytosis and in vivo pharma results in decrease of particle size and improved stability; cokinetics in rats'. 2009. Acta Pharm Sin, 44: 1421-1428. 4. Decrease of TG content and therefore, TG/ART-207 ratio Parag Aggarwal et al. “Nanoparticle interaction with plasma from ~2 to 1 results in decreased particle size, improved 45 proteins as it relates to particle biodistribution, biocompat stability, but lower ART-207 incorporating capacity of for- ibility and therapeutic efficacy”. 2009. Advanced Drug mulation. Delivery Reviews 61, 428-437. TABLE 8a. Formulation composition. Components Weighed, ng (per 100 ml
Date Lotti Material PC TG FC CE U Vit E ART P188 DMPC PS
15 Jan. 2013 OO2.115.4 TSPM S123 1390 70 212 10 10 648 1OOO O O
TG/ART-207 ratio appears to be an important pre-requi- TABLE 8b site in determining ART-207 incorporation capacity of the formulation; Higher TG/ART-207 ratio results in increased 60 - Ratios for major formulation components. ART-207 incorporation capacity of the formulation but compromised particle stability, and lower ratio results in TG.ART-2O7 PC.ART-2O7 PCTG FCfCE improved formulation stability but lower ART-207 incorpo- 2.15 7.91 3.69 O.33 ration capacity. There are at least two possible routes to optimizing ART-207 incorporation, particle size, and stabil- 65 ity of emulsion preparations: Optimizing ratios for major Coarse Suspension was prepared and MF processed formulation components. (loti 002.116.1). US 9,468,603 B2 35 36 TABLE 8 c Particle size, ART-207 content, and particle stability of resultant emulsion. Particle size ART-2O7 Formulation Stability Manufacturing by intensity, Content, Recovery, Days past, Particle size Date Lotti ill mg/ml % mfg ill
16 Jan 2013 002116.1 62 6.07 93.7 4 146
MFP. In FIG. 15, particle size reached 60 nm after 30 min lation was incorporated into lipid particles (8c). Particle size of processing at ~60° C. At this point temperature was analysis of ART-207 containing formulation. The resultant lowered to -20° C. to determine if the decrease in particle ART-207 containing emulsion was unstable. In FIG. 16 and size can be further facilitated at lower temperatures. Addi 15 Table 8c, particle size increased from 62 to 146 nm just after tional 20 min of processing resulted in significant particle four days. size increase from 60 to 73 nm Increasing temperature back Addition of 1% (V/W) of P188 had destabilizing effect on to ~60° C. resulted in particle size decrease to 56 nm and the resultant emulsion. Preparation of lipid-based drug-free reaching resistance point (R2). MFP was stopped and fil and ART-207 containing formulations for maximum toler tered. Increase of particle size from 56 to 62 nm was ated dose (MTD) studies. observed after filtration. HPLC Analysis. ART-207 content in resultant emulsion determined by Experiment 9 HPLC (Taxane Prodrug.M) was 6.07 mg/ml. Data indicate that 93.7% of the drug used for preparation of this formu Preparation of Drug-Free Lipid Emulsion TABLE 9a Formulation composition. Components Weighed. Ing (per 100 ml
Date Lotti Material PC TG FC CE U Wit E ART P188 DMPC PS
15 Jan 2013 OO2.118.1 TSPM S209 1387 69 213 10 10 O O O O
35 TABLE 9b Ratios for maior formulation components.
40 TG.ART-2O7 PC.ART-2O7 PCTG FCfCE NA NA 3.75 O.33
Coarse Suspension was prepared and MF processed (loth 002.118.0). TABLE 9c Particle size, ART-207 content and particle stability of resultant emulsion. Particle size ART-2O7 Formulation Stability Manufacturing by intensity, Content, Recovery, Days past Particle size Date Lotti ill mg/ml % mfg ill
19 Jan 2013 OO2.118.00 47 NA NA 60 79
MFP. In FIG. 17, particle size reached 45 nm after 35 min of processing at ~60° C. MFP was stopped and material was 60 filtered. Increase of particle size from 45 to 47 nm was observed after filtration. HPLC Analysis. ART-207 content in resultant emulsion determined by HPLC (Taxane Prodrug.M) was below 65 detection limit. Particle size analysis of drug-free formulation. The resul tant emulsion was stable for at least 13 days. In FIG. 18 and US 9,468,603 B2 37 38 Table 9c, particle size did not increase for 13 days. For the geted doses of ART-207. The projected doses of formulated next 47 days (60 days total) particle size increased to 79 nm ART-207 for MTD study were the following: 64.4, 46,3422 (FIG. 18 and Table 9 c). mg per kg (mpk). All test articles were administered to mice Data shows that processing of drug-free high PC and TG via intravenous (iv) injections. The injection volumes to containing formulation results in Small and relatively stable 5 achieve projected doses noted in Table 11a. particles. TABLE 11a Experiment 10 ART-207 content, projected doses, and inlection volumes. 10 ART-2O7 Injection Volume, required Dose, ml (per 20 g Preparation of ART-207 (Loti AW-001-243) Lotti Material mg/ml mpk of mouse weight) Containing Lipid Emulsion 002.118.00 Vehicle Control NA NA O.21 002.119.4 Formulated ART-2O7 6.13 64.4 O.21 Manufacture of ART-207 containing emulsion for Maxi 002.119.3 Formulated ART-2O7 4.38 46 O.21 mum Tolerated Dose study. TABLE 10a Formulation composition. Components Weighed, ng (per 100 ml
Date Lotti Material PC TG FC CE U Vit E ART P188 DMPC PS
15 Jan. 2013 OO2.115.2 TSPM S2OO 1389 7O 213 10 10 673 O O O
TABLE 1 Ob TABLE 11a-continued Ratio for major formulation components. ART-207 content, projected doses, and injection volumes. 30 TG ART-2O7 PC.ART-2O7 PCTG FCICE ART-2O7 Injection Volume, required Dose, ml (per 20 g 2.07 7.73 3.74 O.33 Lotti Material mg/ml mpk of mouse weight)
002.119.2 Formulated ART-2O7 3.22 34 O.21 002.119.1 Formulated ART-2O7 2.11 22 O.21 150 ml of coarse suspension was prepared and MF 35 processed (lotiiO02.119.4). TABLE 1 Oc
Particle size, ART-207 content, and particle stability of resultant emulsion.
Particle size ART-2O7 Formulation Stability
Manufacturing by intensity, Content, Recovery, Days past Particle size Date Lotti ill mg/ml % mfg ill
20 Jan. 2013 002119.4 78 6.13 91.8 22 218
50 MFP Preparation of dilutions of emulsion to achieve In FIG. 19, particle size reaches R1 at ~106 nm after 45 min of processing at ~60° C. Lowering the temperature to targeted concentrations of ART-207. The calculated targeted ~20° C. resulted in lowering particle size to 72 nm MFP was concentrations of formulated ART-207 for MTD study stopped and filtered. Increase of particle size from 72 to 78 55 were the following: 6.13, 4.38, 3.22, and 2.11 mg/ml. nm was observed after filtration. To achieve 4.38, 3.22, and 2.11 mg/ml targeted concentra HPLC Analysis. ART-207 content in resultant emulsion determined by HPLC (Taxane Test.M, Rev 0) was 6.13 tions of ART-207, emulsion loti 002-119-4 (6.13 mg/ mg/ml. Data indicate that 91% of the drug used for prepa ml of ART-207) determined by HPLC (Taxane Test.M, ration of this formulation was incorporated into lipid par Rev 0) was diluted 1.4, 1.9 and 2.9 fold with acetic ticles (Table 10c). Particle size analysis of ART-207 con- 60 taining formulation. The resultant ART-207 containing acid buffered saline (pH 5.5) to calculated concentrations: emulsion was unstable. In FIG. 20 and Table 10c, particle 4.38 mg/ml (lotiiO02-119-3), 3.22 mg/ml (lotiiO02 size increased from 78 to 218 nm over 22 days. 119-2), and 2.11 mg/ml (lotiiO02-119-1), respectively. All The outcome of this experiment was similar to results of experiment #5 which indicate: a) consistency in coarse 65 preparations were filtered and ART-207 content of undiluted emulsions preparation and processing, and b) yet unresolved lot and its diluted derivatives analyzed by HPLC (Table stability issue for ART-207 containing formulations. Tarand 11b). US 9,468,603 B2 40 TABLE.11b.
ART
Calculated HPLC Recovery Date Lotti Material Comments mg/ml mg/ml % 15 Jan. 2013 OO2.115.2 TSPM** 6.73 NA NA 20 Jan. 2013 002.119.4 Emulsion DF* = 0.0 6.13 6.13 1OO.O 20 Jan. 2013 002.119.3 Emulsion DF* = 1.4 4.38 3.98 90.8 20 Jan. 2013 002.119.2 Emulsion DF* = 1.9 3.22 3.02 93.8 20 Jan. 2013 002.119.1 Emulsion DF* = 2.9 2.11 2.01 95.3
DF-Dilution Factor; TSPM** total solids pre-mix.
Dilutions of starting lot #002-119-4 (6.13 mg/ml of ART 15 Approximately 20 ml of the following lots were shipped 207) down to targeted concentrations of ART-207 will result to the study site; Loti 002-118-00, Drug-free formulation in Subsequent lowering of lipid content in resultant lots (vehicle control); Loth002-119-4, Formulated ART-207 002-119-3,002-119-2 and 002-119-1. “Dilution vs indepen (6.13 mg/ml); Loth002-119-3, Formulated ART-207 (3.98 dent formulation' was based on the following rationale: a) mg/ml); Lothi002-119-2, Formulated ART-207 (3.02 Independent formulation of different concentrations of ART mg/ml); Loti 002-119-1, Formulated ART-207 (2.01 mg/ml). 207 using identical lipid formula yields emulsions with 10 ml samples of each lot were retained. ART-207 significantly different particle size, whereas dilution does quantification in emulsion preparation resulted in improved not affect particle size (Table 11 d); this approach provides “Taxane Test.M. Rev 1 method (see above “Analytical targeted concentrations of ART-207 that are incorporated in Development' section). The revised ART-207 concentra similar size particles, and b) should toxic effect of lipid 25 tions in shipped samples in the Table 11f. Table 11f shows occur it will be well represented by vehicle control that has that concentrations of ART-207 determined by Tax highest lipid content in the series and is identical to loti 002 ane Test.M. Rev 1 method were significantly higher in all 119-4 (6.13 mg/ml of ART-207) in lipid content (Table 11c). tested lots. TABLE 11 c
Components Weighed, mg (per 100 ml)
Vit TS TL Date Lotti Material DF* PC TG FC CE U E ART P188 DMPC PS 90 %
15 Jan. 2013 OO2.118.1 TSPM** NAA S209 1387 69 213 1O 10 O O O O 6.9 6.9 19 Jan. 2013 OO2.118.OO REM*** NAA S209 1387 69 213 1O 10 O O O O 6.9 6.9 15 Jan. 2013 OO2.115.2 TSPM NAA S2OO 1389 70 213 1O 10 673 O O O 7.6 6.9 20 Jan 2013 OO2.119.4 REM O.O S2OO 1389 70 213 1O 10 673 O O O 7.6 6.9 20 Jan 2013 OO2.119.3 REM 1.4 NAA NAA NAA NAA NAA NAA NA O O O 54 49 20 Jan 2013 OO2.119.2 REM 1.9 NAA NAA NAA NAA NAA NAA NA O O O 4.0 3.6 20 Jan 2013 OO2.119.1 REM 2.9 NAA NAA NAA NAA NAA NAA NA O O O 2.6 2.4
DF-Dilution Factor; TSPM** total solids pre-mix: REM- resultant emulsion
TABLE 11d TABLE 11 f 50 Particle size of resultant emulsion preparations. HPLC Analyses of ART-207 Content, mg/mL Particle size, TS TL Taxane Test.M., Rev O Taxane Test. M, Rev. 1 Date Lotti Material DF: ill % 9/o 55 Material On the day of mfg 9 Feb. 2013 15 Jan 2013 OO2.118.1 TSPM** NA NA 6.9 6.9 19 Jan 2013 OO2.118.00 REM*** NA 49.O 6.9 6.9 OO2-118-OO BDL BDL 15 Jan 2013 OO2.115.2 TSPM NA NA 7.6 6.9 OO2-119-1 2.01 2.26 20 Jan 2013 OO2.119.4 REM O.O 86.7 7.6 6.9 OO2-119-2 3.02 340 20 Jan 2013 OO2.119.3 REM 1.4 86.4 5.4 4.9 60 OO2-119-3 3.98 4.49 20 Jan 2013 OO2.119.2 REM 1.9 83.6 4.0 3.6 OO2-119-4 6.13 6.91 20 Jan 2013 OO2.119.1 REM 2.9 81.7 2.6 2.4
DF-Dilution Factor; TSPM** total solids pre-mix: 65 The treatment of animals was carried out from 22 Jan. REM- resultant emulsion 2013 to 26 Jan. 2013. The revised on 9 Feb. 2013 doses used in animal studies are presented in the Table 11g. US 9,468,603 B2 41 42 TABLE 11g TABLE 11 i-continued
Revised ART-207 doses. Particle size, nm ART-2O7 Injection Volume, Day of mg/ml, Dose, ml (per 20 g of 5 Shipment Returned Retained Lotti Material revised mpk mouse weight) to MTD from MTD at Arbor study site, study site, Therapeutics, 002.151.5 Vehicle Control NA NA O.21 Material (Jan. 19, 2013) (Jan. 29, 2013) (Jan. 29, 2013) OO2.1194 Formulated ART-2O7 6.91 72.6 O.21 OO2.119.3 Formulated ART-2O7 4.49 47.1 O.21 OO2-119-2 83.6 113.O 106.9 OO2.119.2 Formulated ART-2O7 3.40 35.7 O.21 10 002-119-3 86.4 121.7 115.7 OO2.119.1 Formulated ART-2O7 2.26 23.7 O.21 OO2-119-4 86.7 149.0 136.2
MTD study consisted of two major phases: We extended monitoring of particle size for the emulsions Treatment for five consecutive days (Q1 Dx5): Adminis- used in the MTD Study, both that shipped from the MTD tration of Paclitaxel, formulated ART-207, and vehicle con- 15 dy si d ial ined for 23 davs beginni h trol (drug-free formulation) via intravenous injections: study S1te and material retained for ays beginning On the Assessment of tumor size and mouse weight; Assessment ofs day of manufacture. FIG. 21 shows that particle size of the Vital signs; Monitoring the animals after end of treatment for vehicle control did not change over the period of monitoring, two weeks;s Assessment of tumor size and mouse weight; whereas a substantialicl size increase was observed for ART and assessment of vital signs. Material remaining from the 20 207 containing particles. dosing emulsion solutions was shipped back to Arbor Thera- Although, the current lipid formulation and processing peutics after the end of the treatment phase. ART-207 technology allow incorporation of the targeted amount of content was assessed for all formulations used in MTD drug and reduction to an acceptable particle size, the stabil studies. The assessment of returned material was carried out ity of the particles obtained remains an issue. Substantial side by side with material retained. 25 size increase over time was observed for all drug containing Table 11h shows material evaluated by Taxane Test.M lipid formulations. The formulated ART-207 demonstrated Rev. 1 method, returned from MTD study site, and materials efficacy, selectivity and absence of apparent toxic side effect retained have similar drug content in all tested lots. The data in the MTD (ATL-1 and 2) Studies (Formal report “EVALU indicate good stability of formulated ART-207 and proper ATION OF THE TOLERABILITY OF FORMULATED material handling at the research sites. ART-2O7 WHEN ADMINISTERED INTRAVENOUSLY TABLE 11 h. HPLC Analyses of ART-207 Content, mg/mL. Taxane Test.M Taxane Test.M Taxane Test.M Rev O Taxane Test.M Rev 1 Rev 1 On the day Rew 1 Returned Retained Material of mfg 9 Feb. 2013 Material Material
OO2-118-OO BDL BDL BDL BDL OO2-119-1 2.01 2.26 2.37 2.32 OO2-119-2 3.02 340 3.49 3.SO OO2-119-3 3.98 4.49 4.74 4.71 OO2-119-4 6.13 6.91 6.75 6.58
Particle size of the material remaining from the MTD TO NONTUMORED AND TUMOREDATHYMICNUDE Study and returned to Arbor was assessed on the day of its MICE. Southern Research Institute, Birmingham, Ala. delivery. The assessment of returned material was carried 35255-5305). out side by side with material retained. Stability data are Evaluate the efficacy and tolerability of formulated ART presented in Table 13k and reflect monitoring of particle size 207 when administered intravenously to nontumored female from day 0 to the end of MTD treatment phase. The particle 50 hVmic NCr-nu/ d to female athVmic NCr-nu/ ize of emulsions returned from the study site was slightly att ymic Nur-nu nu mice an tO emale athymic NCr-nu/nu S17 O mice implanted subcutaneously with human MDA-MB-231 higher(manufacturing relative date)to that particle of retained size of material.the vehicle Since control day did 0 mammary tutumor xenograitsft (experimeniment ATL-2).-2). PaclitaxelPaclitaxe not change. A significant size increase was observed for was included as a reference compound. ART-207 containing particles (Table 11i). 55 Tumor Model: Forty-six mice were implanted with fragments of the TABLE 111 human MDA-MB-231 mammary tumor from an in vivo passage. Individual tumors of 30 animals grew to 108-600 Particle size, nm mg in weight (108-600 mm in size) on Day 13 after tumor 60 fragments implantation on day of treatment initiation. sis Returned Retained Selected 30 animals with tumors were assigned to six to MTD from MTD at Arbor treatment groups so that the mean tumor weights in all study site, study site, Therapeutics, groups on Day 13 were as close to each other as possible Material (Jan. 19, 2013) (Jan. 29, 2013) (Jan. 29, 2013) (mean tumor weights ranged from 305 to 313 mg, median OO2-118-OO 49.0 47.1 47.1 65 tumor weights ranged from 245 to 294 mg). The studies OO2-119-1 81.7 106.3 97.4 ATL-1 and ATL-2 consisted each of six groups of five nontumored mice per group for a total of 30 mice on Day 1, US 9,468,603 B2 43 44 the first day of treatment. All treatments were administered assessment. The mean group cutoff is two animals per group. intravenously (IV) once a day for five consecutive days In the graph Day 13 is defined as Day 0 or the day of the first (Q1Dx5) beginning on Day 1. The formulated ART-207 treatment. doses of 72.6, 47.1, 35.7, and 23.7 mg/kg were 3.4.x, 2.2x, 1.7x, and 1.1x molar equivalent of a paclitaxel dose of 15 5 Experiment ATL-2 mg/kg, respectively, based on the molecular weight of formulated ART-207 of 1219.6 and molecular weight of The human MDA-MB-231 mammary tumor xenografts in paclitaxel of 853.9. the Emulsion control group (Group 5) grew progressively in all five mice. The animals had an increase in weight. Experiment ATL-1 10 However, the mouse weight corrected for the weight of tumor did not significantly change over the course of study Animals in Groups 1-4 were treated with formulated (FIG. 23). One animal (animal 1) was euthanized on Day 26 ART-207 at doses of 72.6, 47.1, 35.7, and 23.7 mg/kg/ due to tumor ulceration. injection, respectively, administered to the mice by exact The IV treatment with formulated ART-207 at doses of 15 72.6, 47.1, 35.7 and 23.7 mg/kg/injection on a Q1 Dx5 individual animal’s body weight on each day of treatment schedule (Groups 1-4, respectively) was tolerated without with the injection volume being 0.21 mL/20 g of body deaths. Administration of formulated ART-207 at a dose of weight. Animals in Group 5 (Emulsion control) were treated 72.6 mg/kg/injection resulted in a maximum mean body with a drug free formulation (injection volume of 0.21 weight loss of 4% (0.9 g), observed on Days 26 and 28. mL/20 g of body weight) Animals in Group 6 were treated Animals in the groups treated with formulated ART-207 at with paclitaxel at a dose of 15 mg/kg/injection (injection three lower doses gained weight over the experiment. It is volume of 0.1 mL/10 g of body weight). worth mentioning that mean mouse weight corrected for the weight of tumor did not significantly change over the course Experiment ATL-2 of study (FIG. 23). The MTD for formulated ART-207 was 25 above 72.6 mg/kg/injection in this experiment (an equiva Animals in Groups 1-4 were treated with formulated lent of 3.4x of the paclitaxel dose tested). ART-207 at doses of 72.6, 47.1, 35.7, and 23.7 mg/kg/ The treatment with formulated ART-207 was very effec injection, respectively, administered to the mice by exact tive in the Suppression of the tumor growth. Administration individual animal's body weight with the injection volume of formulated ART-207 at all four doses tested resulted in a being 0.21 mL/20 g of body weight. Animals in Group 5 30 dose-dependent, statistically significant inhibition of the (Emulsion control) were treated with a drug free formulation growth of the human MDA-MB-231 mammary tumor xeno (injection volume of 0.21 mL/20g of body weight) Animals grafts. The treatment with a dose of 72.6 mg/kg/injection in Group 6 were treated with paclitaxel at a dose of 15 produced two complete tumor regressions, with one animal mg/kg/injection (injection volume of 0.1 mL/10 g of body remaining tumor-free on Day 47, the day of study termina weight). 