1. 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 zw
H
N
O
O
OMe
x y
XL
Core 1
Core 2
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
3. 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
Relevant Work: Kataoka, Stayton, Kwon, Wooley, McCormick, Bae, Leroux, Armes
Polymer Therapeutics: Duncan, R. Nat. Rev. Drug Discov. 2003, 2, 347-360.
(Traditional Diblock)
3
4. Traditional vs. IVECT™ Micelles
4
Traditional Micelles
(e.g. Diblock polymers)
IVECT-Stabilized Micelles
At Injection
Bloodstream
Circulation
At Tumor Site
Micelles are injected
intravenously
Prior to
Injection
Traditional micelles degrade
immediately upon injection:
Reduced efficacy & increased
side effects
IVECT Micelles are more
stable and remain intact:
Greatly improved pK, tumor
retention & safety
Triggered drug release
Greater tumor accumulation &
improved efficacy
Triggered
Release
5. 5
IVECT™: Polymer Structural Features
Generic Diblock or Triblock
Crosslinkers (XL) = -CO2H,
-(CH2)n-CONHOH, -S(H, X)
N
H
H
N
N
H
Ac
O
O zw
H
N
O
O
OMe
x y
XL
Core 1
Core 2
Polar, charged FG:
metal chelator or -SS-
crosslinker
Hydrophobic Moiety:
aromatic, lipophilic and/or
VDW interactions
Aromatic: stacking,
may provide HBD, HBA
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).
6. Metal-Mediated Crosslinking
Hypothetical crosslinked array via Fe+3 octahedral complex
H
N
N
H
H
N
O
O
O
N
H
H
N
N
H
H
N
O
O
O
O
O O O
O O
O O
NH NH NH
HN
NH HN HN
O O O
O O O
Fe
Fe
H
N
N
H
H
N
O
O
O
N
H
H
N
N
H
H
N
O
O
O
O
O
O
O
OO
O
O
NH
HNNH
NHHN
NH
HN
O
OO
OO
OO
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.
7. Synthesis of Protected Triblock-1
Synthesis on 2.1 Kg scale
in 96% yield
NH2
O
O
Me
270
N
H
H
N
O
CO2Bn
270
H
N
O
CO2Bn
O
O
Me
5 5
O
HN
O
O
CO2Bn
O
HN
O
O
CO2Bn
O
HN
O
O
OAc
O
HN
O
O
CH2Cl2, DMAC: 2,1
25 oC, ~16-24 hr
H
Intermediate Diblock
25 oC, ~30-36 hr
2. Ac2O, NMM,
DMAP, RT, ~ 14hr
1.
N
H
H
N
N
H
H
N
Ac
O
O
O
15 25
CO2Bn
270
H
N
O
CO2Bn
O
O
Me
5 5
OAc
MePEG12K-NH2
dried via azeotropic vacuum dist'n
Protected Triblock
7
GPC
(DMF)
PDI
=1.10
8. 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
N
H
H
N
N
H
H
N
Ac
O
O
O
15 25
CO2Bn
265
H
N
O
CO2Bn
O
OMe
5 5
OBn
N
H
H
N
N
H
H
N
Ac
O
O
O
15 25265
H
N
O
O
O
Me
5 5
OH
NHOH
O
O
NHOH
1. NH2OH (5x), LiOH.H2O (1x),
THF, H2O, RT, ~36 hr
2. Acetone (10x), HOAc (1x),
RT -> reflux -> RT, 14 hrs
3. Ppt'n steps
96.1%, 1.7 Kg scale
ITP-‐102
8
9. Characterization of HATB (ITP-102)
ITP-102 (m-PEG11.7K-b-P-(d-Glu[NHOH]5-co-
Glu[NHOH]5)-b-P-(d-Phe15-co-Tyr[OH]25)-Ac)
HATB
(ITP-‐102)
PDI
=
1.1
Mp
=
21.69K
(theo
MW
=
19.47K)
HMW
Aggregates
GPC (ACN, H2O: 40, 60 w/0.1%TFA); RED = LS, BLUE = dRI
N
H
H
N
N
H
H
N
Ac
O
O
O
15 25265
H
N
O
O
O
Me
5 5
OH
