polyoxazoline: a potent alternative to peg-based ... · a potent alternative to peg-based...

Polyoxazoline:A potent alternative to PEG-basedformulations of hydrophobic drugs

Rainer JordanChair of Macromolecular Chemistry, TU Dresden, Germany

& DelAqua Pharmaceuticals Inc., USA

Robert LuxenhoferFunctional Polymer Materials, Universität Würzburg, Germany


Alexander V. KabanovCenter for Nanotechnology in Drug Delivery, University of North Carolina at Chapel Hill, USA


Professur für Makromolekulare Chemie

PEG –The current gold standard in polymer therapeutics

Most commonly used hydrophilic polymer in life sciences

- Hydrophilic, low toxicity/immunogenicity, low protein interaction.

-> PEGylation improves biocompatibility & reduces toxicity

Widely used in cosmetics, personal care and medicine.

Use/Introduction of PEG is known as „PEGylation“

Knop et al. Angew. Chem. Int. Ed. 2010, 49, 6288.

PEG: R = H

PPG: R = MePoloxamers

(Pluronics)

Arguments for PEG: - Broad commercial availability

- FDA approved conjugates, many derivatives on the FDA X-list

- Vast database on biocompatibility, toxicity, immunogenicity, ...

Frank F. Davis

Abraham Abuchowski

Arguments against PEG: - PEG is limited in terms of functionalization

- PEG-poisoning due to oxidation of the polyether

(Herold, Biochem. Pharmacol. 1989)

- PEG shows limited long-term stability in plasma

(Textor, Biointerphases 2012)

- Development of PEG antibody in humans (approx. 25%)(Armstrong et al. Cancer 2007; Ishida J. Control. Release 2006)


Polymer Therapeutics - Alternatives

Pseudo-polypeptides

Constitutional isomers of polypeptides with a tertiary amide motif

Polypeptoids (POI)

Polypeptide

Poly(2-oxazoline) (POx)

Beta-Polypeptoids (βPOI) Poly(2-oxazine) (POz)

PVP PDMA

(used as blood plasma expander since WWII)


Poly(2-oxazoline)s: Chemistry & Properties

Living cationic ring-opening polymerization (LCROP):

• Strictly linear polymers

• Direct control of polymer chain length by [M]0 / [I]0 - ratio

• Low dispersity Đ = Mw/Mn ~ 1.05 – 1.3

• Quantitative introduction of terminal functional groups

• Introduction of pendant groups via functional monomers and/or polymer analog reactions

• Block, gradient and random copolymerization control of amphiphilicity & distribution of functions

• "no side-reactions"

• typical degree of polymerization n ~150-200

• POx are miscible with most polymers and soluble in many solvents

• POx show a temperature dependent water-solubility:

lower critical solution temperature (LCST) that can be fine-tune between (0) … 4-95… (100)°C

N

C

OR

n

nO N

RInitiator


a,w-functionalized POx

M. Reif, R. Jordan Macromol.Chem.Phys. 2011,212,1815.

Protective groups:tert-butyldiphenylsilyl ethers (TBDPS)& BOC

a,w- & pendant functionalized POx

Living cationic polymerization: Chemistry

R. Luxenhofer, R. Jordan Macromolecules 2006,39,3509.M.D. Bentley, J.M. Harris, K. Yoon, M. Reif, R. Jordan US 14/537,516 2015


a,w-functionalized POx

M. Reif, R. Jordan Macromol.Chem.Phys. 2011,212,1815.

Protective groups:tert-butyldiphenylsilyl ethers (TBDPS)& BOC

a,w- & pendant functionalized POx


R. Luxenhofer, R. Jordan Macromolecules 2006,39,3509.M.D. Bentley, J.M. Harris, K. Yoon, M. Reif, R. Jordan US 14/537,516 2015

BOC protected piperazine:especially efficient terminating agent and useful for defined ligation

same endgroup

different

differentorthogonal



initiator salt

R3

Both termini AND every monomer unitcan bear functional groups.

random or gradientcopolymers

block copolymers

electrophile


initiator salt

R3

Both termini AND every monomer unitcan bear functional groups.

random or gradientcopolymers

block copolymers

POx:defined polymers with high sequence control, multiple functionalization and tuneable solubility

Poly(2-oxazoline)s: Chemistry & Properties


POx: in vivo Biodistribution & Excretion

30 min p.i. 3 h p.i.

bladder kidneys

kidneys

PEtOx48-DOTA[111In]

A. Mitra, et al. Pharm. Res. 2004, 21, 1153.R. Jordan et al., J. Control. Release 2007, 119, 291.

• Rapid distribution in entire organism

• Good and fast blood clearance

• Rapid renal excretion (residual ~ 2 %; 30 min p.i.)