35 tion. Growth of the tumors was statistically different from the growth of the tumors in the Emulsion control group Experiment ATL-1 when individual animals' times to reach two tumor mass doublings were compared (see also formal SRI report). The IV treatment with formulated ART-207 was tolerated Tumor growth continued to be inhibited after the end of the without deaths. The treatment resulted in maximum mean 40 treatment. Three out of five tumors in the group treated with body weight losses of 1% (0.3 g), 5% (1.1 g), 1% (0.3 g), and a dose of 72.6 mg/kg/injection continued to regress until the 3% (0.6 g), when formulated ART-207 was administered at study was terminated, while tumors in the rest of the groups doses of 72.6, 47.1, 35.7, and 23.7 mg/kg/injection, respec started to grow back at different times post treatment in a tively. The maximum tolerated dose for formulated ART-207 dose response manner, higher doses delayed regrowth lon (MTD, defined as the dose that does not produce death or 45 ger. more than 20% body weight loss during and within 14 days The IV treatment with paclitaxel at a dose of 15 mg/kg/ of the end of the treatment) was above 72.6 mg/kg/injection injection on a Q1 Dx5 schedule (Group 6) was toxic, result in this experiment (an equivalent of 3.4x of the paclitaxel ing in death of two out of five animals (with both deaths dose tested). occurring on Day 19) and two more animals being eutha The IV treatment with Emulsion control on a Q1Dx5 50 nized (on Days 20 and 21) due to being moribund. The schedule (Group 5) was tolerated without deaths or body treatment resulted in a mean body weight loss of 1% (0.3 g) weight loss. The IV treatment with paclitaxel at a dose of 15 on the day of the last treatment, Day 17 but individual mg/kg/injection on a Q1DX5 schedule (Group 6) was toler animals lost more weight prior to death. The MTD of ated without deaths and resulted in a maximum mean body paclitaxel was below 15 mg/kg/injection in this experiment. weight loss of 6% (1.3-1.5 g). The MTD for paclitaxel was 55 Tumor of the Surviving animal responded to the treatment above 15 mg/kg/injection in this experiment. Change in and regressed in weight from 180 mg on Day 13 to 32 mg mean body weights over the course of the experiment in all on Day 47. Change in mean body weights over the course of groups shown in FIG. 22. the experiment in all groups is presented graphically in FIG. FIG. 22. Mice were divided into 6 groups (5 mice in each 23. group). All test articles were administered to mice for five 60 FIG. 23. Mice were divided into 6 groups (5 mice in each consecutive days via intravenous (iv) injections. Group #5 group). All test articles were administered to mice for five received drug-free lipid formulation (black dotted line, open consecutive days via intravenous (iv) injections. Group #5 squares) and groups 1-4 received 23.7, 35.7, 47.1 and 72.6 received drug-free lipid formulation (black dotted line, open mg/kg of formulated ART-207 (filled squares). Group #6 squares) and groups 1-4 received 23.7, 35.7, 47.1 and 72.6 received 15 mg/kg of Paclitaxel (blue dotted line, open 65 mg/kg of formulated ART-207 (filled squares). Group #6 circles). Each point on the curves represents mean mouse received 15 mg/kg of Paclitaxel (blue dotted line, open weight of each RX and vehicle treated group at the day of circles). Each point on the curves represents mean mouse US 9,468,603 B2 45 46 weight of each RX and vehicle treated group at the day of of-principle in vivo study is indicative of targeted delivery assessment. Tumored mouse weight was corrected for the and efficacy of our compound with no apparent toxic side weight of tumor. The mean group cutoff is two animals per effects. This was the major cause for not reaching maximum group. In the graph Day 13 is defined as Day 0 or the day tolerated dose in this study. of the first treatment. 5 Response of the SC implanted human MDA-MB-231 mammary tumor Xenografts to the treatment with formulated ART-207, Emulsion control, and paclitaxel is presented Experiment 12 graphically in FIG. 24 as a mean tumor weight change. FIG. 24. Mice were divided into 6 groups (5 mice in each 10 Preparation of ART-207 (Loti AW-001-243) group). All test articles were administered to mice for five Containing Emulsion consecutive days via intravenous (iv) injections. Group #5 received drug-free lipid formulation (black dotted line, open O squares) and groups 1-4 received 23.7, 35.7, 47.1, and 72.6 Testing the effect of the added P188 (0.25%, V/W) on mg/kg of formulated ART-207 (filled squares). Group #6 15 particle size and stability. P188 was added to previously received 15 mg/kg of Paclitaxel (blue dotted line, open processed emulsion lot #002.121.4 (see master Table 3) to circles). “Day 0” (dotted line) represents the average tumor final concentration 0.25% (V/W) while stirring on magnetic weight for all groups assessed at the first day of treatment. stirring plate. The mixture was further processed in micro Each point on the curves represents mean tumor weight of fluidizer. TABLE 12a Formulation composition. Components Weighed, ng (per 100 ml
Date Lotfi Material PC TG FC CE U Vit E ART P188 DMPC PS
15 Jan. 2013 OO2.115.3 TSPM S18S 1393 7O 213 10 10 671 O O O each RX and vehicle treated group at the corresponding day 30 TABLE 12b of assessment. The mean group cutoff is two animals per group. In the graph Day 13 is defined as Day 0 or the day - Ratios for major formulation components of the first treatment. TG.ART-2O7 PCART-2O7 PCTG FCfCE Animal Survival 35 (FIG. 25). In the group treated with Paclitaxel 4 of 5 2.07 7.72 3.72 O.33 animals died before day 11 due to apparent toxicity of the drug. In the vehicle treated group 4 of the animals had to be Coarse Suspension was prepared and MF processed euthanized on day 18 due to extremely large tumor size. In (loth 002. 122.0). TABLE 12c Resultant particle size, ART-207 content, and particle stability. Particle size ART-2O7 Formulation Stability Manufacturing by intensity, Content, Recovery, Days past Particle size Date Lotti ill mg/ml % mfg ill
22 Jan. 2013 OO2.122.O 8O S.63 83.9 57 197
50 the group treated with the lowest (23.7 mpk) dose of MFP. In FIG. 26, particle size reaches R1 at -71 nm after formulated ART-207, 4 animals were euthanized due to large 30 min of processing at ~60° C. MFP was stopped and tumors but on day 30. The animals treated with 35.7, 47.1 material was sterile filtered. Increase of particle size from 71 and 72.6 mpk of ART-207 were alive and active and no to 80 nm was observed after filtration. weight loss was observed during the entire course of the 55 study in contrast to the animals dosed with Paclitaxel. Some HPLC Analysis. ART-207 content in resultant emulsion mice in the 72.6 mpk of ART-207 group became tumor free determined by HPLC (taxane Test M. Rev 0) was 5.63 at day 11 post dosing and stayed tumor free until the end of mg/ml. These data indicate that 83.9% of the drug used for the study (FIG. 25) preparation of this formulation was incorporated into lipid a particles (Table 12c). The lower ART-207 concentration in group).FIG. 25.All Micetest articles were divided were administered into 6 groups to (5 micemice forin each five 60 resultant emulsion could be explained by additional MF consecutive days via intravenous (iv) injections starting processing and possible dilution with buffer used tO equili from day O. Group #5 received drug-free lipid formulation brate the MF prior to processing. Particle size analysis of (black dotted line, open squares) and groups 1-4 received ART-207 containing formulation. The resultant ART-207 23.7, 35.7, 47.1 and 72.6 mg/kg of formulated ART-207 65 containing emulsion was unstable. FIG. 27 and Table 12c (filled squares). Group #6 received 15 mg/kg of Paclitaxel show that particle size increased from 80 to 197 nm over 57 (blue dotted line, open circles). The outcome of this proof- days. US 9,468,603 B2 47 48 Result suggests that addition of 0.25% of P188 at post- particles (Table 13c). Particle size analysis of ART-207 coarse emulsion step does not improve the stability of the containing formulation. FIG. 29 and Table 13c shows that resultant formulation. over 56 days, particle size of loti 002. 123.11 increased from 68 to 73 nm, and loti 002. 123.13 increased from 69 to 70 nm. Experiment 13 5 over 56 days. Particle size of the emulsion loti 002. 123.12 increased from 57 to 67 nm over 30 days. a) Decreasing Preparation of ART-207 (Loti AW-001-243) TG/ART-207 ratio by lowering TG content and subsequently Containing Emulsion raising PC/TG ratio; b) lowering CE and subsequently total Testing the effect of P188 (0.25%, V/W), decreased 10 cholesterol content; and c) addition of P188 (0.25%, V/W) TG/ART-207 ratios, and further decrease of CE and subse- at the TSPM step resulted in fairly stable emulsions. Without quently TC on resultant particle size and stability. P188 was P188, similar manipulation of TG/ART-207 and PC/TG added to the TSPM. ratios (experiment #7) resulted in an unstable emulsion. TABLE 1.3a Formulation composition. Components Weighed, ng (per 100 ml
Date Lotti Material PC TG FC CE U Vit E ART P188 DMPC PS
22 Jan. 2013 OO2.122.1 TSPM SO85 SO1 7O 148 10 10 500 256 O O
TABLE 13b Addition of P188 to TSPM with higher TG/ART-207 and 25 lower PC/TG ratios (experiment #8 and 12) also failed to - Ratos for major formulation components produce stable emulsions. TG ART-2O7 PC.ART-2O7 PCTG FCICE In FIGS. 28 and 29, stopping the MF processing at 60°C. (lot #002. 123.11 and 002. 123.13) resulted in stable emul 1.OO 10.17 10.15 O.47 sions with 68-69 nm particle size, whereas the 20° C. end 30 point (lotiiO02.123.12) resulted in initially smaller ~57 nm Coarse suspension was prepared and MF processed particles that shortly increased their size to ~66 nm and (loth 002. 123.11). stabilized at this level. The ART-207 content measured in TABLE 13c Resultant particle size, ART-207 content, and particle stability. Particle size ART-2O7 Formulation Stability Manufacturing by intensity, Content, Days past Particle size Date Lotti ill mg/ml Recovery, % mfg ill
23 Jan 2013 OO2.123.11 68 3.71 742 56 73 23 Jan 2013 OO2.123.12 57 3.71 742 30 67 23 Jan 2013 OO2.123.13 69 3.71 742 56 70
45 MFP resultant emulsion lotiiO02.123.13 was notably lower (3.7 In FIG. 28, particle size reaches R1 at-68 nm after 20 min mg/ml) than in preparations with higher TG/ART-207 and of processing at -60° C. MFP was stopped, sample WaS lower PC/TG ratios (experiments #4-5, and 10-12), (4.8–6.1 collected and filtered (Table 13c). Filtration did not affect the so mg/ml). In contrast to preparations in experiments #4-5, and particle size. The rest of material was processed further at 10-12, the resultant material obtained in this experiment -20° C. for 10 min (FIG. 28). This resulted in a decrease of (loti 002.123.13) was extremely stable. the particle size to 64 nm and was considered as R2. MFP was stopped, sample was collected and filtered (Table 13c). ART-207 may have destabilizing effect on lipid particles, Filtration resulted in significant decrease of the particle size 55 and therefore stability achieved in loti 002. 123.13 is due to to 57 nm. The temperature was raised to 60° C. and lower drug content. Comparison of loti 002. 123.13 stability remainder of material was processed for 20 min. Further to that of emulsions obtained in experiments #3, 6 and 7 processing resulted in particle size increase to 66 nm and (with similar or even lower drug content) clearly demon reached the R3. MFP was stopped and remainder of material strates improved stability of the current material. collected and filtered (Table 13c). No significant size 60 Experiment suggests that TG/ART-207 and PCITG ratios, increase was observed after filtration. addition of 0.25% of P188 at TSPM step, and processing HPLC Analysis. temperature are important in determining stability of the resultant emulsion preparations, but shows that manipula ART-207 content was determined only in emulsion tions lead to lower drug incorporation. To achieve both high lothi002. 123.13 (Taxane Test.M. Rev 0) and was equal to 65 drug content and stability of lipid formulations, gradual 3.71 mg/ml. Data shows that 74.2% of the drug used for increase of TG/ART-207 ratio above 1.0 and addition of preparation of this formulation was incorporated into lipid shorter chain phospholipid can be considered. US 9,468,603 B2 49 50 Experiment 14 cessing and stability, P188 was added to the rest of the material to final concentration 0.5% (V/W). Further process ing at ~60° C. for 30 more minutes did not result in the Preparation Containingof ART-207 Emulsion (Loti AW-001-243) 5 particle size change. MFP was stopped, sample was col lected and filtered (see also Table 14c). Filtration resulted in decrease of the particle size from 83 to 66 nm. Testing the effect of DMPC and decreased TG/ART-207 HPLC Analysis. ART-207 content was determined only in ratio on ART-207 incorporation, resultant particle size and emulsion lotiiO02.125.22 (Taxane Test.M. Rev 0) and was stability of P188 containing formulation. DMPC was added 10 equal to 3.97 mg/ml. Data indicate that 79% of the drug used to the TSPM instead of mass equivalent of PC. for preparation of this formulation was incorporated into
TABLE 14a Formulation composition. Components Weighed, ng (per 100 ml
Date Lotti Material PC TG FC CE U Vit E ART P188 DMPC PS
24 Jan 2013 OO2.125.1 TSPM 4085 S11 70 150 1 0 10 503 250 1053 O
TABLE 1.4b. lipid particles (Table 14c). Particle size analysis of ART-207 containing formulation. In FIG. 31 and Table 14c, over 54 - Raios for major formulation components - days no particle size increase was observed for TG ART-2O7 PC.ART-2O7 PCTG FCICE lotiiOO2.125.21 or lotiiOO2.125.22. Addition of shorter chain phospholipid (DMPC) along 1.02 10.21 1O.OS O.47 with P188 and lower TG/ART-207 ratio resulted in stable 30 emulsions. The increase of P188 to 0.5% (V/W) does not Coarse Suspension was prepared and MF processed affect the stability of resultant emulsion. The ART-207 (loth.002.125.2). content measured in resultant emulsion lotiiO02.123.22 was
TABLE 14c Resultant particle size, ART-207 content, and particle stability. Particle size ART-2O7 Formulation Stability Manufacturing by intensity, Content, Days past Particle size Date Lotti ill mg/ml Recovery, % mfg ill
25 Jan 2013 OO2.125.21 67 3.97 79.0 S4 62 25 Jan 2013 OO2.125.22 66 3.97 79.0 S4 64
45 MFP higher and equal to 3.97 mg/ml vs. 3.7 mg/ml observed in In FIG. 30, particle size reaches R1 at -99 nm after 10-20 experiment #13 where no DMPC was added to formulation. min of processing at ~60° C. Temperature was lowered to ~20° C. Further processing at lower temperatures resulted in increase of particle size to ~ 119 nm (R2). Gradual raising the 50 Experiment 15 temperature to ~60° C. resulted in particle size decrease to 84 nm after 60 min of processing and reaching R3. A sample Preparation of Drug-Free Emulsion was collected and filtered (Table 14c). Filtration resulted in significant decrease of the particle size from 84 to 67 nm To Investigating the effect of P188 on particle size and investigate effect of increased P188 concentration on pro- stability of drug-free high phospholipid emulsions.