NHOH
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
10. 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
Physical Properties of Empty Micelles
11. 11
Representative Drugs Encapsulated
N
N
N
N
H
N
O
H
N
O
OH
HO O
H2N
NH2
Aminopterin
N
N
O
HO O
O
HO
SN-38 (IT-141)
O
O
O
OH
OH
OH
O
O
O
H
OH
H2NDaunorubicin
(IT-143)
O
O O
S
N
OH
Epothilone D (IT-147)
O
O
O
OH
NH HO
O
O
O
OH
O
O
O
O
O
Paclitaxel
HN
H
N
O
NH
OH
Panobinostat
O
O
O
O O
OO
N
OH
OO
O
O
HO
OH
Everolimus
N
O
N
N
H2N
N
H
N
N
S
AMG-900
N
O
N
H
O
N
H
O
N
H
Cl
CF3
Sorafenib
12. Homologous HA3-20 Crosslinkers
mPEG N
H
O
H
N
O
N
H
O
H
N
Ac
x
y z
OH
O NHOH
SN-38 Formulations & PK Studies
• Polymer 1 demonstrated inferior formulation properties
• subtle core variation can impact phys. props.
• 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 z
Efficiency
(%)
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
N
N
HO O
O
OOH
SN-38
(IT-141)
“HA”
HATB Polymers (1-7)
Mol. Wt. = 18.8K-21.2K
13. Impact of XL Groups: "
Carboxylate vs. Hydroxamate
O
O
O
OH
OH
OH
O
O
O
H
OH
H2N
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
%DaunorubicinRemaining
Buffer pH
IT-143 NHOH
IT-143 Asp
Figure. pH dependent release from crosslinked micelles (3500 MWCO dialysis)
Daunorubicin Formulation & PK Studies
Polymer
Cross-
Linker
Moiety
Fe(III) Ka
(M-1
)
mPEG12K-b-p-[Asp(OH)10]-b-p-
[D-Phe15-co-Tyr25]Ac
Carboxylic
Acid
2.4 x 103
mPEG12K-b-p-[D/L-Glu(NHOH)5/5]-
b-p-[D-Phe15-co-Tyr25]Ac
Hydroxamic
Acid
2.6 x 1011
Rat PK Model (10mg/kg)
Encapsulation
Dialysis
(% remaining)
XL Wt.
Loading
(%)
DLS:
Diameter
(nm)
AUC
(µg*h/mL)
Cmax
(µg/mL)
T1/2
(h)
93 4.3 60 152.0 178.0 3.3
86 3.9 70 329.7 130.0 7.2
13
14. IT-143 Rat PK Study
Administration
AUC (μg*hr/mL)
Cmax (μg/mL)
IT-143
116 -> 688
153.8 -> 160
Conventional Micelle
1.48
2.61
Daunorubicin
1.30
3.29
Plasma PK Parameters
14
(original formulation)
• IT-143 exhibits 90X greater plasma
exposure than free Daunorubicin
• Recent refinements delivered 160 gm
of drug micelle formulation that
exhibited 529X greater plasma
exposure than free Daunorubicin
15. IT-143: Mouse Biodistribution Study
Organ
IT-143
Daunorubicin
Fold Increase
Plasma
44.82
0.77
57.9
Tumor
7.15
0.09
75.6
Liver
116.44
70.22
1.7
Spleen
385.82
546.53
0.7
Kidney
145.62
0.00*
N/A
Small Intestine
69.36
77.71
0.9
Skin
39.19
31.78
1.2
Brain
0.19
0.20
1.0
Heart
47.06
33.81
1.4
Lung
78.31
98.33
0.8
AUC Summary (µg*h/g)
• 10 mg/kg daunorubicin as
free drug and IT-143
administered by single tail
vein injection to A549
xenograft mouse model
IT-143 demonstrates 75 times greater
tumor accumulation of Daunorubicin
compared to free drug
* Metabolites were observed, but Daunorubicin was not
identified by HPLC
15
