Polyoxazoline: A potent alternative to PEG-based ... · A potent alternative to PEG-based...
Transcript of 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
& DelAqua Pharmaceuticals Inc., USA
Alexander V. KabanovCenter for Nanotechnology in Drug Delivery, University of North Carolina at Chapel Hill, USA
& DelAqua Pharmaceuticals Inc., 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)
Professur für Makromolekulare Chemie
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)
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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
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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
Professur für Makromolekulare Chemie
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
BOC protected piperazine:especially efficient terminating agent and useful for defined ligation
same endgroup
different
differentorthogonal
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Living cationic polymerization: Chemistry
initiator salt
R3
Both termini AND every monomer unitcan bear functional groups.
random or gradientcopolymers
block copolymers
electrophile
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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
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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)
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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)
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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
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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!
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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
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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.
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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.
Professur für Makromolekulare Chemie
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:
+
Professur für Makromolekulare Chemie
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)
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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
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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.
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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.
Professur für Makromolekulare Chemie
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
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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
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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
)
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Start Day 6, Control Day 6, Taxol Day 6, POxsol
Only POxsol shows complete tumor regressionfor MTD and MTD/2 and 7/7 survivors
Kabanov, Luxenhofer, Jordan et al. Biomaterials 2016, 101, 296.
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
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Tumor Inhibition
Kabanov, Luxenhofer, Jordan et al. Biomaterials 2016, 101, 296.
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
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Tumor Inhibition
Kabanov, Luxenhofer, Jordan et al. Biomaterials 2016, 101, 296.
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
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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:
Kabanov, Luxenhofer, Jordan et al. Biomaterials 2016, 101, 296.
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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
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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
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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
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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
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• 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.
POx DDS-Systems:Variation of the drug & drug combinations
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Biomaterials 2019, 192, 1.
POx DDS-Systems:Variation of the drug & drug combinations
• POx formulation of PTX & cisPt prodrug• LC>50wt.% stable formulations• combi drug formulation outperforms seperate drug formulations
PTX/cisPtcombi
PTX+cisPt
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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
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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