TABLE 1.5a Formulation composition. Components Weighed, mg (per 100 ml
Date Lotti Material PC TG FC CE U Vit E ART P188 DMPC PS 8 Feb. 2013 OO2.1311 TSPM S190 701 69 148 10 10 O 251 O O US 9,468,603 B2 51 52 TABLE 1.5b. Ratios for maior formulation components. TG ART-2O7 PC.ART-2O7 PCTG FCICE NA NA 7.40 O.47
Coarse Suspension was prepared and MF processed (loth 002. 131.2). TABLE 1.5c Particle size, ART-207 content, and particle stability of resultant emulsion.
Particle size ART-2O7 Formulation Stability
Manufacturing by intensity, Content, Days past Particle size Date Lotti ill mg/ml Recovery, % mfg ill
9 Feb. 2013 OO2.1312 42 NA NA 39 49
MFP The initial particle size increase in the first 2-3 days was observed for almost all preparations and may reflect equili In FIG. 32, particle size reached 41 nm after 20 min of brating and stabilizing of emulsion preparations after high processing at ~60° C. MFP was stopped and filtered. No 25 pressure and often different from the ambient temperature particle size change was observed after filtration. processing. HPLC Analysis. Experiment 16 ART-207 content in resultant emulsion determined by HPLC Taxane Test.M. Rev 1) was below detection limit. 30 Preparation of ART-207 (Loti AW-004-13) Particle size analysis of drug-free formulation. The resultant Containing Emulsion emulsion was stable for 39 days (FIG.33 and Table 15c). In FIG. 33, particle size increased from 42 to 45 nm in the first Testing the effect of gradual increase of TG/ART-207 two days. From day 3 to day 39 the increase in particle from ratio (from 1 to 1.35) on ART-207 incorporation, particle 45 to 49 mm. size and stability of formulation. TABLE 16a Formulation composition. Components Weighed, mg (per 100 ml)
Date Lotti Material PC TG FC CE U Vit E ART P188 DMPC PS
9 Feb. 2013 002.134.1 TSPM S22O 697 69 149 10 10 S29 2SO O O
TABLE 16b Ratios for major formulation components. 50 TG.ART-2O7 PC.ART-2O7 PCTG FCfCE
1.32 9.87 7.49 O46
Coarse Suspension was prepared and MF processed (loti 002.134.2). TABLE 16c Resultant particle size, ART-207 content, and particle stability.
Particle size ART-2O7 Formulation Stability Manufacturing by intensity, Content, Days past Particle size Date Lotti ill mg/ml Recovery, % mfg ill
9 Feb. 2013 OO2.1342 76 4.71 89.0 39 84 US 9,468,603 B2 53 54 MFP MFP. In FIG. 34, particle size reaches R1 at ~81 nm after 30 min In FIG. 36, particle size reached -70 nm after 10 min of of processing at ~60° C., and the temperature was lowered processing at ~60° C. MFP was stopped, sample was col to -20°C. Further processing at lower temperatures resulted lected and filtered (Table 17c). Decrease in the particle size in increase of particle size to -84 nm (R2). Raising the 5 from 70 to 58 nm was observed after sterile filtration. temperature to ~60° C. resulted in decrease of the particle Although the first resistance point (R1) was not reached, the size to 76 nm after 50 min of processing and reaching R3. particle size was Small enough to collect the sample for stability assay. Further processing of remainder material at The sample was collected and filtered. No change of the 60° C. did not result in change of particle size (R1). The particle size was observed after filtration. temperature was lowered to -20° C. Processing at lower HPLC Analysis. 10 temperatures for 30 more minutes did not result in change in ART-207 content was determined in resultant emulsion particle size (R2). MFP was stopped, sample was collected (Taxane Test.M. Rev 1) and was equal to 4.71 mg/ml. Data and filtered (see Table 17c). Filtration resulted in dramatic indicate that 89% of the drug used for preparation of this decrease of the particle size from 68 to 42 nm. The tem formulation was incorporated into lipid particles (Table perature was raised to 60° C. and remainder of material was 16c). Particle size analysis of ART-207 containing formu- 15 processed for additional 20 min. Additional processing lation. In FIG. 35, initial rise of the particle size from 76 to resulted in decrease of particle size from 68 to 66 nm and 82 nm takes place in the first two days, whereas during next reached the R3. MFP was stopped and remainder of material 37 days particle size did not change. collected and filtered (Table 17c). Decrease of particle size Thus, increasing of TG/ART-207 ratio form 1 to 1.35 from 66 to 59 nm was observed after filtration. resulted in increased ART-207 incorporation capacity of 20 HPLC Analysis. formulation without affecting its stability. The resultant ART-207 content was determined in all resultant emul particles were larger than particles formed at lower sions (Taxane Test.M. Rev 1) and was equal to 4.3 mg/ml TG/ART-207 ratio. (loti 002.136.21), 4.22 mg/ml (lotiiO02.136.22), and 4.5 mg/ml (lotiiO02.136.23). Data indicate that 81.9, 80.4, and 25 85.7% of the drug used for preparation of this formulation Experiment 17 was incorporated into lipid particles of lots 002.136.21, 002.136.22 and 002.136.23, respectively (Table 17 c). Particle size analysis of ART-207 containing formulation. Preparation of ART-207 (Loti AW-004-13) In FIG. 37, after initial 1-3 nm increase of the particle size Containing Emulsion 30 observed in the first two days for the lots #002.136.21 and 002.136.23, particle size did not change for the next 34 days. Testing effect of DMPC and an increase of TG/ART-207 The particle size of emulsion lot #002. 136.22 increased by ratio (from 1 to 1.34) on ART-207 incorporation, particle 7 nm during the first two days of storage, and from 49 to 55 size, and stability of resultant emulsion. DMPC was added nm over 34 days. All resultant emulsions can be character to the TSPM instead of mass equivalent of PC. ized as stable. TABLE 17a Formulation composition. Components Weighed, ng (per 100 ml
Date Lotti Material PC TG FC CE U Vit E ART P188 DMPC PS
11 Feb. 2013 OO2.135.1 TSPM 418O 70S 68 147 10 10 525 300 1098 O
45 TABLE 17b Although processing at higher temperatures resulted in bigger particles relative to lower temperature processed Ratios for major formulation components. material, the stability of material processed at ~60° C. is TG.ART-2O7 PC.ART-2O7 PCTG FCICE higher compared to ~20° C. processed emulsions. Lots 50 #002.136.21 and 002. 136.23 processed at 60° C. were 1.34 1O.OS 7.49 O46 markedly more stable over the entire course of monitoring, whereas lot #002.136.22 (processed at -20° C.) started Coarse Suspension was prepared and MF processed increasing particle size from 42 to 53 nm on the day 1 and (loth 002. 136.2). stabilized only on day 10 (FIG. 37). Increasing of TG/ART TABLE 17c Resultant particle size, ART-207 content, and particle stability. Particle size ART-2O7 Formulation Stability Manufacturing by intensity, Content, Days past Particle size Date Lotti ill mg/ml Recovery, % mfg ill
12 Feb. 2013 002.136.21 58 4.30 81.9 36 61 12 Feb. 2013 002.136.22 42 4.22 80.4 36 55 12 Feb. 2013 002.136.23 59 4SO 85.7 36 60 US 9,468,603 B2 55 56 207 ratio form 1 to 1.34 and addition of DMPC resulted in smaller particles and essentially the same ART-207 incor poration capacity and stability of the resultant formulation relative to emulsion obtained in experiment #16.
Experiment 18
Preparation of Drug-Free Lipid Emulsion 10
Investigate effect of phosphotidyl serine (PS) on particle size and stability of drug-free formulation. PS may be used as a formulation component instead of DMPC and P188. TABLE 1.8a. Formulation composition. Components Weighed, ng (per 100 ml
Date Lotti Material PC TG FC CE U Vit E ART P188 DMPC PS
14 Feb. 2013 OO2.137.1 TSPM S36O 1408 70 150 1 0 10 O O O 2OO
25 TABLE 1.8b. Ratios for major formulation components. TG.ART-2O7 PC.ART-2O7 PCTG FCICE 30 NA NA 3.95 O.47
Coarse Suspension was prepared and MF processed (loth 002. 137.3). TABLE 18C Particle size, ART-207 content, and particle stability of resultant emulsion. Particle size ART-2O7 Formulation Stability Manufacturing by intensity, Content, Days past Particle size Date Lotti ill mg/ml Recovery, % mfg ill 15 Feb. 2013 OO2.137.3 49 NA NA 33 51
45 MFP. In FIG.38, particle size reached 49 nm after 20 min Experiment 19 of processing at ~60° C. MFP was stopped and material Preparation of ART-207 (Loti AW-004-13) was filtered. No particle size change was observed after Containing Lipid Emulsion filtration. 50 Investigating the effect of phosphotidyl serine (PS) on HPLC Analysis. ART-207 content in resultant emulsion particle size and stability of ART-207 containing formula determined by HPLC (Taxane Test.M. Rev 1) was below tions. The combination of P188 and DMPC, and also lower detection limit. Particle size analysis of drug-free formula (1.34) TG/ART-207 ratio were used in experiment #17 to tion. The resultant emulsion was stable. No significant 55 achieve stability and target particle size of resultant emul increase of particle size over 33 days (FIG. 39 and Table Sion. We also added PS to TSPM that did not contain P188 18c). and DMPC, and had higher (2.7) TG/ART-207 ratio. TABLE 19 Formulation composition. Components Weighed. Ing (per 100 ml
Date Lotti Material PC TG FC CE U Vit E ART P188 DMPC PS
14 Feb. 2013 OO2.137.2 TSPM S390 1391 7O 148 10 10 524 O O 200 US 9,468,603 B2 57 58 TABLE 1.9b Ratios for maior formulation components. TG.ART-2O7 PC.ART-2O7 PCTG FCICE
2.65 10.67 4.O2 O.47
Coarse Suspension was prepared and MF processed (loth 002. 137.4). TABLE 19C Particle size, ART-207 content, and particle stability of resultant emulsion. Particle size ART-2O7 Formulation Stability Manufacturing by intensity, Content, Days past Particle size Date Lotti ill mg/ml Recovery, % mfg ill
15 Feb. 2013 OO2.13741 53 4.79 912 33 108 15 Feb. 2013 OO2.13742 84 S.O1 95.4 33 134 15 Feb. 2013 OO2.13743 S4 4.89 93.1 33 183
MFP. In FIG. 40, particle size reached R1 at -86 nm after indicate that 91.2, 95.4 and 93.1% of the drug used for 30 min of processing at ~60° C. Since particle size was far preparation of this formulation was incorporated into lipid from targeted no sample was collected, and temperature was 25 particles of lots 002. 137.41, 002. 137.42, and 002. 137.43, lowered to -20° C. Particle size decrease from 86 to 50 nm. respectively (Table 19c). was observed during further processing at -20°C. MFP was Particle size analysis of ART-207 containing formulation. stopped, a sample was collected and filtered (Table 19c). No FIG. 41 and Table 19c show that regardless of high ART-207 change in particle size was observed after filtration. incorporation capacity, all resultant emulsions were Although the first resistance point (R1) was not reached, the 30 unstable. After 33 days, the particle size increased in all particle size was Small enough to collect the sample for processed lots. stability assay (R2). The temperature was raised to 60° C. Although processing at higher temperatures results in and the remainder of material was processed for 20 more bigger particles relative to lower temperature processing, the minutes. There was increase of particle size from 50 to 82 stability of material processed at ~60°C. is higher compared nm and reached the R3. MFP was stopped, sample was 35 to -20° C. processed emulsions. For example, collected and filtered (FIG. 43 and Table 19c). No change in loti 002. 137.42 processed at 60° C. was relatively stable for particle size was observed after filtration. Temperature was 18 days of storage, whereas lots #002. 137.41 and lowered to ~25° C. and further processing of remainder 002. 137.43 were increasing particle size from day 1 (FIG. material for 20 min resulted in particle size decrease from 82 41). Drug incorporation capacity of this PS containing to 53 nm and reached R4. MFP was stopped, a sample was 40 formulation was high probably due to high (2.7) TG/ART collected and sterile filtered (FIG. 40 and Table 19c). No 207 ratio. Addition 2.5% (W/W) of negatively charged PS to change in particle size was observed after filtration. material with high TG/ART-207 ratio does not improve the HPLC Analysis. ART-207 content was determined in all stability of resultant emulsion. Experiment 20. Preparation resultant emulsions (Taxane Test.M. Rev 1) and was equal of drug-free emulsion. Investigate effect of P188, DMPC, to 4.79 mg/ml (loth002. 137.41), 5.01 mg/ml 45 and low TG on particle size and stability of drug-free (loti 002. 137.42), and 4.89 mg/ml (loth002. 137.43). Data formulation. TABLE 20a Formulation composition. Components Weighed, mg (per 100 ml
Date Lotti Material PC TG FC CE U Vit E ART P188 DMPC PS
21 Feb. 2013 002.139.1 TSPM 421 O 700 70 150 10 10 O 297 1060 O
TABLE 20b Ratios for major formulation components. 60 TG.ART-2O7 PC.ART-2O7 PCTG FCfCE
NA NA 7.53 O.47
65 Coarse Suspension was prepared and MF processed (loth 002. 137.3). US 9,468,603 B2 59 60 TABLE 20c Particle size, ART-207 content, and particle stability of resultant emulsion. Particle size ART-2O7 Formulation Stability Manufacturing by intensity, Content, Days past Particle size Date Lotti ill mg/ml Recovery, % mfg ill 21 Feb. 2013 002.139.2 38 NA NA 27 43
MFP. FIG. 42 shows that particle size reached 39 nm after MFP. 20 min of processing at ~60° C. MFP was stopped and In FIG. 44, particle size reached R1 at ~68 nm after 20 filtered. No particle size change noted after filtration. min of processing at ~60° C. MFP was stopped, sample was HPLC Analysis. is collected and filtered (see also Table 21c). Decrease of ART-207 content in resultant emulsion determined by particle size from 68 to 58 nm noted after filtration. The HPLC (Taxane Test.M. Rev 1) was below detection limit. temperature was lowered to ~23° C. and the remainder of Particle size analysis of drug-free formulation. The resultant material was processed for 20 more minutes. This resulted in emulsion was stable. No significant increase of particle size decrease of particle size 65 nm and reached the R2. MFP from 38 to 42.5 nm observed during 27 days (FIG. 43 and 20 was stopped, a sample was collected and filtered (FIG. 44 Table 20 c). and Table 21c). Decrease of particle size from 65 to 46 nm noted after filtration. Then temperature was raised to ~60° C. Experiment 21 and further processing of remainder material for 20 min resulted in increase of particle size 68 nm and reached R3. 25 MFP was stopped, a sample was collected and filtered (FIG. 44 and Table 21c). Decrease of particle size from 68 to 59 Preparation of ART-207 (Loti AW-004-13) nm was observed after filtration. Temperature was lowered Containing Emulsion to -20° C. and the remainder of material was processed for an additional 20 min. This did not change the particle size Repeat of experiment #17, to confirm favorable effect of 30 reached at R4. MFP was stopped, sample was collected and DMPC/P188 and 1.34 TG/ART-207 ratio on ART-207 incor- filtered (FIG. 44 and Table 21c). Decrease of particle size poration, particle size, and stability of resultant emulsion. from 67 to 49 nm after filtration. TABLE 21a Formulation composition. Components Weighed, ng (per 100 ml
Date Lotti Material PC TG FC CE U Vit E ART P188 DMPC PS 21 Feb. 2013 OO2.140.1 TSPM 423O 703 7O 150 1 0 10 527 298 1080 O
TABLE 21b. HPLC Analysis. ART-207 content was determined in all resultant emul - Ratios for major formulation components "sions (Taxane Test.M. Rev 1) and was equal to 4.42 mg/ml TG.ART-2O7 PC.ART-2O7 PCTG FCICE (loti 002.140.21), 4.1 mg/ml (lotiiO02.140.22), 4.5 mg/ml (loti 002.140.23), and 4.35 mg/ml (loth.002.140.24). Data 1.33 10.08 7.55 O.47 indicate that 84.2, 78.1, 85.7 and 82.9% of the drug used for 50 preparation of this formulation was incorporated into lipid Coarse Suspension was prepared and MF processed particles of lots 002.140.21, 002.140.22, 002.140.23, and (loti 002.140.2). 002.140.24 respectively (Table 21c). TABLE 21c Particle size, ART-207 content, and particle stability of resultant emulsion. Formulation Stability Particle size ART-2O7 Days Particle Manufacturing by intensity, Content, Recovery, past size Date Lotti ill mg/ml % mfg ill
21 Feb. 2013 002.140.21 58 4.42 84.2 27 58 21 Feb. 2013 002.140.22 46 4.10 78.1 27 S4 21 Feb. 2013 002.140.23 59 4SO 85.7 27 58 21 Feb. 2013 002.140.24 49 4.35 82.9 27 55 US 9,468,603 B2 61 62 Particle size analysis of ART-207 containing formulation. similar TG/ART-207 ratios. The range of TG/ART-207 In FIG. 45, emulsion lots #002.140.21 and 002.140.23 ratios ~1.3-1.4 results in reproducible ART-207 incorpora processed at 60° C. were highly stable. An initial ~5-7 mm tion capacity and stability of resultant emulsions. particle size increase (in the first 5 days of storage) was observed in Lots HO02.140.22 and 002.140.24. Both lots stabilized at ~53-55 nm by day 6. Preparation of Drug-Free and Art-207 Containing Processing at higher temperatures results in bigger par Formulations for Efficacy and Stability Studies ticles relative to lower temperature processing, stability of Experiment 22 material processed at ~60° C. was higher compared to -20° 10 C. processed emulsions. In terms of particle size and sta Preparation of Drug-Free Emulsion Vehicle bility, this result is similar to outcome of experiment #17. Control Drug incorporation capacity of lot #002.140.2 formulation was similar to that obtained in other experiments with Preparation of drug-free emulsion for efficacy study. TABLE 22a Formulation composition. Components Weighed, mg (per 100 ml
Date Lotti Material PC TG FC CE U Vit E ART P188 DMPC PS
13 Mar. 2013 OO2.15O1 TSPM 4344 700 71 151 11 10 O 3O3 1076 O
25 TABLE 2.2b Ratios for major formulation components. TG.ART-2O7 PC.ART-2O7 PCTG FCfCE 30 NA NA 7.74 O.47
Coarse Suspension was prepared and MF processed (loth 002.151.5). TABLE 2.2c Particle size, ART-207 content, and particle stability of resultant emulsion. Formulation Stability Particle size ART-2O7 Days Particle Manufacturing by intensity, Content, Recovery, past size Date Lotti ill mg/ml % mfg ill
14 Mar. 2013 OO2.151.5 37 NA NA 42 47
MFP. In FIG. 46 that particle size reached 41 nm after 20 min of processing at ~60° C. MFP was stopped and material 50 filtered. Decrease of particle size from 41 to 37 nm was observed after filtration. HPLC Analysis. ART-207 content in resultant emulsion determined by HPLC (Taxane Test.M. Rev 2) was below detection limit. Particle size analysis of drug-free formula 55 tion. The resultant emulsion was stable for 33 days. In FIG. 50, particle size did not change for 33 days. For the next 9 days (42 days total) particle size increased to 47 nm (FIG. 47 and Table 22c).