• Very low kidney uptake

• Low organ deposition (24h p.i.) for PMeOx and PEtOx

• Liver uptake 39 times lower as P(HPMA)

• Low to none muscle & skin accumulation (a PEG problem)

g-Camera Imaging

PMeOxpoly(2-methyl-2-oxazoline)

PEtOxpoly(2-ethyl-2-oxazoline)


R. Luxenhofer, G. Sahay, A. Schulz, D. Alakhova, T. K. Bronich, R. Jordan, A. V. Kabanov, J. Control. Release 2011, 153, 73.

Cytotoxicity of POx

Different immortal cell lines (human breast cancer: MCF7, MCF7-ADR, immortalized canine kidney cells: MDCK)after 2 and 24 h incubation with POx-solution @ 37°C:

non-ionic POx

POx withquaternized chain end

polymer concentration (wt%)

P(MeOx-BuOx-MeOx)

Summary:

• All water soluble POx homo- andblock copolymers: low cytotoxicity

• Quaternization: considerable cytotoxicity foundin fact they are antimicrobial(Tiller et al. Biomacromolecules 2005)


Endocytosis of POx

R. Luxenhofer, G. Sahay, A. Schulz, D. Alakhova, T. K. Bronich, R. Jordan, A. V. Kabanov, J. Control. Release 2011, 153, 73.

Endocytosis of TRITC-labeled POx (c=1g/L) by MDR cancer cells (MCF7-ADR)

hydrophilic

hydrophobic

TRITC label

diblockstriblocks

Homopolymer

Summary:

• Uptake can be very rapid:100% labeled cells after 10 min (P(EtOx-BuOx))

• Endocytosis strongly depend on

• block size, composition, sequence

• POx ends up in perinuclear region

• Uptake similar to pluronics but much faster

• Mechanism of uptake unclear – in part caveolae


Immunogenicity of POx

Viegas, et al.Bioconjug. Chem. 2011, 22, 976.

Low Immunogenicity (POx-BSA)

Low Complement activation (POx)

R. Luxenhofer et al. Biomaterials 2010, 31, 4972.

P1-3: PMeOx-BuOx-MeOxP4: PEtOx-BuOx

„Stealth Effect“ of POx liposomes

Woodle et al. Bioconj. Chem. 1994, 5, 493.

Even amphiphilic POx and POx/drug formulations showonly minor activation of thecomplement system

POx

POx & PEG!


POx as Biomaterials

Biodistributionin vivo (mouse)

Cytotoxicityin vitro (MCF7, MDCK)

hydrophilic POx• immediate whole body distribution• low unspecific organ deposition• fast blood clearance by renal excretionJ. Control. Release 2007, 119, 291

uncharged hydrophilic & amphiphilic POx• non-cytotoxicJ. Control. Release 2011, 153, 73

Cell-uptakein vitro (MCF7, MDCK)

hydrophilic POx• low uptake

amphiphilic POx• very fast uptakeJ. Control. Release 2011, 153, 73

„Stealth-Effect“in vitro & in vivo

hydrophilic POx• non-immunogenic

amphiphilic POx• low to none complement activationBiomaterials 2010, 31, 4972

POxylated liposomes: Woodle et al. Bioconj. Chem. 1994, 5, 493.

POxylated proteins : Viegas et al. Bioconjug. Chem. 2011, 22, 976.

Tong et al. Mol. Pharmaceutics 2013, 10, 360.

POxylated nanoparticles : Manzenrieder et al. Angew. Chem. Int. Ed. 2011, 50, 2601.

Bludau et al. Eur. Polym J. 2016.

Immunogenicityin vitro


Poly(2-oxazoline)s as Polymer Therapeutics

Reviews: R. Luxenhofer, A. V. Kabanov, R. Jordan et al. Macromol. Rapid Commun. 2012, 33, 1613.R. Luxenhofer, A. V. Kabanov, R. Jordan et al. Biomaterials 2018, 178, 204.


