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

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  

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  

IVECT™: Polymer Structural Features

Generic Diblock or Triblock Crosslinkers (XL) = -CO2H, -(CH2)n-CONHOH, -S(H, X)

NH

HN N

HAc

O

O zw

HN

OOOMe

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).

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

NH

HN N

H

HN

Ac

O

O

O

15 25

CO2Bn

265

HN

O

CO2Bn

OOMe

5 5

OBn

NH

HN N

H

HN

Ac

O

O

O

15 25265

HN

OOOMe

5 5

OHNHOH

O

O

NHOH

1. NH2OH (5x), LiOH.H2O (1x), THF, H2O, RT, ~36 hr2. Acetone (10x), HOAc (1x), RT -> reflux -> RT, 14 hrs3. Ppt'n steps

96.1%, 1.7 Kg scale

ITP-­‐102  

8  

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

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

Physical Properties of Empty Micelles

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Representative Drugs Encapsulated

N

N

N

NHN

O

HN

O

OH

HO O

H2N

NH2

Aminopterin

NN

O

HO O

OHO

SN-38 (IT-141)

O

O

O

OH

OH

OH

O

O

O H

OHH2NDaunorubicin

(IT-143)

O

O O

S

N

OH

Epothilone D (IT-147)

O

O

O

OH

NH HO

OO

O

OH

O OO

O

O

Paclitaxel

HN

HN

O

NHOH

Panobinostat

O

OO

O O

OON OH

OOO

OHO

OH

Everolimus

N

O

N

N

H2N

NH

NN

S

AMG-900

N

ONH

O

NH

O

NH

Cl

CF3

Sorafenib

Homologous HA3-20 Crosslinkers

mPEG NH

O HN

ONH

O HN

Acx

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).

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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)

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

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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

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(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

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  

Acknowledgements Bradford Sullivan Tomas Vojkovsky Kevin Rodriquez

Brian Burke Adam Carie Tyler Ellis

Habib Skaff Kevin Sill

16