60 Experiment 23 Preparation of ART-207 (Loti AW-004-24) Containing Emulsion 65 Manufacture of ART-207 containing emulsion prepara tion for efficacy study. US 9,468,603 B2 63 64 TABLE 2.3a Formulation composition. Components Weighed, ng (per 100 ml
Date Lotti Material PC TG FC CE U Vit E ART P188 DMPC PS
13 Mar. 2013 OO2.150.4 TSPM 42O3 699 7O 150 1 0 10 532 300 1083 O
10 TABLE 23b Ratios for major formulation components. TG.ART-2O7 PC.ART-2O7 PCTG FCICE 15 1.31 9.94 7.56 O.47
150 ml of coarse suspension was prepared and MF processed (loti 002.151.8). TABLE 23c Particle size, ART-207 content, and particle stability of resultant emulsion. Formulation Stability Particle size ART-2O7 Days Particle
Manufacturing by intensity, Content, Recovery, past size Date Lotti ill mg/ml % mfg ill
14 Mar. 2013 OO2.151.8 62 S.26 98.9 42 67
MFP emulsion was stable. In FIG. 49 and Table 23c, particle size In FIG. 48, particle size reaches 78 nm after 30 min of did not change over 33 days. During the next 9 days (42 days processing, at ~60° C. Additional 30 min of processing at is total) a increase of -3.5-4 nm was observed. ~60° C. did not significantly reduce the particle size and was considered as R2. The temperature was lowered to 20° C. MF processing of formulation with TG/ART-207 ratio and processed for additional 45 min. Processing at 20° C. equal 1.31 resulted in stable emulsion with 98.9% ART-207 resulted in increase of particle size and was considered as incorporation capacity. R2. MFP was stopped and material was sterile filtered. Decrease of the particle size from 76 to 61 nm was observed 40 Experiment 24 after filtration. HPLC Analysis. ART-207 content in resultant emulsion Preparation of ART-207 (Loti AW-004-24) determined by HPLC (Taxane Test.M, Rev 2) was 5.26 Containing Backup Emulsion for Efficacy Study mg/ml. Data indicate that 98.9% of the drug used for preparation of this formulation was incorporated into lipid 45 Manufacturing ART-207 containing emulsion with particles (Table 23c). Particle size analysis of ART-207 increased TG/ART-207 ratio to 1.4 as a candidate for effi containing formulation. The resultant ART-207 containing cacy study. TABLE 24a Formulation composition. Components Weighed, mg (per 100 ml
Date Lotti Material PC TG FC CE U Vit E ART P188 DMPC PS
15 Mar. 2013 OO2.152.1 TSPM 4277. 698 70 150 1 0 1 0 SO1 301 1067 O
TABLE 24b 60 Ratios for major formulation components. TG.ART-2O7 PC.ART-2O7 PCTG FCfCE
1.39 10.67 7.66 O.47
65 130 ml of coarse suspension was prepared and MF processed (loti 002.153.1). US 9,468,603 B2 65 66 TABLE 24c Particle size, ART-207 content, and particle stability of resultant emulsion. Formulation Stability
Particle size ART-2O7 Days Particle
Manufacturing by intensity, Content, Recovery, past size Date Lotti ill mg/ml % mfg ill
15 Mar. 2013 OO2.153.1 66 4.68 93.5 41 69
MFP. FIG.50 shows that particle size reaches 78 nm after MF processing of formulation with TG/ART-207 ratio 39 min of processing at ~60° C. Temperature was lowered 15 equal 1.39 resulted in stable emulsion with 93.5% ART-207 to 20° C. and material was processed for additional 39 min incorporation capacity. MF processing at 20° C. did not result in particle size decrease and was considered as R2. MFP was stopped and filtered. Decrease of the particle size from 78 to 66 nm noted after filtration. 2O Experiment 25 HPLC Analysis. ART-207 content in resultant emulsion determined by HPLC (Taxane Test.M, Rev 2) was 4.68 mg/ml. Data indicate that 93.5% of the drug used for preparation of this formulation was incorporated into lipid Preparation of ART-207 (Loti AW-004-24) particles (Table 24c). 25 Containing Backup Emulsion for Efficacy Study Particle size analysis of ART-207 containing formulation. The resultant ART-207 containing emulsion was stable. FIG. To manufacture ART-207 containing emulsion with fur 51 and Table 24c show that particle size did not significantly ther increased TG/ART-207 ratio to 1.5 as a candidate for change over 41 days. efficacy study. TABLE 25a Formulation composition. Components Weighed, mg (per 100 ml
Date Lotti Material PC TG FC CE U Vit E ART P188 DMPC PS
15 Mar 2013 OO2.152.2 TSPM 432O 698 70 150 10 10 47S 304 1163 O
40 TABLE 25b. Ratios for major formulation components. TG.ART-2O7 PC.ART-2O7 PCTG FCfCE
45 1.47 11.54 7.85 O.47
130 ml of coarse suspension was prepared and MF processed (loti 002.153.2). TABLE 25c Particle size, ART-207 content, and particle stability of resultant emulsion. Formulation Stability Particle size ART-2O7 Days Particle Manufacturing by intensity, Content, Recovery, past size Date Lotti ill mg/ml % mfg ill
15 Mar. 2013 OO2.153.2 53 4.38 92.2 49 62
MFP. In FIG. 52, particle size reaches 72 nm after 52 min of processing at ~60° C. Then temperature was lowered to 20° 65 C. and material was processed for additional 26 min. MF processing at 20° C. resulted in slight decrease of the particle size to 66 nm and reaching R2. MFP was stopped and US 9,468,603 B2 67 68 material was sterile filtered. Decrease of the particle size stability of formulated ART-207 and also proper material from 66 to 53 nm was observed after filtration. handling at the research sites. HPLC Analysis. ART-207 content in resultant emulsion determined by HPLC (Taxane Test.M, Rev 2) was 4.38 TABLE 25e mg/ml. 92.2% of the drug used for preparation of this formulation was incorporated into lipid particles (Table HPLC Analyses of ART-207 Content, mg/mL. 25c). Particle size analysis of ART-207 containing formu On the day of Returned Retained lation. The resultant ART-207 containing emulsion was Material mfg Material Material fairly stable. FIG. 53 and Table 25c show that particle size OO2.151.5 BDL BDL BDL 10 increased from 53 to 62 nm over 49 days. OO2.151.8 S.26 S.11 S.26 MF processing of formulation mix with TG/ART-207 ratio equal 1.47 resulted in stable emulsion with 92.2% Particle size of the material remaining from the Efficacy ART-207 incorporation capacity. Selection of emulsions for Study and material returned to Arbor was assessed. The efficacy study. ART-207 containing emulsion lot #002.151.8 15 assessment of returned material was carried out side by side was selected for efficacy study based on particle size (62 nm) with material retained. Stability data are presented in Table and ART-207 content (5.26 mg/ml). Drug-free emulsion lot 25f and reflect monitoring of particle size over 42 days. The #002.151.5 was selected for efficacy study as a vehicle particle size of drug-free emulsions returned from the study control (Table 25a and 25d). site was identical to that of retained material. The particle size of the ART-207 containing emulsions returned from the TABLE 25d study site was 10 nm bigger relative to that of retained Resultant emulsions on the day of manufacturing. Particle size, material. ART-207 content, total solids and lipid concentration (WV). TABLE 25f Particle ART-207, 25 size, HPLC TS TL Particle size, nm Date Lotti Material ill mg/ml % 9/o 14 Mar. 2013 002.151.5 Emulsion 37 NA 6.7 6.3 Retained Day of Shipment Returned from at Arbor 14 Mar. 2013 002.151.6 Emulsion 38 NA 7.8 to study site, the study site, Therapeutics, 14 Mar. 2013 002.151.7 Emulsion 72 5.30 7.1 7.4 30 14 Mar. 2013 002.151.8 Emulsion 62 S.26 7.1 6.2 Material (Mar. 18, 2013) (Apr. 25, 2013) (Apr. 25, 2013) 15 Mar. 2013 002.153.1 Emulsion 66 4.68 7.1 6.3 OO2.151.5 37.4 45.7 46.6 15 Mar. 2013 002.153.2 Emulsion 53 4.38 7.2. 6.4 OO2.151.8 61.9 77.6 66.5
Targeted doses of ART-207. The projected doses of for 35 We extended monitoring of particle size for the emulsions mulated ART-207 for the efficacy study were the following: used in the efficacy study, both that shipped from the study 105.2, 78.9 and 52.6 mg per kg (mpk). All test articles were site and material retained at Arbor for 55 days. FIG. 54 administered to mice via intravenous (iv) injections. The shows that particle size of the vehicle control did not injection volumes to achieve projected doses are shown in significantly change over the period, whereas ART-207 Table 25e. 40 containing emulsions were stable for 42 days. Particle size increase was observed for ART-207 containing emulsions TABLE 25e starting from day 42. Possible bacterial contamination of ART-207 content, projected doses, and iniection volumes. ART-207 emulsions. (Previously monitored emulsions showed a similar rapid rise in particle size when they Injection Volume, 45 ART-207 Dose, ml (per 20 g of became contaminated due to handling.) Lotti Material mg/ml mpk mouse weight) Regardless of the observed change in particle size of ART-207 containing emulsions starting from day 42, both OO2.151.5 Vehicle Control BDL NA 0.4 OO2.151.8 Formulated ART-2O7 S.26 52.6 O.2 emulsions shipped to/from the study site and retained OO2.151.8 Formulated ART-2O7 S.26 78.9 O.3 50 samples were stable during animal treatment period. The OO2.151.8 Formulated ART-2O7 S.26 105.2 0.4 treatment of animals with ART-207 containing emulsion was completed 22 days past the manufacturing date. In study Approximately 45 ml of loti 002.151.5 and -(a) 80 ml of ATL-3 formulated ART-207 clearly demonstrated similar or lot #002.151.8 were shipped to the study site: higher tumor Suppression effect, caused significantly less LotfiO02.151.5—Drug-free formulation (vehicle control): 55 weight loss, and provided a two fold increase in animal Loth002.151.8 Formulated ART-207 (5.26 mg/ml). -10 survival rate compared to Paclitaxel treated animals (see ml samples of each lot were retained. also formal report “EVALUATION OF THE ANTITUMOR The Efficacy study consisted of two major phases similar EFFICACY OF FORMULATED ART-2O7 WHEN to those described above for the MTD study: Material ADMINISTERED INTRAVENOUSLY TO ATHYMIC remaining was returned to Arbor Therapeutics after the end 60 NUDEMICE IMPLANTED SUBCUTANEOUSLY WITH of the treatment phases. ART-207 content was assessed HUMAN MDA-MEB-231 MAMMARY TUMOR XENO (Taxane Test.M. Rev 2) for all formulations used in the GRAFTS. Southern Research Institute, Birmingham, Ala. efficacy study. The assessment of returned material was 35255-5305) carried out side by side with material retained. Table 25e Experiment ATL-3 was performed to evaluate the antitu shows that material evaluated on the day of manufacturing, 65 mor activity of the intravenous (IV) treatment with formu returned from the study site, and material retained have lated ART-207 when administered on two different sched similar drug content in all tested lots. The data indicate good ules to female, athymic NCr-nu/nu mice implanted US 9,468,603 B2 69 70 subcutaneously (SC) with human MDA-MB-231 mammary tolerated without deaths and was associated with a maxi tumor Xenografts. Paclitaxel was included as a reference mum mean body weight loss of 2% (0.4 g), observed on Day compound. Tumor Model: 4. The treatment with formulated ART-207 at a dose of 52.6 One-hundred and fifty-eight mice were implanted with mg/kg/injection was very effective in the inhibition of the fragments of the human MDA-MB-231 mammary tumor growth of the MDA-MB-231 mammary tumor xenografts. from an in Vivo passage. Individual tumors of 90 animals The treatment produced nine complete tumor regressions, grew to 88-216 mg in weight (88-216 mm3 in size) on Day with six animals remaining tumor-free on Day 56. Growth of 10 after tumor fragments implantation, the day of treatment the tumors in the group treated with formulated ART-207 at initiation. The selected 90 animals with tumors were a dose of 52.6 mg/kg/injection was statistically different assigned to nine treatment groups so that the mean tumor 10 from the growth of the tumors in the control group, when weights in all groups on Day 10 were as close to each other individual animals' times to reach three tumor mass dou as possible (mean tumor weights ranged from 147 to 154 blings were compared (Group 1 vs. Group 4: P-0.001) (FIG. mg, median tumor weights ranged from 144 to 153 mg). 55-57). Different formulated ART-207 doses were achieved by The IV treatment with Emulsion Control on a Q1 Dx5 administering different injection volumes of the 5.26 mg/mL 15 schedule (Group 5) was tolerated without deaths or mean formulation. The formulated ART-207 doses of 105.2, 78.9, body weight loss. The median tumor reached three tumor and 52.6 mg/kg were 4.9x, 3.7x, 2.5x molar equivalent of a mass doublings in 9.2 days, and reached 1,680 mg in weight paclitaxel dose of 15 mg/kg, respectively, based on the on Day 11. Growth of the tumors in untreated control group molecular weight of formulated ART-207 of 1219.6 and and the group treated with Emulsion Control on a Q1 Dx5 molecular weight of paclitaxel of 853.9. The time points are schedule was not statistically different, when individual indicated starting from the first day of treatment with animals' times to reach three tumor mass doublings were formulated ART-207, vehicle control and Paclitaxel. compared (Group 1 vs. Group 5: P=0.810) (FIG. 55-57). Study Design: The IV treatment with paclitaxel at a dose of 15 mg/kg/ The study consisted of nine groups often mice per group injection on a Q1Dx5 schedule (Group 6) resulted in death for a total of 90 mice bearing human MDA-MB-231 mam 25 of one out often animals (with death occurring on Day 2). mary tumor on the first day of treatment, Day 0 Mice in One more animal was euthanized on Day 12 due to ataxia. Group 1 were untreated until formulated ART-207 treatment Treatment resulted in a mean body weight loss of 10% (2.2 was added starting on Day 11: formulated ART-207 at a dose g), observed on Day 6. Treatment with paclitaxel at a dose of 78.9 mg/kg/injection was administered intravenously (IV) of 15 mg/kg/injection was effective in the inhibition of the once a day for five consecutive days (Q1Dx5) on Days 30 growth of the MDA-MB-231 mammary tumor xenografts. 