POx: Amphiphilicity

PMeOx PEtOx PiPOx PnPOx PBuOx PNOx PPhOx

hydrophilic hydrophobicTcp≈70°C Tcp≈40°C Tcp≈25°C

amphiphilic monomer unit

(polysoap)

amphiphilicity

amphiphilicity

≈PEG

M. B. Foreman, J. P. Coffman, M. J. Murcia, S. Cesana, R. Jordan, G. S. Smith, C. A. Naumann, Langmuir 2003, 19, 326.R. Luxenhofer, R. Jordan, Materials Matter 2013, 8, 70-73.

R. Luxenhofer, R. Jordan, Materials Matter 2013, 8, 70-73.


T. B. Bonne, et al. Colloid Polym. Sci. 2004, 282, 833.

T. B. Bonne, et al. Macromol. Chem. Phys. 2007, 208, 1402.

R. Ivanova, et al. Macromol. Chem. Phys. 2008, 209, 2248.

A. Schulz, et al. ACS Nano 2014, 8, 2686.

S. Jaksch, et al. Coll. Polym. Sci. 2014, 292, 2413

S. Jaksch et al. Macromol Chem. Phys. 2016, 217, 1448.

Amphiphilic POx Micelles

Amphiphilic POx is a non-ionic polysoap:

• very low CMC ≈ 10-6 to 10-5 mol/L

• defined small and stable micelles

FCS & DLS:

P(NOx7-MOx40)

P(NOx10-MOx32)

P(MOx30-NOx7-MOx26)

P(NOx6-g-MOx40)

P(NOx7-MOx40)

FCS & SANS:

Ideal system for defined micellar drug formulations:

+


Commercial PTX Formulations

Paclitaxel(PTX)

Action:PTX associates and stabilize microtubuli➡︎ supression of cell devision➡︎ apoptosisOne of the most frequently used anti-cancer drug inovarian, breast, lung, prostate cancer & on stents

Problem:Solubility ≦ 1 µg/mL

Status

(Sales)Excipient Drug loading Concentration in

final solutionComments

Taxol® Market CremophorEL / EtOH

< 1% 0.3 - 1.2 mg/mL

(6 mg/mL)▪extensive premedication necessary

▪dose limiting toxicity byexcipient

Abraxane® Market Human serum albumin

10 %(w/w) 5 mg/mL ▪HSA is derivative of human blood

NK105 Phase III(stopped)

PEGylatedsynthetic polypeptide

30 %(w/w) 0.12 mg/mL

(3 mg/mL) ▪Very low final concentration

Opaxio®

(formerly Xyotax)

Phase III poly(l-glutamic acid)

26 %(w/w) 4 mg/mL

(9 mg/mL)

▪Polymer ill-defined▪Pluronic F68 in formulationhttp://www.ema.europa.eu/humandocs/PDFs/EPAR/opaxio/73102609en.pdf

Genexol® Phase PEGylated polyester (PLA)

17 %(w/w) 0.6 - 3 mg/mL

(6 mg/mL)


PTX Formulation with POx

R. Luxenhofer, A. Schulz, C. Roques, S. Li, T. Bronich, E. Batrakova, R. Jordan, A. Kabanov, Biomaterials 2010, 31, 4972.A. Kabanov, R. Jordan, R. Luxenhofer, US 9,402,908, RU 2523714, JP 5671457, EP 09769029.1, IN 7778/CHENP/2010, BR PI0915401-9, CN 200980123536.X.

Dh = 24 nm

cryoTEM


PTX Formulation with POx

• Formulation with PTX gives a stable micellar solution of defined micelles

• Micelles are very small (Dh > 30 nm)

• No further exipients needed (e.g. no cryoprotectants)

• Formulation can be fully reconstructed multiple times

• POx is thermally stable e.g. @ 200°C for 24 h

• POx (POx/drug film) can be sterilized (to remove endotoxines)

• POx/PTX formulation is stable for > 7 month

Taxolday 1

Taxol day 2

POx-PTX day 2

R. Luxenhofer, A. Schulz, C. Roques, S. Li, T. Bronich, E. Batrakova, R. Jordan, A. Kabanov, Biomaterials 2010, 31, 4972.A. Kabanov, R. Jordan, R. Luxenhofer, US 9,402,908, RU 2523714, JP 5671457, EP 09769029.1, IN 7778/CHENP/2010, BR PI0915401-9, CN 200980123536.X.


