Design, Synthesis, and Structure-Function Studies of Novel Triblock Copolymers J. Edward Semple*, Bradford T. Sullivan, Brian K. Burke, Tomas Vojkovsky and Kevin N. Sill Intezyne Technologies, Tampa, FL 2017 ACS National Meeting - Denver PMSE.519 N H H N N H Ac O O z w H N O O O Me x y XL Core 1 Core 2
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Design, Synthesis, and Structure-Function Studies of Novel Triblock Copolymers
J. Edward Semple*, Bradford T. Sullivan, Brian K. Burke, Tomas Vojkovsky and Kevin N. Sill Intezyne Technologies, Tampa, FL
2017 ACS National Meeting - Denver
PMSE.519
NH
HN N
HAc
O
O zw
HN
OOOMe
x y
XL
Core 1
Core 2
Typical drug distribu0on profile
Ideal distribu0on profile using a targeted delivery system
Drug (shown in blue) is distributed to all areas of the body
Drug is localized at diseased site leading to increased efficacy and reduced side effects
Targeted Drug Delivery
Wide-range of therapeutics can be targeted using the IVECTTM Method*
*IVECT™ = Intezyne’s Versatile Encapsulation and Crosslinking Technology. Intezyne patents: Sill, K.; Skaff, H. et al. US8980326B2 (2015), US 8263663B2 (2012), US 7638558B2 (2009). Reviews: Boehme, D. et al. J. Pept. Sci. 2015, 21, 186-200; Beech, J. et al. Curr. Pharm. Design 2013, 19, 6560-6574 2
Polymer Micelles for Drug Delivery Core/Shell Morphology - Drug protected in core - Relatively large payload of therapeutics - PEG shell imparts aqueous solubility and “stealth” properties Improved Safety Profile - Reduced side-effects Improved Pharmacokinetics - Ideal micelle size is above renal (< 40 nm) and
below hepatic (>150 nm) clearance thresholds - Individual polymer components are cleared through the kidneys Tumor Targeting - Passively targets solid tumors due to EPR Effect - Can be modified to actively target receptors on diseased cells
Hydrophilic PEG moiety:imparts high aqueous solubility
Intezyne patents: Sill, K.; Skaff, H. et al. US8980326B2 (2015), US 8263663B2 (2012), US 7638558B2 (2009), US 7638558B2 (2009).
Metal-Mediated Crosslinking Hypothetical crosslinked array via Fe+3 octahedral complex
HN N
H
HN
O
O
O
NH
HN N
H
HN
O
O
O
O
O O O
O OO O
NH NH NH
HN NH HN HN
O O O
O O OFeFe
HNN
H
HN
O
O
O
NH
HNN
H
HN
O
O
O
O
OO
O
OOOO
NHHNNH
NHHNNH
HN
OOO
OOOO
Fe
O
Fe
6
• Previous series utilized carboxylate (poly-Asp[OH]) in the crosslinking block
• Hydroxamate-metal complexes more stable than carboxylate-metal complexes • Binding constants (Ka) for acetohydroxamate with Fe(III) = 2.6 x 1011 M-1 while acetate = 2.4 x 103 M-1
(aqueous, Whitesides et al., LANGMUIR, 1995, 11, 813-824).
Sill, K. N. et al. US20140113879A1 (2014), US20130280306A1 (2013). Coordination chemistry and chemical biology of hydroxamic acids: Codd, R. Coord. Chem. Rev. 2008, 252, 1387-1408.
Synthesis of Protected Triblock-1
Synthesis on 2.1 Kg scale in 96% yield
NH2OOMe
270NH
HN
O
CO2Bn
270
HN
O
CO2Bn
OOMe
5 5
OHN O
O
CO2Bn
OHN O
O
CO2Bn
OHN O
O
OAc
OHN O
O
CH2Cl2, DMAC: 2,125 oC, ~16-24 hr
H
Intermediate Diblock
25 oC, ~30-36 hr
2. Ac2O, NMM, DMAP, RT, ~ 14hr
1.
NH
HN N
H
HN
Ac
O
O
O
15 25
CO2Bn
270
HN
O
CO2Bn
OOMe
5 5
OAc
MePEG12K-NH2 dried via azeotropic vacuum dist'n
Protected Triblock
7
GPC (DMF) PDI =1.10
Synthesis of Hydroxamic Acid Triblock (HATB)-2
- Successful synthesis of ITP-102 on 1.7 Kg scale with overall 92.5% yield - Both Tech Transfer and GMP runs proceeded without issues and
delivered nearly identical lots of pure HATB final product
GPC (ACN, H2O: 40, 60 w/0.1%TFA); RED = LS, BLUE = dRI
NH
HN N
H
HN
Ac
O
O
O
15 25265
HN
OOOMe
5 5
OHNHOH
O
O
NHOH
9
Glu sidechains
PEGs
Tyr + Phe
Backbone Amide NH Tyr-(OH) +
-CONHOH
Tyr + Phe sidechains
Backbone methine
MeO-
1H-NMR (DMSO-d6, 400 MHz)
H2O
DMSO
Impact of Mixed Core Stereochemistry
10
• CMC: shift towards higher concentrations for D,L mixed core polymers • DLS: micelle size for D,L-core polymers is 2.2-2.6x smaller than all L-polymers • Turbidity data shows dramatic differences in physical appearance of the polymer micelles (cf. photo) • Results are consistent with literature precedent-
• CD studies show disruption of α-helical structure when D-AAs are incorporated into polymers • As little as 3% D-AA can disrupt α-helix; by ~ 8% observe disordered (random coil) conformations
• Mixed stereochemistry in polymer backbone results in greatly enhanced drug loading efficiency
• In polymers 2-7, increasing #HA repeats from 3 to 7 led to significant increase of AUC • Increase from 7 to 10 (or 20) HA units resulted in marginal improvement of PK properties • Studies with several other oncology drugs led to selection of Polymer 5 (x =10) for advanced preclinical development (ITP-102).
12
Polymer #
1
2
3
4
5
6
7
#HA repeats
(x)
10
3
5
7
10
15
20
Rat PK (10mg/kg)
y zEfficiency
(%)
Weight Loading
(%)Diameter
(nm)
Micelle Turbidity
(RTU)AUC
(µg*h/µL)
10 30 40 1.5 >300 120 ND
15 25 68 6.4 116 25 46.7
15 25 72 6.8 118 21 ND
15 25 70 6.2 119 18 72.5
15 25 75 6.3 114 17 75.7
15 25 68 6.0 117 16 ND
15 25 70 6.6 120 15 90.7
NN
HO O
O
OOHSN-38
(IT-141)
“HA” HATB Polymers (1-7)
Mol. Wt. = 18.8K-21.2K
Impact of XL Groups: "Carboxylate vs. Hydroxamate O
O
O
OH
OH
OH
O
O
OH
OHH2N
Daunorubicin (IT-143)
• Polymers differ only in XL moiety • Similar micelle properties • Both IT-143 formulations demonstrated
pH-dependent drug release from the micelle in biologically relevant range
• HATB analog demonstrated superior PK in rats-over 100% increase of exposure (AUC) and terminal T1/2 vs. carboxylic acid.
0
20
40
60
80
100
3 4 5 6 7 7.4 8
% D
au
no
rub
icin
Re
ma
inin
g
Buffer pH
IT-143 NHOH
IT-143 Asp
Figure. pH dependent release from crosslinked micelles (3500 MWCO dialysis)