11-15. Animals in Groups 2, 3, and 4 were treated with The treatment produced five complete tumor regressions, formulated ART-207 which was administered IV on a with four animals remaining tumor-free on Day 56. Growth Q1Dx5 schedule starting on Day 0 (Days 0-4) at doses of of the tumors in the group treated with paclitaxel at a dose 105.2, 78.9, and 52.6 mg/kg/injection, respectively. Animals of 15 mg/kg/injection on a Q1 Dx5 schedule was statistically in Group 5 (Emulsion control) were treated IV on Q1 Dx5 35 different from the growth of the tumors in the control group, schedule starting on Day 0 with a drug free formulation when individual animals' times to reach three tumor mass Animals in Group 6 were treated IV on Q1 Dx5 schedule doublings were compared (Group 1 vs. Group 6: P-0.001) starting on Day 0 with paclitaxel at a dose of 15 mg/kg/ (FIG. 55-57). Re-growth of the tumors in the group treated injection Animals in Group 7 were treated with formulated with formulated ART-207 at a dose of 52.6 mg/kg/injection ART-207 which was administered IV once every four days 40 (group 4) was slower than in the group treated with pacli for a total of three injections (Q4DX3) starting on Day 0 taxel (group 6) at a dose of 15 mg/kg/dose when both were (Days 0, 4, and 8) at a dose of 78.9 mg/kg/injection Animals administered IV on a Q1 DX5 schedule (Group 4 median in Group 8 (Emulsion control) were treated IV on a Q4Dx3 tumor growth delay of 45.9 days, 6 tumor-free mice on Day schedule starting on Day 0 with a drug free formulation 56 vs. Group 6 median tumor growth delay of 31.8 days, 4 Animals in Group 9 were treated IV on a Q4DX3 schedule 45 tumor-free mice on Day 56); the difference in tumor growth starting on Day 0 with paclitaxel at a dose of 18.9 mg/kg/ was not statistically different between the two groups, when injection. individual animals' times to reach three tumor mass dou Q1Dx5 schedule The IV treatment with formulated ART blings were compared (Group 4 vs. Group 6: P=0.133) (FIG. 207 at a dose of 105.2 mg/kg/injection on a Q1 Dx5 schedule 55). (Group 2) was lethal, resulting in death of eight out often 50 FIG.55 Mice were divided into 5 groups (10 mice in each animals (with deaths occurring on Days 5 and 6) and one group). All test articles were administered to mice for five more animal being euthanized on Day 5 due to being consecutive days via intravenous (iv) injections starting moribund. The treatment was associated with a maximum from day O. Groups #2-4 received 105.2 (solid green), 78.9 mean body weight loss of 8% (1.9 g), observed on Day 4. (solid red), and 52.6 (solid yellow) mg/kg of formulated The one Surviving mouse in the group was tumor-free on 55 ART-207. Group #5 received drug-free lipid formulation Day 56 (FIG. 55-57). (black dotted line, open squares). Group #6 received 15 The IV treatment with formulated ART-207 at a dose of mg/kg of Paclitaxel (blue dotted line, open circles). Each 78.9 mg/kg/injection on a Q1 Dx5 schedule (Group 3) was point on the curves represents mean tumor weight of each toxic, resulting in death of six out often animals (with deaths drug and vehicle treated group at the corresponding day of occurring on Days 5 and 6) and a maximum mean body 60 assessment. The mean group cutoff is two animals per group. weight loss of 7% (1.5 g), observed on Day 4. Growth of the FIG. 56 Mice were divided into 5 groups (10 mice in each tumors of four surviving mice was inhibited by the formu group). All test articles were administered to mice for five lated ART-207 treatment, with two animals being tumor-free consecutive days via intravenous (iv) injections starting on Day 56 and two other tumors weighing 32-40 mg on Day from day O. Groups #2-4 received 105.2 (solid green), 78.9 56 (FIG. 55-57). 65 (solid red), and 52.6 (solid yellow) mg/kg of formulated The IV administration with formulated ART-207 at a dose ART-207. Group #5 received drug-free lipid formulation of 52.6 mg/kg/injection on a Q1Dx5 schedule (Group 4) was (black dotted line, open squares). Group #6 received 15 US 9,468,603 B2 71 72 mg/kg of Paclitaxel (blue dotted line, open circles). Each FIG. 59. Mice were divided into 2 groups (10 mice in each point on the curves represents mean mouse weight of each group). All test articles were administered to mice for five drug and vehicle treated group at the day of assessment. consecutive days via intravenous (iv) injections starting Tumored mouse weight was corrected for the weight of from day O. Group #1 received no injections (black solid tumor. The mean group cutoff is two animals per group. 5 line, open squares) from day 0 to day 11 and then a new FIG. 57 Mice were divided into 6 groups (10 mice in each sequence of Q1 Dx5 with 78.9 mg/kg was begun. Group #5 group). All test articles were administered to mice for five received drug-free lipid formulation (black dotted line, open consecutive days via intravenous (iv) injections starting squares). Each point on the curves represents mean tumor from day O. Groups i2-4 received 105.2 (solid green), 78.9 weight of each drug and vehicle treated group at the corre 10 sponding day of assessment. The mean group cutoff is two (solid red), and 52.6 (solid yellow) mg/kg of formulated animals per group. ART-207. Group #5 received drug-free lipid formulation The data obtained demonstrate effective suppression of (black dotted line, open squares). Group #6 received 15 advanced tumors by formulated ART-207 while its toxic mg/kg of Paclitaxel (blue dotted line, open circles). Each 15 effect was notably decreased. The decreased lethality asso point on the curves represents mean tumor weight of each ciated with the administration of high doses of ART-207 into drug and vehicle treated group at the corresponding day of animals with advanced tumors Supports the selective cellular assessment. The mean group cutoff is two animals per group. uptake of formulated ART-207 by tumor tissue and provides To elucidate the effect of tumor size on the toxicity of a the opportunity to safely adjust the dose in patients with high (78.9 mg/kg) ART-207 dose, and to test the ability of various tumor sizes. The continuous regression of the tumors formulated ART-207 to suppress the growth of advanced following a single course of dosing Suggests that tumors, an experiment on mice with the mean tumor size pseudoLDL nanoparticle formulated ART-207 shows an ~1460 mg was carried out. The human MDA-MB-231 extended efficacy. The slower regrowth of tumors treated mammary tumor Xenografts in the untreated control group with formulated ART-207 in both the initial ATL 3 study (Group 1) grew progressively in all ten mice until Day 11. 25 with a single Q1 DX5 course (FIG. 55) and in the second study where animals with much larger tumors were dosed The animals gained weight until Day 11. The median tumor (FIG. 59. This can be explained by tumor up take and reached three tumor mass doublings in 10.1 days and sequestration of prodrug/drug into tumor cells not actively reached 1.461 mg (mean group tumor weight) in weight on dividing but still growing. When those cells reach entry into Day 11 (see formal report). Three animals were euthanized 30 cell division the sequestered drug exerts its toxic effect. on Day 11 due to tumor ulceration. The remaining seven (Paclitaxel is only effective in cells undergoing division.) mice were treated IV with formulated ART-207 at a dose of Q4DX3 schedule The IV administration of formulated 78.9 mg/kg/injection on a Q1 Dx5 schedule starting on Day ART-207 at a dose of 78.9 mg/kg/injection on a Q4Dx3 11. Two out of seven animals died (with deaths occurring on schedule (Group 7) was tolerated without deaths and Day 18) and animals experienced a maximum mean body 35 resulted in a mean body weight loss of 2% (0.4 g), observed weight loss of 15% (3.5 g), observed on Day 18 (FIG. 58). on Day 8. Treatment with formulated ART-207 at a dose of The lethality associated with the administration of formu 78.9 mg/kg/injection was very effective in the inhibition of lated ART-207 in this group was less (2 out of 7 mice died, the growth of the MDA-MB-231 mammary tumor xeno 29%) than the lethality associated with the administration of 40 grafts. The treatment produced three complete tumor regres formulated ART-207 in Group 5 at the same dose, (6 out of sions, with all three animals remaining tumor-free on Day 10 mice died, 60%) (Table 25g). 56. Growth of the tumors in the group treated with formu lated ART-207 at a dose of 78.9 mg/kg/injection was statis TABLE 25g tically different from the growth of the tumors in the 45 untreated control group (Group 1 vs. Group 7: P<0.001) and Dependence of formulated ART-207 toxicity on tumor size. from the Emulsion Control group (Group 7 vs. Group 8: Toxicity Driven P<0.001), when individual animals' times to reach three Tumor size, mg Dose, mpk Death rate, 9% tumor mass doublings were compared (FIGS. 60a and 60b). The IV treatment with Emulsion Control on a Q4Dx3 150 78.9 60 50 schedule (Group 8) was tolerated without deaths or mean 1461 78.9 33 body weight loss. The median tumor reached three tumor mass doublings in 10.7 days, and reached 1,163 mg in FIG. 58. Mice were divided into 2 groups (10 mice in each weight on Day 11. Growth of the tumors in untreated control group). All test articles were administered to mice for five group and the group treated with Emulsion Control admin consecutive days via intravenous (iv) injections starting 55 istered on a Q4DX3 schedule was not statistically different from day O. Group #1 received no injections (black solid (Group 1 vs. Group 8: P=0.252) nor was the growth of the line, open squares) from day 0 to day 11 and then a new tumors in the two Emulsion Control groups (when admin sequence of Q1 Dx5 with 78.9 mg/kg was begun. Group #5 istered on Q1 DX5 and Q4DX3 schedules, Group 5 vs. Group received drug-free lipid formulation (black dotted line, open 8: P=0.348), when individual animals times to reach three squares). Each point on the curves represents mean mouse 60 tumor mass doublings were compared (FIGS. 60a and 60b). weight of each drug and vehicle treated group at the day of The IV treatment with paclitaxel at a dose of 18.9 assessment. Tumored mouse weight was corrected for the mg/kg/injection on a Q4DX3 schedule (Group 9) was toler weight of tumor. The mean group cutoff is two animals per ated without deaths or mean body weight loss (FIG. 60b). group. The formulated ART-207 treatment resulted in an The treatment with paclitaxel at a dose of 18.9 mg/kg/ effective inhibition of the tumor growth: the median tumor 65 injection was very effective in the inhibition of the growth weight decreased from 1,029 mg on Day 11 (based on seven of the MDA-MB-231 mammary tumor xenografts (FIG. mice) to 104 mg on Day 60 (FIG. 59). 60a). The treatment produced one complete tumor regres US 9,468,603 B2 73 74 sion. Growth of the tumors in the group treated with TABLE 25h. paclitaxel at a dose of 18.9 mg/kg/injection on a Q4Dx3 schedule was statistically different from the growth of the Comparative Effect of Formulated ART-207 and Paclitaxel on End tumors in the untreated control group (Group 1 vs. Group 9: of Study Animal Survival and Number of Tumor-Free Animals. P<0.001) and from the Emulsion Control group (Group 8 vs. Group 9: P-0.001), when individual animals' times to reach Tumor three tumor mass doublings were compared. Tunor Particle Survival free FIG. 60a. Mice were divided into 3 groups (10 mice in Study size, Treatment Dose, size, rate, mice, each group). All test articles were administered to mice on 10 # Compound mg Schedule mpk ill % % day 0, 5, and 9 via intravenous (iv) injections. Group if7 1 Formulated 270 Q1 Dx5 72.6 120 1OO 40 received 78.9 mg/kg of formulated ART-207 (solid red line, ART-2O7 filled squares). Group #8 received drug-free lipid formula Paclitaxel 270 Q1 Dx5 15 NA 2O O tion (black dotted line, open squares). Group #9 received 15 2 Formulated 150 Q1 Dx5 52.6 65 8O 60 18.9 mg/kg of Paclitaxel (blue dotted line, open circles). ART-2O7 Each point on the curves represents mean tumor weight of Paclitaxel 150 Q1 Dx5 15 NA 40 40 each drug and vehicle treated group at the corresponding day of assessment. The mean group cutoff is two animals per group. FIG. 60b. Mice were divided into 3 groups (10 mice in Experiment 26 each group). All test articles were administered to mice on day 1, 5, and 9 via intravenous (iv) injections. Group if7 25 Preparation of ART-207 (Loti AW-004-24) received 78.9 mg/kg of formulated ART-207 (solid red line, Containing Emulsion filled squares). Group #8 received drug-free lipid formula tion (black dotted line, open squares). Group #9 received Purpose: To systematically investigate effect of TG/ART 18.9 mg/kg of Paclitaxel (blue dotted line, open circles). 207 ratio—on particle size, stability and ART-207 incorpo Each point on the curves represents mean mouse weight of ration capacity of formulation. TABLE 26a Formulation composition. Components Weighed, mg (per 100 ml
Date Lotti Material PC TG FC CE U Vit E ART P188 DMPC PS
21 Mar. 2013 OO2.155.1 TSPM 43OO 490 69 148 11 10 505 300 1107 O each drug and vehicle treated group at the day of assessment. TABLE 26b Tumored mouse weight was corrected for the weight of tumor. The mean group cutoff is two animals per group. Ratios for maior formulation components. Both the MTD (ATL-12)) and Efficacy (ATL-3) studies 45 TG.ART-2O7 PC.ART-2O7 PCTG FCfCE clearly demonstrated similar or higher tumor Suppression 0.97 10.72 11.03 O.47 effect of formulated ART-207 compared to Paclitaxel at concentrations below the MTD. In contrast to Paclitaxel the Coarse Suspension was prepared and MF processed tumor suppression effect of formulated ART-207 was not (loth 002.156.1). TABLE 26c Particle size, ART-207 content, and particle stability of resultant emulsion. Formulation Stability Particle size ART-2O7 Days Particle Manufacturing by intensity, Content, Recovery, past size Date Lotti ill mg/ml % mfg ill
22 Mar. 2013 OO2.156.1 63 4.OS 80.3 42 59 accompanied by any significant weight loss. Relative to MFP. FIG. 61 shows that particle size reaches R1 at 69 mm Paclitaxel, the treatment with formulated ART-207 consis- is after 30 min of processing at ~60° C. MFP was stopped and tently resulted in a significant increase in animal Survival material was sterile filtered. Decrease of the particle size rate and higher number of tumor free animals (Table 25h). from 69 to 62 nm was observed after filtration. US 9,468,603 B2 75 76 HPLC Analysis. ART-207 content in resultant emulsion additional 20 min. MF processing at 20°C. resulted in slight determined by HPLC (Taxane Test.M, Rev 2) was 4.05 increase of the particle size to 78 nm and reached R2. MFP mg/ml. Data shoes 83% of the drug used for preparation of this formulation was incorporated into lipid particles (Table was stopped and material was sterile filtered. Decrease of the 26c). Particle size analysis of ART-207 containing formu particle size from 78 to 62 nm was observed after filtration. lation. The resultant ART-207 containing emulsion was HPLC Analysis. highly stable. In FIG. 62 and Table 26c, particle did not increase over 42 days. ART-207 content in resultant emulsion determined by MF processing of formulation with TG/ART-207 ratio HPLC (Taxane Test.M. Rev 2) was 4.8 mg/ml. The data equal to 0.97 resulted in highly stable -60 nm particles. 10 indicate that 94% of the drug used for preparation of this ART-207 incorporation capacity of this formulation was 83%. The lower drug incorporation capacity was expected formulation was incorporated into lipid particles (Table based on the TG/ART-207 ratio of 1. 27c). Particle size analysis of ART-207 containing formu lation. ART-207 containing emulsion was highly stable. In 15 Preparation of Art-207 Containing Formulation for FIG. 64 and Table 27c that particle size did not change over PK/PD Studies 43 days. TMF processing of formulation with TG/ART-207 ratio Experiment 27 equal to 1.17 resulted in highly stable -60 nm particles. ART-207 incorporation capacity of this formulation was 94%. Higher drug incorporation capacity (relative to formu Preparation of ART-207 (Loti AW-004-24) Containing Emulsion lation described in experiment 26) was expected based on 25 the higher TG/ART-207 ratio. A sample of>30 ml of the lot #002.156.3 was shipped to the study site; ~10 ml sample of To prepare of ART-207 containing formulation for PK/PD studies and to systematically investigate effect of TG/ART the lot HO02.156.3 was retained. 207 ratio on particle size, stability, and ART-207 incorpo The material was characterized on the day of shipment. ration capacity of formulation. Data noted in Table 27d. TABLE 27a
Formulation composition.