Drug Solubilization

0 10 20 30 40 50

POxsol

Taxol

Abraxane

NK 105

Opaxio

Genexol

Drug Loading Capacity, LC (wt%)

Drug Concentration (mg/mL)

Taxol®

1%1:100

Abraxane®

10%1:10

50%1:1

POxsol

POxsol

➢ 50 mg/mL PTX solubilized in 50 mg/mL POx

➢ 10 mg/mL PTX injectable but range for more

➢ Solution stabile for at least 7 month

➢ Solubility increase: x 50.000

0 10 20 30 40 50

POxsol

Taxol

Abraxane

NK 105

Opaxio

Genexol formulation

injected

R. Luxenhofer, A. Schulz, C. Roques, S. Li, T. Bronich, E. Batrakova, R. Jordan, A. Kabanov, Biomaterials 2010, 31, 4972.A. Kabanov, R. Jordan, R. Luxenhofer, US 9402908, RU 2523714, JP 5671457, EP 09769029.1, IN 7778/CHENP/2010, BR PI0915401-9, CN 200980123536.X.


Paclitaxel(PTX)

2-nonyl-2-oxazoline (NOx) block:- water insoluble, strongly hydrophobic- needs long hydrophilic PMeOx blocks

to give micelles- block copolymers form stable aggregates

➢ mainly hydrophobic interactions in themicellar core

2-butyl-2-oxazoline (BuOx) block:- first water insoluble POx- short pendant alkyl chain

➢ hydrophobic & polar interactions in themicellar core

Why is the loading capacity for PTX so high?

Polymer composition Mna

[kg/mol]

Mnb

[kg/mol]

Đ PTX

[wt%]

#MU/

PTX

P1 P[MeOx34-b-NOx12-b-MeOx34] 8.3 8.7 1.28 24 3.6

P2 P[MeOx33-b-BuOx26-b-MeOx45] 10.0 11.4 1.14 49 2.3

A. Schulz, et al. ACS Nano 2014, 8, 2686-2696.

P1 P2

LC


P1: PMeOx-NOx-MeOx P2: PMeOx-BuOx-MeOx

30-150°

exp. fit

30-150°

exp. fit

Addition of PTX leads to smaller & defined micelles

PTX loaded Micelles

ABA Triblock POx: Size of Polymeric Micelles by DLS (37°C, buffer, @90°)

P2

onlymicelles

aggregates&

micelles

A. Schulz, et al. ACS Nano 2014, 8, 2686-2696.

90°;

RILT

15 nm

24 nm

≈8 nm

27 nm

P2P1

aggregates&

micelles

onlymicelles


Maximum Tolerated Dose (MTD):Increased Saftey

Kabanov, Luxenhofer, Jordan et al. Biomaterials 2016, 101, 296.

Multiple Dose Schedule:4 times every 4th day injection of given PTX dose. Groups of 3 NCI-Nu/Nu mice.

Single dose: 200 mg/kg PTX in POx was well tolerated. No weight loss or change in animal activity.

Exipient alone: Cremophor EL: 2/3 or 3/3 aminal death.POx: 0/3 for 150 – 1000 mg/kg: - no change in animal activity,

- no signs of toxicity,- no weight loss- injections well tolerated by animals.

Taxol:high viscosity,inflammation at tail skin„shock“ after injectionsx ∼7

x ∼2

POxsol

(P2 & PTX)

MT

D (

mg

/kg

)


Start Day 6, Control Day 6, Taxol Day 6, POxsol

Only POxsol shows complete tumor regressionfor MTD and MTD/2 and 7/7 survivors


Tumor Inhibition

Early stage cancer model: Xenograft of fast growing, very agressive ovarian cancer (A2780) in nude NCI female mice, n=7 for each group

Vtumor≈ 100-200 mm3 ≈1500 mm3 ≈ 800 mm3 no tumor


Tumor Inhibition


POxsol vs Taxol® POxsol vs Abraxane®

Abraxane®

Taxol®

POxsol

Taxol®:

- delayed tumor growth only within treatment time

Abraxane®:

- shows tumor regression within treatment time but - cancer regrowth - severe side effects (paralysis, weight loss)

POxsol:

- complete tumor remission within treatment time for all animals- no noticeable side effects other than drug action- no cancer reoccurrence- full survival

POxsol

Taxol®Control


Tumor Inhibition


Late stage cancer model (A2780, Vtumor≈400 mm3)