Components Weighed, mg (per 100 ml)
Date Lotti Material PC TG FC CE U Vit E ART P188 DMPC PS
25 Mar. 2013 OO2.156.2 TSPM 434O 600 70 150 1 0 10 511 300 1126 O
TABLE 27b. TABLE 27d 45 Ratios for maior formulation components. Formulated ART-207. Content and particle size. TG.ART-2O7 PC.ART-2O7 PCTG FCICE Lotti Material ART-207, mg/ml Particle size, nm. 1.17 10.70 9.11 O.47 002.156.3 Formulated ART-207 4.49 61.9 50 Coarse Suspension was prepared and MF processed Table 27d shows that ART-207 content as determined by (loth 002.156.3). HPLC was slightly lower compared to that on the day of TABLE 27c Particle size, ART-207 content, and particle stability of resultant emulsion. Particle size ART-2O7 Formulation Stability Manufacturing by intensity, Content, Recovery, Days past Particle size Date Lot # ill mg/ml 90 mfg ill
25 Mar. 2013 OO2.156.3 62 4.8O 94.O 39 61
MFP. FIG. 63 shows that particle size reaches R1 at 75 nm 65 manufacture, whereas particle size did not change. The after 50 min of processing at ~60° C. Temperature was treatment of animals was performed via single intravenous lowered to 20° C. and material was processed for an injection. US 9,468,603 B2 77 78 TABLE 27e tumor (experiment ATL-5 only) after a single intravenous (IV) injection of formulated paclitaxel or ART-207 to non ART-207 content and doses. tumored female athymic NCr-nu/nu mice and female athy ART-2O7 Dose, Injection Volume, ml mic NCr-nu/nu mice bearing a subcutaneous (SC) human Lotti Material mg/ml mg/kg (per 20g of mouse weight) 5 MDA-MB-231 mammary tumor. 002.156.3 Formulated 4.49 70 O.31 Tumor Model; Experiment ATL-4. Animals were nontu mored. Experiment ATL-5 Material unused for dosing of animals was shipped back 10 to Arbor Therapeutics after the end of the treatment. ART 207 content was assessed. The assessment of material Mice were implanted with fragments of the human MDA shipped back was carried out side by side with material MB-231 mammary tumor from an in vivo passage. The day retained. Table 27f shows that material returned from the of tumor fragments implantation was designated as Day 0. PK/PD study site evaluated by Taxane Test.M Rev. 2 15 Individual tumors of 30 animals grew to 908-1,437 mg in method and material retained have similar drug content. weight (908-1,437 mm3 in size) on Day 23 after tumor ART-207 content was not significantly lower compared to fragments implantation, one day before the treatment day. that on the day of shipment. Decrease of ART-207 content Thirty selected animals were assigned to ten treatment relative to that on the day of manufacture was observed groups so that the mean tumor weights in all groups on Day (Tables 27c and 270. 23 were as close to each other as possible (mean tumor weights ranged from 1,056 to 1,178 mg, median tumor TABLE 27f weights ranged from 908 to 1,152 mg). Treatment in both experiments ATL-4 and ATL-5 was administered on the HPLC Analyses of ART-207 Content, mg/mL. same day. Taxane Test.M 25 Rev 2 Experiment ATL-4 On the day of Taxane Test.M Rev 2 Taxane Test.M Rev 2 Material shipment Returned Material Retained Material The study consisted of ten groups of three nontumored mice per group for a total of 30 mice on Day 1, one day OO2.156.3 4.49 4.34 4.30 before the treatment. All treatments were administered as a 30 single IV injection on Day 2. Animals in Groups 1-5 were Particle size of the material remaining from the PK/PD treated with formulated ART-207 at a dose of 70 mg/kg. Study and that returned to Arbor was assessed on the day of Animals in Groups 6-10 were treated with paclitaxel at a its delivery. Assessment of returned material was carried out dose of 18.9 mg/kg. The formulated ART-207 dose of 70 side by side with material retained. Data are presented in mg/kg was 2.6x molar equivalent of a paclitaxel dose of 18.9 Table 27g and reflect monitoring of particle size from day 0 mg/kg, based on the molecular weight of formulated ART to the end of PK/PD treatment phase. The particle size of 207 of 1219.6 and molecular weight of paclitaxel of 853.9. emulsions received back from the study site was slightly (not significantly) higher relative to that of retained material. Experiment ATL-5 Particle size of the formulated ART-207 did not change from its day of manufacture until the dosing date of the PK/PD 40 The study consisted often groups of three mice per group study (Tables 27c and 27g). There was no significant dif for a total of 30 mice bearing human MDA-MB-231 mam ference of the particle size of returned and retained material mary tumor on Day 23 after tumor fragment implantation, as well. one day before the treatment. All treatments were adminis TABLE 27g Particle size, nm Day of Shipment to study Returned from study site, Retained at Arbor Material site, (14 May 2013) (31 May 2013) Therapeutics, (31 May 2013) OO2.156.3 61.9 67.1 65.8
The formulated ART-207 demonstrated selectivity in the tered as a single IV injection on Day 24. Animals in Groups PK/PD Study (ATL-4 and 5). Data suggest selective cellular 55 1-5 were treated with formulated ART-207 at a dose of 70 uptake of formulated ART-207 by the organs actively mg/kg. Animals in Groups 6-10 were treated with paclitaxel expressing LDL-receptors (see also formal report "DETER at a dose of 18.9 mg/kg. The formulated ART-207 dose of 70 MINATION OF PLASMA AND TISSUE CONCENTRA mg/kg was 2.6x molar equivalent of a paclitaxel dose of 18.9 TIONS OF ART-2O7 AND PACLITAXEL, IN NONTU mg/kg. MORED AND TUMORED MICE AFTER A SINGLE 60 Plasma levels of Paclitaxel, ART-207, and ART-207 INTRAVENOUS INJECTION WITH FORMULATED derived Paclitaxel. For mice administered a single IV dose ART-207 OR PACLITAXEL. Southern Research Institute, of formulated ART-207, a mean peak plasma concentration Birmingham, Ala. 35255-5305) (Cmax) of ART-207 of 640 ug/mL was observed at 5 Experiments ATL-4 and ATL-5 minutes (0.083 hours) after dosing (earliest time point); the 65 mean concentration of paclitaxel in plasma at this time was To determine the concentration of paclitaxel and ART-207 2.248 g/mL (2237 ng/mL). ART-207 subsequently was derived paclitaxel in plasma, brain, heart, liver, lungs and eliminated from plasma with an apparent terminal elimina US 9,468,603 B2 79 80 tion half-life of 2.8 hrs; the apparent terminal elimination FIG. 67. Tumored mice were subdivided into 5 groups (3 half-life of paclitaxel in plasma for animals administered mice per each group). All mice were injected intravenously formulated ART-207 was slower and was 11.5 hr. The low with 70 mg/kg of formulated ART-207. Blood samples rate of clearance of ART-207 (22.0 mL/hr/kg) and small collected at 5, 30, 60,240, and 480 min after single bolus volume of distribution at steady state of ART-207 (88.0 injection. Each time point represents 3 mice. Tissue con centration is expressed as area under the concentration curve mL/kg) were indicative of limited metabolism/elimination/ (AUC) from 0 to last time point analyte was quantifiable (8 tissue distribution of the compound. The AUClast for ART hrs). Areas under the mean concentration versus time curve 207 in plasma was 2794 hrug/mL and that for paclitaxel was from 0 to the time of the last quantifiable sample (AUC last) 14.4 hrug/mL, indicating that the systemic exposure to 10 calculated using the linear/log trapezoidal rule. Difference ART-207 was approximately 200-fold greater than that to between Paclitaxel plasma levels of non-tumored and paclitaxel (FIG. 65). For mice administered a single IV dose tumored mice injected with formulated ART-207 was sig of paclitaxel, a mean peak plasma concentration of pacli nificant. When the Volume of Distribution of formulated taxel of 32.9 g/mL was observed at 5 minutes (0.083 hours) 15 ART-207 and Paclitaxel were evaluated post iv. dosing, the after dosing (earliest time point); thereafter, paclitaxel was Volume of Distribution of formulated ART-207 was signifi eliminated from plasma with an apparent terminal elimina cantly lower (6 fold) than that of Paclitaxel indicating tion half-life of 1.0 hour. AUClast for paclitaxel in plasma formulated ART-207 remains within the vascular system and was 37.7 hrug/mL. The total body clearance of paclitaxel is not distributed into tissues. The long circulating pseudo (500 mL/hr/kg) and volume of distribution of paclitaxel at LDL nanoparticulate formulation can continue to concen steady State (658 mL/kg) were higher than the corresponding trate into tumor tissues. This is consistent with the observed values determined for ART-207. Although the relative tissue extended efficacy of formulated ART-207 in both ATL 2 and distribution of paclitaxel was similar to that observed for ATL 3 studies. Also, tumor tissue cellular up take of the mice given formulated ART-207, mean peak tissue concen formulation into growing but not dividing tumor cells would trations of paclitaxel were higher for mice administered 25 provide a reservoir of prodrug? drug to exert a cytotoxic paclitaxel than for mice administered a 2.6-fold higher molar effect once those cells begin cell division. The cytotoxic equivalent dose of formulated ART-207. effect of taxanes is only present for dividing cells. A reservoir of drug in growing but not dividing cells is Change in Mean Concentration of ART-207 and Pacli desirable since not all tumor cells are dividing at any given taxel in Plasma Over Time 30 FIG. 65 Tumored and/or non-tumored mice were subdi time so that when these quiescent cells do begin to divide vided into 5 groups (3 mice per each group). All mice were leading to tumor recurrence, they are killed by the reservoir injected intravenously with 70 mg/kg of formulated ART of drug presence. 207. Blood samples were collected at 5, 30, 60,240 and 480 Organ distribution of Paclitaxel in mice injected with min after single bolus injection. Each time point represents 35 Paclitaxel and ART-207. Organs were harvested and then 3 mice. Plasma concentration is expressed as area under the analyzed without prior perfusion or rinsing in PBS or saline. concentration curve (AUC) for each time point analyzed. With the small volume of distribution observed and reported Based on AUClast values (areas under the mean concentra for formulated ART-207, we will focus our data analysis on tion versus time curve from 0 to the last quantifiable distribution of ART-207 derived Paclitaxel which is a more sample), the systemic exposure to Paclitaxel in mice injected 40 specific indicator of formulated ART-207 tissue distribution with Paclitaxel was similar for non-tumored and tumored and processing that result in local release of the Paclitaxel mice. The difference between Paclitaxel levels of Non moiety. Non-tumored mice Tissue levels of Paclitaxel were tumored and Tumored mice injected with Paclitaxel was not significantly lower in mice injected with ART-207 relative to significant (FIG. 66). Data indicated that absence or pres 45 that in mice injected with Paclitaxel (FIG. 68). ence of tumor did not affect plasma Paclitaxel content, and FIG. 68. Non-tumored mice were subdivided into 5 therefore inability of Paclitaxel to selectively target tumor groups (3 mice per each group). All mice were injected tissue. intravenously with 70 mg/kg of formulated ART-207 or 18.9 FIG. 66. Non-tumored mice were subdivided into 5 mg/kg of Paclitaxel. Blood samples and organs were col groups (3 mice per each group). All mice were injected 50 lected at 5, 30, 60, 240, and 480 min after single bolus intravenously with 18.9 mg/kg of Paclitaxel. Blood samples injection. Each time point represents 3 mice. Tissue con were collected at 5, 30, 60, 240, and 480 min after single centration is expressed as area under the concentration curve bolus injection. Each time point represents 3 mice. Tissue (AUC) from 0 to last time point analyte was quantifiable (8 concentration is expressed as area under the concentration 55 hrs). Areas under the mean concentration versus time curve curve (AUC) from 0 to last time point analyte was quanti from 0 to the time of the last quantifiable sample (AUClast) fiable (8 hrs). Areas under the mean concentration versus were calculated using the linear/log trapezoidal rule. Dif time curve from 0 to the time of the last quantifiable sample ference between Paclitaxel levels of non-tumored mice (AUClast) were calculated using the linear/log trapezoidal injected with Paclitaxel or formulated ART-207 was signifi 60 cant for all assessed tissues (p<0.05). Tumored mice Pacli rule. Plasma levels of ART-207 derived Paclitaxel in ART taxel levels were lower in all non-target organs of mice 207 treated tumored animals are significantly lower com injected with ART-207 relative to that in mice injected with pared to that in ART-207 treated non-tumored mice (FIG. Paclitaxel, whereas Paclitaxel concentration in tumor tissue 67). Data demonstrate significant impact of tumor on plasma was significantly higher in mice injected with formulated levels of ART-207 derived paclitaxel suggesting improved 65 ART-207 (FIGS. 69, 70). Results are in agreement with targeting capability of the formulated ART-207 relative to Paclitaxel plasma concentration data and Suggest selective Paclitaxel. targeting of tumor site by formulated ART-207. US 9,468,603 B2 81 82 FIG. 69. Tumored mice were subdivided into 5 groups (3 sample (AUClast) were calculated using the linear/log trap mice per each group). All mice were injected intravenously ezoidal rule. In both non-tumored and tumored mice treated with 70 mg/kg of formulated ART-207 or 18.9 mg/kg of with formulated ART-207 the highest and comparable con Paclitaxel. Blood samples and organs were collected at 5, centrations of Paclitaxel were observed in the liver, lung and 30, 60, 240 and 480 min after single bolus injection. Each tumor tissues (FIGS. 68, 69), which are the major LDL time point represents 3 mice. Tissue concentration is receptor expressing sites. Data obtained Suggest selective expressed as area under the concentration curve (AUC) from cellular uptake of formulated ART-207 by the organs 0 to last time point analyte was quantifiable (8 hrs). Areas actively expressing LDL-receptors and Support LDL-recep under the mean concentration versus time curve from 0 to tor dependent mechanism for cellular internalization of the time of the last quantifiable sample (AUClast) were 10 formulated ART-207. The PK/PD data are in agreement with calculated using the linear/log trapezoidal rule. Difference the results from the efficacy and toxicity studies which show between Paclitaxel levels of non-tumored mice injected with similar or higher potency of formulated ART-207 compared Paclitaxel or formulated ART-207 was significant for all to Paclitaxel while lower toxicity is observed for formulated assessed tissues (p<0.05). ART-2O7. FIG. 70. Tumored mice were subdivided into 5 groups (3 15 mice per each group). All mice were injected intravenously Experiment 28 with 70 mg/kg of formulated ART-207 or 18.9 mg/kg of Paclitaxel. Tumors were collected at 5, 30, 60,240 and 480 Preparation of ART-207 (Loti AW-004-24) min after single bolus injection. Each time point represents Containing Emulsion 3 mice. Tissue concentration is expressed as area under the concentration curve (AUC) from 0 to last time point analyte To systematically investigate effect of TG/ART-207 ratio was quantifiable (8 hrs). Areas under the mean concentration (Table 28b) on particle size, stability and ART-207 incor versus time curve from 0 to the time of the last quantifiable poration capacity of the formulation. TABLE 28a. Formulation composition. Components Weighed, mg (per 100 ml
Date Lot # Material PC TG FC CE U Vit E ART P188 DMPC PS
26 Mar. 2013 OO2.158.1 TSPM 4345 698 70 1 SO 11 10 S29 301 1130 O
TABLE 28b. 35 Ratios for major formulation components. TG.ART-2O7 PC.ART-2O7 PCTG FCfCE
1.32 10.35 7.84 O.47 40 Coarse Suspension was prepared and MF processed (loth 002. 158.2). TABLE 28c Particle size, ART-207 content, and particle stability of resultant emulsion. Particle size ART-2O7 Formulation Stability Manufacturing by intensity, Content, Recovery, Days past Particle size Date Lot # ill mg/ml 90 mfg ill
27 Mar. 2013 OO2.158.2 80 4.74 89.6 37 72
MFP. In FIG. 71, particle size reaches 102 nm after 10 min of processing at ~60° C. MFP was stopped and material was sterile filtered. Decrease of the particle size from 102 to 81 nm was observed after filtration. 60 HPLC Analysis. ART-207 content in resultant emulsion determined by HPLC (Taxane Test.M. Rev 2) was 4.74 mg/ml. Data indicate that 89.6% of the drug used for preparation of this formulation was incorporated into lipid particles (Table 28c). Particle size analysis of ART-207 65 containing formulation. The ART-207 containing emulsion was stable. In FIG. 72 and Table 28c, particle size did not increase over 37 days. US 9,468,603 B2 83 84 MF processing of formulation with TG/ART-207 ratio equal to 1.32 resulted in highly stable -70 nm particles. ART-207 incorporation capacity of this formulation was 89.6%. Drug incorporation capacity did not increase com pared to the formulation with TG/ART-207 ratio equal to 5 1.17 (experiment 27).
Experiment 29 10 Preparation of ART-207 Containing Emulsion
To systematically investigate effect of TG/ART-207 ratio is (Table 33b) on particle size, stability and ART-207 incor poration capacity of the formulation. TABLE 29a Formulation composition. Components Weighed, ng (per 100 ml
Date Lot # Material PC TG FC CE U Vit E ART P188 DMPC PS
28 Mar. 2013 OOS.1.1 TSPM 434O 8SO 70 1 SO 11 10 530 300 1130 O
TABLE 29b.
Ratios for major formulation components. 30 TG ART-2O7 PC.ART-2O7 PCTG FCICE
1.60 10.32 6.44 O.47
Coarse Suspension was prepared and MF processed 35 (loth 005.1.2). TABLE 29c Particle size, ART-207 content, and particle stability of resultant emulsion. Particle size ART-2O7 Formulation Stability Manufacturing by intensity, Content, Recovery, Days past Particle size Date Lot # nm. mg/ml 90 mfg ill 29 Mar. 2013 OO5.1.2 74 4.74 89.5 35 78
MFP compared to formulation with TG/ART-207 ratio equal to In FIG. 73, particle size reaches 77 nm after 10 min of 1.17 and 1.32 (experiment 27 and 28, respectively). Data processing at ~60° C. MFP was stopped and filtered. No 50 suggest that the optimum for TG/ART-207 ratio is in the change of the particle size was observed after filtration. range of 1.2-1.3. HPLC Analysis. ART-207 content in resultant emulsion determined by HPLC (Taxane Test.M. Rev 2) was 4.74 mg/ml. The data indicate that 89.5% of the drug used for 55 preparation of this formulation was incorporated into lipid Experiment 30 particles (Table 29c). Particle size analysis of ART-207 containing formulation. The resultant ART-207 containing Preparation of ART-207 (Loti AW-004-24) emulsion was fairly stable. In FIG.74 and Table 29c, particle Containing Emulsion size increased by 4 nm over 35 days. 60 MF processing of formulation with TG/ART-207 ratio To generate -90-100 nm ART-207 containing particles equal to 1.6 resulted in relatively stable -75 nm particles. A and to assess their drug-incorporation capacity and stability. slight increase of particle size observed for loti 005.1.2 over Since resultant particle size in experiments 28 and 29 was monitoring period suggests initial signs of instability accom 65 significantly below targeted (~90-100 nm) the MF process panying higher TG/ART ratio. ART-207 incorporation ing pressure was lowered to 10,000 PSI. The TG/ART ratio capacity of this formulation was 89.5% and did not increase was 1.35. US 9,468,603 B2 85 86 TABLE 30a Formulation composition. Components Weighed, ng (per 100 ml
Date Lot # Material PC TG FC CE U Vit E ART P188 DMPC PS
29 Mar. 2013 OOS.2.1 TSPM 42SO 73S 70 150 1 0 10 543 300 1123 O
10 TABLE 3Ob For exact composition and details see Appendix, Master Tables 2 and 3. Ratios for major formulation components. TG/ART-207 Ratio. The concentration of TG and specifi TG ART-2O7 PC.ART-2O7 PCTG FCICE cally TG/ART-207 ratio is the major factor that determines 15 stability of drug-containing emulsion. The optimum range of 1.35 9.90 7.31 O.47 TG/ART-207 ratios is 0.97-1.4. FIG. 77 shows that increase of the TG/ART-207 ratio above 0.97 facilitates ART-2O7 Coarse Suspension was prepared and MF processed incorporation while resulting in stable particles. ART-207 (loth 005.2.2). incorporation reaches the maximum and plateaus at TABLE 3Oc Particle size, ART-207 content, and particle stability of resultant emulsion. Particle size ART-2O7 Formulation Stability Manufacturing by intensity, Content, Recovery, Days past Particle size Date Lot # nm. mg/ml 90 mfg ill 29 Mar. 2013 OOS.2.2 102 4.85 89.3 35 101
30 MFP TG/ART-207 ratio -1.3-1.4. Further increase of TG/ART In FIG. 75 that particle size reaches 105 nm after 10 min 207 ratio does not enhance drug incorporation capacity of of processing at ~60° C. MFP was stopped and material the formulation and moreover, results in unstable nanopar filtered. No significant change of the particle size was ticles. 35 FIG. 77. Effect of TG/ART-207 ratio on ART-207 incor observed after filtration. poration capacity of formulation (black Solid line, left axis) HPLC Analysis. and particle size increase (black dotted line, right axis). ART-207 content in resultant emulsion determined by ART-207 incorporation capacity (i.e. drug content of resul HPLC (Taxane Test.M. Rev 2) was 4.85 mg/ml. This data tant emulsion) is expressed as percent of amount of drug indicate that 89.3% of the drug used for preparation of this weighed for preparation of this emulsion. Particle size formulation was incorporated into lipid particles (Table 40 increase is expressed as percent of particle size change over 30c). Particle size analysis of ART-207 containing formu time relative to that on the day of manufacturing. lation. The resultant ART-207 containing emulsion was Processing Temperature. highly stable. FIG. 76 and Table 30c show that particle size Although processing of material at lower temperatures did not change over 35 days. (-20° C.) yields smaller particles, processing at 60° C. MF processing of formulation with TG/ART-207 ratio 45 results in more stable emulsions. FIG. 78 shows that pro equal to 1.35 resulted in highly stable ~105 nm particles. cessing at ~20° C. results in ~55 nm drug-containing nano ART-207 incorporation capacity of this formulation was particles, whereas 60° C. processing yields ~65 nm drug 89.3% as expected for this TG/ART-207 ratio. Data obtained containing nanoparticles. ~15% particle size increase during indicate that the current formulation has the capacity to the storage was observed only in the material processed at generate stable particles in at least 40-100 nm range. 50 -20° C. (FIG. 78). Both “smaller” and “bigger” nanopar Composition and Ratios. Unique composition with spe ticles contained similar amounts of ART-207. cific ratios of formulation components that produces stable FIG. 78. Dependence of particle size (black solid line, left drug-free and drug-containing nanoparticles in the range axis) and percent of particle size increase during the storage from 40 to 100 nm. The optimal range of total solids (black dotted line, right axis) on processing temperature. calculated as Weight/Volume (W/V) percent is 6.5-7.5% 55 Development of lipid-based formulations for incorpora (TS, 96). The optimal range of total lipids calculated as tion of 287. Optimization of 287 incorporation capacity, Weight/Volume (W/V) percent is 5.5-6.5% (TL, %). Ratios particle size and stability. For all examples described below, for major components are given in the Tables 30d. see also Master Tables 1, 2, and 3 (Appendix II). Experiment 31 TABLE 3Od 60 Preparation of 287 (Loti ISI-30052013-1) Ratios for major formulation components. Containing Lipid Emulsion TGART2O7 PCART2O7 PCTG FCCE PCTC TGTC Investigate the capacity of formulation developed for O.97-16 7.5-12 3.5-11 O.3-0.6 17-25 2-7 65 preparation of ART-207-containing nanoparticles to incor porate structurally different 287, a lipophilic drug, not a prodrug derivative. US 9,468,603 B2 87 88 TABLE 31a Formulation composition. Components Weighed, mg (per 100 ml
Date Lotfi Material PC TG FC CE U Vit E ART P188 DMPC PS
O4 Jun. 2013 OOS.16.1 TSPM S370 606 71. 149 10 10 SO8 300 O O
10 TABLE 31b. step at the end of MF processing. The HPLC data are in line - - with notable decrease of the particle size after filtration. Data - Ratos for major formulation components Suggest particle size heterogeneity of resultant emulsion and TGART 287 PCART 287 PCTG FCICE retention of large 287 containing particles on the filter. No 15 decrease of 287 content from 2.81 to 2.64 mg/ml (6%) was 1.19 10.57 8.86 O.47 observed in resultant emulsion after 28 days of storage at room temperature (Table 31c). Particle size analysis of 287 Coarse Suspension was prepared and MF processed containing formulation. FIG. 85 and Table 39d show that (loth 005. 16.2). Buffer: 10 mM Tris-HCl pH 7.4. particle size increased from 53.2 to 72.6.4 nm over 6 days TABLE 31c 287 content and Stability of resultant emulsion. 287 Formulated 287 Stability Manufacturing Content, Recovery 1, Days past Content, Recovery 2, Date Lot # mg/ml % mfg mg/ml % O4 Jun 2013 OO5.16.2 2.81 55.3 28 2.64 94.0
30 Recovery 1, 96 relative to amount of 287 used for and then stabilized in the range of 67 nm No further change formulation; Recovery 2, 96 relative to 287 content deter- of particle size observed up to day 28 (FIG. 80). mined in emulsion on the day of manufacturing; Particle size analysis data are in line with HPLC data indicates: a) decrease of nanoparticle size and 287 content TABLE 31d 35 after final filtration step; and b) relative stability of resultant Particle size and stability of resultant emulsion. emulsion and formulated 287. Emulsion was prepared in 10 Particle size Formulation Stability mM Tris-HCl buffer, pH 7.4. Manufacturing by intensity, Days past Particle size 40 Date Lotti ill mfg ill Experiment 32
4 Jun. 2013 OOS.16.2 56.4 28 674 Preparation of 287 Containing Lipid Emulsion MFP. In FIG. 79, particle size reached R1 at -80.6 nm 45 after 100 min of processing at 55-65° C. MFP was stopped Investigate the capacity of DMPC-containing formulation and filtered. A decrease of particle size from 80.6 to 56.4 nm developed for preparation of ART-207-containing stable was observed after filtration (FIG. 79). nanoparticles to incorporate structurally different 287. TABLE 32a Formulation composition. Components Weighed, mg (per 100 ml
Date Lot # Material PC TG FC CE U Vit E ART P188 DMPC PS
OS Jun. 2013 OOS.17.1 TSPM 436O 602 71 150 1 0 10 SO7 3O2 1089 O
HPLC Analysis. 287 content in resultant emulsion deter- TABLE 32b mined by HPLC (Taxane Prodrug.M) was 2.81 mg/ml. Data 60 indicate that only 55.3% of the drug used for preparation of Ratios for major formulation components. this formulation was incorporated into lipid particles (Table 31c). HPLC determined drug content of the unfiltered emul- TGART 287 PCART 287 PCTG FCfCE sion was 4.40 mg/ml. Data indicate that 86.6% of the drug used for preparation of this formulation was incorporated 65 1.19 10.75 9.OS O.47 into lipid particles and suggests that Substantial portion (-36%) of formulated 287 was lost during final filtration US 9,468,603 B2 89 90 Coarse Suspension was prepared and MF processed (loti 005.14.2). Buffer: Acetate (10 mM) buffered saline pH 5.5. TABLE 32c 287 content and stability of resultant emulsion. 287 Formulated 287 Stability Manufacturing Content, Recovery 1, Days past Content, Recovery 2, Date Lot # mg/ml % mfg mg/ml O6 Jun 2013 OOS.18.1 281 SS.4 26 2.82 100.4
Recovery 1, 96 relative to amount of 287 used for 15 days of storage at room temperature (Table 32c). Particle formulation; Recovery 2, 96 relative to 287 content deter size analysis of 287 containing formulation. No significant mined in emulsion on the day of manufacturing. particle size change observed during 26 days (FIG. 82 and Table 32c). Data indicate high stability of the resultant TABLE 32d emulsion similar to that of ART-207 containing emulsions when the same lipid formulation was used. Particle size and stability of resultant emulsion. Particle size analysis data are in line with HPLC data indicating: a) notable decrease of nanoparticle size and 287 Particle size Formulation Stability content after final filtration step; and b) high stability of Manufacturing by intensity, Days past Particle size resultant emulsion and formulated 287. Emulsion was pre Date Lotti ill mfg ill pared in 10 mM Tris-HCl buffer, pH 7.4. Addition of DMPC 25 markedly improves Stability of 287-containing emulsion. A 6 Jun 2013 OOS.18.1 47.5 26 51.7 similar effect of DMPC was observed for ART-207-contain ing emulsions. MFP In FIG. 81, particle size reached R1 at -99 nm after Composition and Ratios. Unique composition with spe 30 min of processing at ~60° C. Lowering the temperature 30 cific ratios of formulation components that produces stable to -20° C. resulted in increase of the particle size to 161.7 drug-free and drug-containing nanoparticles in the range nm Raising the temperature to 60° C. and processing for 30 from 40 to 100 nm. The optimal range of total solids min resulted in particle size decrease to 97 nm and reaching calculated as Weight/Volume (W/V) percent is 6.9-7.1% R2 (FIG. 81). MFP was stopped and material was filtered. (TS, 96). The optimal range of total lipids calculated as Decrease of particle size from 97.7 to 47.5 nm was observed 35 Weight/Volume (W/V) percent is 6.1-6.3% (TL, %). Data after filtration (FIG. 81). indicate that lipid formulation optimized for ART-207 is HPLC Analysis. 287 content in resultant emulsion deter Suitable for generation of stable nanoparticular emulsions mined by HPLC (Taxane Prodrug.M) was 2.81 mg/ml. Data containing a broad variety of lipophilic structurally diverse indicate that only 55.4% of the drug used for preparation of compounds. Examples of ratios for major components are in this formulation was incorporated into lipid particles (Table 40 the Table 32e. 32c). HPLC determined drug content of the unfiltered emul sion was 4.56 mg/ml. Data indicate that 89.9% of the drug TABLE 32e used for preparation of this formulation was incorporated into lipid particles and suggests that Substantial portion Ratios for maior formulation components. (-38%) of formulated 287 was lost during final filtration 45 TG287 PC 287 PCTG FCICE PCTC TGTC step. HPLC data are in line with notable decrease of the particle size after filtration. Data Suggest particle size het O.97-1.6 7.5-12 3.5-11 O.3-0.6 17-25 2-7 erogeneity of resultant emulsion and retention of large 287 containing particles on the filter. No change of 287 content For exact composition and details see Master Tables 5 and or particle size was observed in resultant emulsion after 26 6. MASTER TABLE 1. Total Solids Pre-Mixes (TSPM) and corresponding resultant emulsions. Preparation Processing Material date Lotti Material date Lotti
TSPM 17 Dec. 2012 OO2.102.1 Emulsion 18 Dec. 2012 OO2.103.1 TSPM 17 Dec. 2012 OO2.102.2 Emulsion 19 Dec. 2012 OO2.104.1 TSPM 18 Dec. 2012 OO2.103.2 Emulsion 19 Dec. 2012 OO2.105.1 TSPM 9 Jan 2013 OO2.107.2 Emulsion 9 Jan 2013 OO2.108.1 TSPM 9 Jan 2013 OO2.108.2 Emulsion 9 Jan 2013 OO2.109.1 TSPM 10 Jan 2013 OO2.109.2 Emulsion 10 Jan 2013 OO2.110.1 TSPM 10 Jan 2013 OO2.109.3 Emulsion 11 Jan. 2013 OO2.1111 TSPM 11 Jan. 2013 OO2.110.4 Emulsion 11 Jan. 2013 OO2.111.2 TSPM 15 Jan 2013 OO2.115.4 Emulsion 16 Jan 2013 OO2.116.1 TSPM 15 Jan 2013 OO2.115.1 Emulsion 19 Jan 2013 OO2.118.0 TSPM 15 Jan 2013 OO2.118.1 Emulsion 19 Jan 2013 OO2.118.00 TSPM 15 Jan 2013 OO2.115.2 Emulsion 20 Jan. 2013 OO2.1194 US 9,468,603 B2 91 MASTER TABLE 1-continued
Total Solids Pre-Mixes (TSPM) and corresponding resultant emulsions. Preparation Processing Material date Lotti Material date Lotti