Control

POxsol

POxsol vs Taxol® POxsol vs Abraxane®

Taxol®: no control of tumor growth

Abraxane®: @ MTD complete tumor remission but side effects

@MTD/2 tumor regrowth and side effects

POxsol: complete remission of tumor @MTD and @MTD/2 and no regrowth for 120 d

Abraxane®

Abraxane®

POxsol


Tumor Inhibition

Orthotopic cancer model- (LCC6-MDR in mammary fat pad, Vtumor≈300 mm3)- POxsol vs. Taxol vs. Abraxane

POxsol shows best tumor growth inhibition and improved survival

Orthotopic syngeneic cancer model- (T 11 OST), very aggressive, clinically faithful, mimics claudin-lowbreast cancer (Vtumor≈10-50 mm3), immunocompetent mice- POxsol vs. Taxol

POxsol is the only single drug formulation tried to date thatshows significant tumor growth inhibition and improved survival

Advanced cancer models:



POx DDS-Systems for Taxanes:Variation of the hydrophobic block

Y. Seo, A. Schulz, Y. Han, Z. He, H. Bludau, X. Wan, J. Tong, T. K. Bronich, R. Luxenhofer, R. Jordan, A. V. Kabanov, Polym. Adv. Technol. 2015, 26, 837.

tested with drugs:

- paclitaxel (PTX)

- docetaxel (DTX)

POxsol


POx DDS-Systems:Variation of the hydrophobic block

R. Luxenhofer, R. Jordan, A. V. Kabanov et al. Polym. Adv. Technol. 2015, 26, 837.

tested with:

- paclitaxel (PTX)

- docetaxel (DTX)

„POxsol“ is the best performer with taxanesPOxsol


POx DDS-Systems:Variation of the drug

Z. He, A. Schulz, X. Wan, J. Seitz, H. Bludau, D.B. Darr, C.M. Perou, R. Jordan, I. Ojima, A.V. Kabanov, R.

Luxenhofer, J. Control. Release 2015, 208, 67

• Loading capacity (LC) for all taxanes: between 42 and 47 wt%

• MTD for all taxanes significantly improved

(POxsol)

from the Ojima lab


POx DDS-Systems:Variation of the drug & drug combinations

Y. Han, Z. He, A. Schulz, T.K. Bronich, R. Jordan, R. Luxenhofer, A.V. Kabanov Mol. Pharmaceutics 2012, 9, 2302.

Single and Multi-drug formulationsDrug loading: up to 50 wt%

• High loading capacities were also observed for drug combinations in combinations with PTX or 17-AAG

• Solubilization of the drug combinations were similar or better than those observed for single drugs

• Micelles containing ternary drug combinations are stable for at least 14 days

• Drug synergy when co-delivered

• Currently 60 other drugs successfully testedas single formulations

single double triple


• POx formulation of Etoposide & cisPt prodrug• LC>50wt.% stable formulation• combi drug formulation outperforms seperate drug formulations

ETO/Ptcombi

ETO+Pt

ETO/Ptcombi

ETO+Pt

ACS Nano 2018, 12, 2426.



Biomaterials 2019, 192, 1.


• POx formulation of PTX & cisPt prodrug• LC>50wt.% stable formulations• combi drug formulation outperforms seperate drug formulations

PTX/cisPtcombi

PTX+cisPt


Summary

▪ Amphiphilic POx as a tunable DDS system for micellar drug delivery

▪ Extremely high drug loading because of hydrophobic and polar interactions

▪ Increase of solubility up to x 50.000

▪ Facile drug formulation with no further excipients

▪ Stabile micellar solution of small and defined micelles of low viscosity

▪ Improved Safety: Very high MTD

▪ High animal acceptance of POx & POx/drug with no observable side effects

▪ Complete tumor regression in xenografts

▪ Significant efficacy in orthotopic cancer models

▪ 60 other drugs successfully formulated

▪ Successful formulation of multiple drugs: combination chemotherapy

▪ POx-based drug formulation was independently evaluated by:

Poly(2-oxazoline)s as a Micellar DDS


Thank you!

Yingchao Han

Zhijian He

Robert Luxenhofer

Anita (Schulz)

Luxenhofer

Herdis Bludau

Elena Batrakova

Tatiana Bronich

Alexander V.

Kabanov

Rainer Jordan

XiaomengWang

Youngee Seo

Lisa Holz

ErikWegener

SarahNaumann

polyoxazoline: a potent alternative to peg-based ... · a potent alternative to peg-based...

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