TSPM 15 Jan 2013 OO2.115.3 Emulsion 21 Jan 2013 OO2.121.4 TSPM 15 Jan 2013 OO2.115.3 Emulsion 22 Jan 2013 OO2.122.0 TSPM 22 Jan. 2013 OO2.122. Emulsion 23 Jan 2013 OO2.1231 TSPM 22 Jan. 2013 OO2.122. Emulsion 23 Jan 2013 OO2.123.12 TSPM 22 Jan. 2013 OO2.122. Emulsion 23 Jan 2013 OO2.123.13 TSPM 24 Jan. 2013 OO2.125. Emulsion 25 Jan 2013 OO2.125.2 TSPM 24 Jan. 2013 OO2.125. Emulsion 25 Jan 2013 OO2.125.22 TSPM 8 Feb. 2013 002.131. Emulsion 9 Feb. 2013 OO2.131.2 TSPM 9 Feb. 2013 002.134. Emulsion 9 Feb. 2013 OO2.134.2 TSPM 11 Feb. 2013 OO2.135. Emulsion 12 Feb. 2013 OO2.136.2 TSPM 11 Feb. 2013 OO2.135. Emulsion 12 Feb. 2013 OO2.136.22 TSPM 11 Feb. 2013 OO2.135. Emulsion 12 Feb. 2013 OO2.136.23 TSPM 14 Feb. 2013 OO2.137. Emulsion 15 Feb. 2013 OO2.137.3 TSPM 14 Feb. 2013 OO2.137.2 Emulsion 15 Feb. 2013 OO2.137.4 TSPM 14 Feb. 2013 OO2.137.2 Emulsion 15 Feb. 2013 OO2.137.42 TSPM 14 Feb. 2013 OO2.137.2 Emulsion 15 Feb. 2013 OO2.137.43 TSPM 21 Feb. 2013 002.139. Emulsion 21 Feb. 2013 OO2.139.2 TSPM 21 Feb. 2013 002.140. Emulsion 21 Feb. 2013 OO2.140.2 TSPM 21 Feb. 2013 002.140. Emulsion 21 Feb. 2013 OO2.140.22 TSPM 21 Feb. 2013 002.140. Emulsion 21 Feb. 2013 OO2.140.23 TSPM 21 Feb. 2013 002.140. Emulsion 21 Feb. 2013 OO2.140.24 TSPM 13 Mar. 2013 OO2.150. Emulsion 14 Mar. 2013 OO2.151.5 TSPM 13 Mar. 2013 OO2.150.2 Emulsion 14 Mar. 2013 OO2.151.6 TSPM 13 Mar. 2013 OO2.150.3 Emulsion 14 Mar. 2013 OO2.151.7 TSPM 13 Mar. 2013 OO2.1504 Emulsion 14 Mar. 2013 OO2.151.8 TSPM 15 Mar. 2013 OO2.152. Emulsion 15 Mar. 2013 OO2.153.1 TSPM 15 Mar. 2013 OO2.152.2 Emulsion 15 Mar. 2013 OO2.153.2 TSPM 21 Mar. 2013 OO2.155. Emulsion 22 Mar. 2013 OO2.156.1 TSPM 25 Mar. 2013 OO2.156.2 Emulsion 25 Mar. 2013 OO2.156.3 TSPM 26 Mar. 2013 OO2.158. Emulsion 27 Mar. 2013 OO2.158.2 TSPM 28 Mar. 2013 OOS.1.1 Emulsion 29 Mar. 2013 OOS.1.2 TSPM 29 Mar. 2013 OOS.2.1 Emulsion 29 Mar. 2013 OOS.2.2 TSPM 1 Apr. 2013 005.2.1 Emulsion 1 Apr. 2013 OOS.23
MASTER TABLE 2 Composition of TSPM (Total Solids Pre-Mix). TS, TL, Components Weighed, ng (per 100 ml % %
Date Lot # PC TG FC CE U VitE ART P188 DMPC PS WV WV
7 Dec. 2012 OO2.102.1 233O 1239 206 1S3 10 O O O O O 39 3.9 7 Dec. 2012 OO2.102.2 2338 1247 203 152 10 O O O O O 4.0 3.9 8 Dec. 2012 OO2.103.2 2176 1145 184 141 10 O 32O O O O 4.0 3.6 9 Jan. 2013 OO2.107.2 4157 1397 7O 347 10 O 648 O O O 6.6 6.O 9 Jan. 2013 OO2.108.2 SO2S 1397 71. 216 10 O 648 O O O 7.4 6.7 O Jan. 2013 OO2.109.2 S1 SS 1390 7O 213 10 O 648 O O O 7.5 6.8 O Jan. 2013 OO2.109.3 S17O 1392 7O 213 10 O 213 O O O 7.1 6.8 1 Jan 2013 OO2.110.4 5900 652 69 150 10 O 648 O O O 7.4 6.8 5 Jan. 2013 OO2.115.4 S123 1390 70 212 10 O 648 1OOO O O 8.5 6.8 5 Jan 2013 OO2.115. S2OO 1390 7O 213 10 O O O O O 6.9 6.9 5 Jan 2013 OO2.118. S209 1387 69 213 10 O O O O O 6.9 6.9 5 Jan. 2013 OO2.115.2 S2OO 1389 7O 213 10 O 673 O O O 7.6 6.9 5 Jan. 2013 OO2.115.3 S18S 1393 7O 213 10 O 671 O O O 7.6 6.9 5 Jan. 2013 OO2.115.3 S18S 1393 7O 213 10 O 671 250 O O 7.8 6.9 22 Jan 2013 OO2.122. SO8S SO1 7O 148 10 O 500 256 O O 6.6 5.8 22 Jan 2013 OO2.122. SO8S SO1 7O 148 10 O 500 256 O O 6.6 5.8 22 Jan 2013 OO2.122. SO8S SO1 7O 148 10 O 500 256 O O 6.6 5.8 24 Jan 2013 OO2.125. 4085 S11 70 150 10 O 503 25O 1053 O 6.6 5.9 24 Jan 2013 OO2.125. 4085 S11 70 150 10 O 503 475 1053 O 6.9 5.9 8 Feb. 2013 002.131. S190 701 69 148 10 O O 251 O O 6.4 6.1 9 Feb. 2013 002.134. 522O 697 69 149 10 O 529 250 O O 6.9 6.1 11 Feb. 2013 OO2.135. 418O 70S 68 147 10 O 525 3OO 1098 O 7.0 6.2 11 Feb. 2013 OO2.135. 418O 70S 68 147 10 O 525 3OO 1098 O 7.0 6.2 11 Feb. 2013 OO2.135. 418O 70S 68 147 10 O 525 3OO 1098 O 7.0 6.2 14 Feb. 2013 OO2.137. S36O 1408 70 1 SO 10 O O O O 200 7.2 7.2 14 Feb. 2013 OO2.137.2 S390 1391 7O 148 10 O 524 O O 200 7.7 7.2 14 Feb. 2013 OO2.137.2 S390 1391 7O 148 10 O 524 O O 200 7.7 7.2 14 Feb. 2013 OO2.137.2 S390 1391 7O 148 10 O 524 O O 200 7.7 7.2 21 Feb. 2013 002.139. 421.O 700 70 150 10 O O 297 1060 O 6.5 6.2 US 9,468,603 B2 93 MASTER TABLE 2-continued Composition of TSPM (Total Solids Pre-Mix). TS, TL, Components Weighed, ng (per 100 ml % %
Date Lot # PC TG FC CE U VitE ART P188 DMPC PS WV WV 21 Feb. 2013 OO2.140.1 423O 703 7O 150 10 O 527 298 O8O O 7.1 6.2 21 Feb. 2013 OO2.140.1 423O 703 7O 150 10 O 527 298 O8O O 7.1 6.2 21 Feb. 2013 OO2.140.1 423O 703 7O 150 10 O 527 298 O8O O 7.1 6.2 21 Feb. 2013 OO2.140.1 423O 703 7O 150 10 O 527 298 O8O O 7.1 6.2 13 Mar. 2013 OO2.150.1 4344 700 71 151 11 O O 3O3 O76 O 6.7 6.3 13 Mar. 2013 OO2.150.2 SOST 840 85 180 13 2 O 361 269 O 7.8 7.4 13 Mar. 2013 OO2.150.3 4233 70S 70 150 10 O S32 300 O83 O 7.1 6.2 13 Mar. 2013 OO2.150.4 42O3 699 7O 150 10 O S32 300 O83 O 7.1 6.2 15 Mar. 2013 OO2.152.1 4277. 698 70 150 10 O SO1 301 O67 O 7.1 6.3 15 Mar. 2013 OO2.152.2 432O 698 70 150 10 O 47S 304 163 O 7.2 6.4 21 Mar. 2013 OO2.155.1 4300 490 69 148 11 O 505 300 107 O 6.9 6.1 25 Mar. 2013 OO2.156.2 434O 600 70 150 10 O 511 300 126 O 7.1 6.3 26 Mar. 2013 OO2.158.1 4345 698 70 1 SO 11 O 529 301 130 O 7.2 6.4 28 Mar. 2013 OO5.1.1 4340 850 70 1 SO 11 O 530 300 130 O 7.4 6.5 29 Mar. 2013 OOS.2.1 4250 735 70 150 10 O S43 300 123 O 7.2 6.3 1 Apr. 2013 005.2.1 4250 735 70 150 10 O S43 300 123 O 7.2 6.3
MASTER TABLE 3 Major components ratios. Particle size, stability and ART-207 content of resultant emulsion. Formulation Ratios of major components On the day of MFG Stability TGf PC Particle ART-207 ART-207 Days Particle MFG ART- ART- PC FC size content recovery, past size Date Lot # 2O7 2O7 TG CE mg/ml % MFG in 18 Dec. 2012 OO2.103.1 NA NA 1.88 1.35 63 NA NA 36 79 19 Dec. 2012 OO2.104.1 NA NA 1.87 1.34 45 NA NA 35 82 19 Dec. 2012 OO2.105.1 3.58 6.8O 1.90 1.30 82 3.1 98.1 35 196 9 Jan 2013 OO2.108.1 2.16 6.42 2.98 O.2O 129 4.8 742 14 184 9 Jan 2013 OO2.109.1 2.16 7.75 3.60 O.33 74 5.5 84.9 33 169 10 Jan 2013 OO2.110.1 2.15 7.96 3.71 O.33 70 6.1 94.4 13 156 11 Jan 2013 OO2.1111 6.54 24.27 3.71 O.33 47 1.9 87.3 24 66 11 Jan 2013 OO2.111.2 1.01 9.10 9. OS O46 66 3.9 59.9 38 75 16 Jan 2013 OO2.116.1 2.15 7.91 3.69 O.33 62 6.1 93.7 4 146 19 Jan 2013 OO2.118.0 NA NA 3.74 O.33 47 NA NA 60 70 19 Jan. 2013 002.118.00 NAA NA 3.75 O.33 47 NA NA 60 79 20 Jan 2013 OO2.119.4 2.07 7.73 3.74 O.33 78 6.9 1OO.O 22 218 21 Jan 2013 OO2.121.4 2.07 7.72 3.72 O.33 110 6.1 90.2 O NA 22 Jan 2013 OO2.122.O 2.07 7.72 3.72 O.33 8O S.6 83.9 57 197 23 Jan 2013 OO2.1231 1.00 O.17 10.15 O.47 68 3.7 742 56 73 23 Jan 2013 OO2.123.12 100 O.17 10.15 O.47 57 3.7 742 30 67 23 Jan 2013 OO2.123.13 1.00 O.17 10.15 O.47 69 3.7 742 56 70 25 Jan 2013 OO2.125.2 1.02 O.21 1O.OS O.47 67 4.0 79.0 S4 62 25 Jan 2013 OO2.125.22 1.02 O.21 1O.OS O.47 66 4.0 79.0 S4 64 9 Feb. 2013 002.131.2 NA NA 740 O.47 42 NA NA 39 49 9 Feb. 2013 002.134.2 1.32 9.87 7.49 O46 76 4.7 89.0 39 84 12 Feb. 2013 002.136.2 1.34 O.OS 7.49 O46 58 4.3 81.9 36 61 12 Feb. 2013 002.136.22 1.34 O.OS 7.49 O46 42 4.2 80.4 36 55 12 Feb. 2013 002.136.23 1.34 O.OS 7.49 O46 59 4.5 85.7 36 60 15 Feb. 2013 OO2.137.3 NA NA 3.95 O.47 49 NA NA 33 51 15 Feb. 2013 OO2.137.4 2.65 0.67 4.02 O.47 53 4.8 912 33 108 15 Feb. 2013 OO2.137.42 2.65 0.67 4.02 O.47 84 S.O 95.4 33 134 15 Feb. 2013 OO2.137.43 2.65 0.67 4.02 O.47 S4 4.9 93.1 33 183 21 Feb. 2013 002.139.2 NA NA 7.53 O.47 38 NA NA 27 43 21 Feb. 2013 002.140.2 1.33 O.O8 7.55 O.47 58 4.4 84.2 27 58 21 Feb. 2013 002.140.22 1.33 O.O8 7.55 O.47 46 4.1 78.1 27 S4 21 Feb. 2013 002.140.23 1.33 O.O8 7.55 O.47 59 4.5 85.7 27 58 21 Feb. 2013 002.140.24 1.33 O.O8 7.55 O.47 49 4.4 82.9 27 55 14 Mar. 2013 OO2.1515 NA NA 7.74 O.47 37 NA NA 42 47 14 Mar. 2013 OO2.151.6 NA NA 7.53 O.47 38 NA NA 42 43 14 Mar. 2013 OO2.151.7 1.33 9.99 7.54 O.47 72 5.3 99.6 42 71 14 Mar. 2013 OO2.151.8 1.31 9.94 7.56 O.47 62 5.3 98.9 42 67 15 Mar. 2013 OO2.153.1 1.39 10.67 7.66 O.47 66 4.7 93.5 41 69 15 Mar. 2013 OO2.153.2 1.47 11.54 7.85 O.47 53 4.4 92.2 53 64 22 Mar. 2013 OO2.156.1 0.97 10.72 11.03 O.47 63 4.1 80.3 42 59 25 Mar. 2013 OO2.156.3 1.17 10.70 9.11 O.47 62 4.8 94.O 43 61 27 Mar. 2013 OO2.158.2 1.32 10.35 7.84 O.47 8O 4.7 89.6 37 72 US 9,468,603 B2 95 96 MASTER TABLE 3-continu ed Maior components ratios. Particle size, stability and ART-207 content of resultant emulsion. Formulation Ratios of major components On the day of MFG Stability TGf PC Particle ART-207 ART-207 Days Particle MFG ART- ART- PC FC size content recovery, past size Date Lot # 2O7 2O7 TG CE ill mg/ml % MFG nm.
29 Mar. 2013 OOS.1.2 1.60 10.32 6.44 O.47 74 4.7 89.5 35 78 29 Mar. 2013 OOS.2.2 1.35 9.90 7.31 O.47 102 4.9 89.3 35 101 1 Apr. 2013 005.2.3 1.35 9.90 7.31 O.47 92 4.3 89.3 26 95
Example composition and ratios for major components 1s embodiments, aspects and variations. It is intended that the for formulations incorporating 287 are given in the Master following claims are interpreted to include all such modifi Tables 4 and 5 below: cations, permutations, additions and combinations and cer tain sub-combinations of the embodiments, aspects and MASTER TABLE 4 variations are within their scope. The entire disclosures of all documents cited throughout this application are incorpo Total Solids Pre-Mixes (TSPM) and corresponding resultant emulsions. rated herein by reference. Material Preparation date Loth Material Processing date Loth We claim: TSPM 30 May 2013 005.14.1 Emulsion 31 May 2013 005.14.2 TSPM 4 Jun. 2013 005.16.1 Emulsion 4 Jun. 2013 005.16.2 1. A stable, synthetic low density lipoprotein (LDL) solid TSPM 5 Jun. 2013 005.17.1 Emulsion 6 Jun. 2013 005.18.1 25 nanoparticle comprising: a) a lipophilic anti-cancer agent of the formula 1a, 1b or formula 2a: MASTER TABLE 5 Composition of TSPM (Total Solids Pre-Mix). TS, Components Weighed. Ing (per 100 ml %
Date Lot # PC TG FC CE U Vit E ART P188 DMPC PS WV WV 30 May 2013 005.14.1 5300 596 71 150 10 10 SO1 300 O O 6.9 6.1 O4 Jun. 2013 OOS.16.1 S370 606 71. 149 10 10 SO8 300 O O 7.O 6.2 OS Jun. 2013 OOS.17.1 4360 602 71 1SO 10 10 507 302 1089 O 7.1 6.3
MASTER TABLE 6 Major components ratios. Particle size, stability and 287 content of resultant emulsion. Formulation On the day of MFG Stability Ratios of major components Particle 287 287 Days Particle
MFG TG, PC PC FC size content recovery, past size Date Lot # 287 287 TG CE mm mg/ml % MFG mm
30 May 2013 005.14.1 1.19 10.58 8.89 O.47 S3.2 2.73 54.5 32 67.2 O4 Jun 2013 OOS.16.1 119 10.57 8.86 0.47 56.4 2.81 55.3 28 67.4 OS Jun. 2013 OOS.17.1 1.19 10.75 9. OS O.47 47.5 2.80 55.2 26 51.7
In addition to the above representative experiments and formulations of ART-207 and compounds disclosed 1 herein, the above procedures are also performed on selected compounds disclosed in the present application, and the 60 X results are substantially consistent with the results described Q T above. While a number of exemplary embodiments, aspects and R1 os-au variations have been provided herein, those of skill in the art 65 will recognize certain modifications, permutations, additions and combinations and certain Sub-combinations of the US 9,468,603 B2 97 98 -continued (propylene oxide)) flanked by two hydrophilic chains of 1.1 polyoxyethylene (poly(ethylene oxide)), or mixtures thereof. 10. A stable, synthetic low density lipoprotein (LDL) 5 nanoparticle comprising: a) a lipophilic anti-cancer agent of the formula 1a, 1b or formula 2a:
10 RI-X, R2 15 R1 N-au Y wherein: 1.1 for formula 1a or 1b R' is hydrogen, C-C alkyl or R Cs-C alkyl; and R is Cs-C alkyl; and for formula 2a: R is C-C alkyl; and X is hydrogen or is selected Q R2 from the group consisting of Cl, Brand I; and R is a hydroxyl bearing cancer chemotherapeutic agent (HBCCA) selected from the group consisting of a O Z C taxane, abeo-taxane, camptothecin, epothilone, cucur bitacin, quassinoid and an anthracycline; 25 r b) phospholipids (PL) wherein the phospholipids is Y Selected from the group consisting of phosphotidylcho X line, phosphotidylethanolamine, symmetric or asym 2 metric 1,2-diacyl-sn-glycero-3-phosphorylcholines, 30 1,2-dimyristoyl-sn-glycero-3-phosphorylcholine, 1,2- --O T dimyristoyl-sn-glycero-3-phosphorylethanolamine, egg phospholipids, egg phosphatidylglycerol, dipalmi toylphosphatidyl glycerol, egg lecithin, soy lecithin, wherein: lecithin (NOS) and mixtures thereof; and for formula 1a or 1b R' is hydrogen, C-C alkyl or c) triglycerides (TG); 35 Cs-C alkyl; and R is Cs-C alkyl; and for formula wherein the LDL nanoparticle has a mean particle size of 2a: R is C-C2 alkyl; and X is hydrogen or is selected 40-80 nm. from the group consisting of Cl, Brand I; and 2. The LDL nanoparticle of claim 1, wherein the LDL R is a hydroxyl bearing cancer chemotherapeutic agent nanoparticle has a mean size distribution of 60 nm. (HBCCA) selected from the group consisting of a 3. The LDL nanoparticle of claim 1, further comprising 40 taxane, abeo-taxane, camptothecin, epothilone, cucur cholesterol ester (CE) or cholesterol (C), or mixtures of bitacin, quassinoid and an anthracycline; cholesterol ester and cholesterol. b) phospholipids (PL) wherein the phospholipids is 4. The LDL nanoparticle of claim 3, wherein the choles Selected from the group consisting of phosphotidylcho terol ester is selected from the group consisting of C. line, phosphotidylethanolamine, symmetric or asym esters of cholesterol, and mixtures thereof; and the triglyc 45 metric 1,2-diacyl-sn-glycero-3-phosphorylcholines, erides is selected from the group consisting of soybean oil. 1,2-dimyristoyl-sn-glycero-3-phosphorylcholine, 1.2- triolein, glyceryl tripalmitate and mixtures thereof. dimyristoyl-sn-glycero-3-phosphorylethanolamine, 5. The LDL nanoparticle of claim 1, further comprising an egg phospholipids, egg phosphatidylglycerol, dipalmi agent selected from the group consisting of triolein, BHT. toylphosphatidyl glycerol, egg lecithin, soy lecithin, ubiquinol, ubiquinol 10, vitamin E, alpha-tocopherol, 50 lecithin (NOS) and mixtures thereof; and gamma-tocopherol, lycopene, retinyl derivative and betac c) triglycerides (TG) prepared by the process comprising: arotene, or mixtures thereof. 1) combining the lipophilic anti-cancer agent, phos 6. The LDL nanoparticle of claim 1, wherein the lipo pholipids and triglycerides to form a mixture; philic anti-cancer agent has a log P greater than 6.0 or 2) homogenizing the mixture by dissolution in a vola greater than 8.0. 55 tile solvent; 7. The LDL nanoparticle of claim 3, wherein the weight 3) removing the solvent; ratio of PL:TG:CE:C ranges from 73:12:2:1 to 78:12:2:1; 4) forming a coarse emulsion by blending of the optionally further comprising an additive selected from the mixture in a buffer to form an emulsion mixture: group consisting of triolein, BHT, ubiquinol, ubiquinol 10, 5) microfluidizing the emulsion mixture in a microflu Vitamin E, alpha-tocopherol, gamma-tocopherol, lycopene, 60 idizer apparatus for a sufficient amount of time to retinyl derivative and betacarotene, or mixtures thereof. produce a particle preparation of 100 nm or less; and 8. The LDL nanoparticle of claim 7, wherein the weight 6) sterilizing the nanoparticle preparation through a ratio of PL:TG:CE:C is 77:10:2:1. 0.22 micron filter to obtain the synthetic LDL nano 9. The LDL nanoparticle of claim 1, further comprising a particles with a mean particle size range of 40 nm to poloxamer that are nonionic triblock copolymers composed 65 80 nm. of a central hydrophobic chain of polyoxypropylene (poly