BLOCK COPOLYMERS OF POLY(N-2-HYDROXYPROPYL

METHACRYLAMIDE) AND POLY(PROPYLENE GLYCOL)

– THE WAY TO INHIBIT P-GLYCOPROTEIN?

BLOCK COPOLYMERS OF POLY(N-2-HYDROXYPROPYL

METHACRYLAMIDE) AND POLY(PROPYLENE GLYCOL)

– THE WAY TO INHIBIT P-GLYCOPROTEIN?

Alena Braunová, Libor Kostka, Lucie Cuchalová, Zuzana Hvězdová, Olga

Janoušková, Michal Pechar, Tomáš Etrych and Karel Ulbrich

Institute of Macromolecular Chemistry AS CR, Heyrovský Sq. 2, 162 06 Prague 6, Czech Republic

MULTIDRUG RESISTANCE (MDR)

• „the resistance of tumour cells to more than one chemotherapeutic agent“ Mosby's Medical Dictionary, 8th edition. © 2009, Elsevier.

• „the adaptation of tumour cells or infectious agents to resist chemotherapeutic agents“Jonas: Mosby's Dictionary of Complementary and Alternative Medicine. (c) 2005, Elsevier.

• „the insensitivity of various tumours to a variety of chemically related anticancer drugs; mediated by a process of inactivating the drug or removing it from the target tumour cells“Farlex Partner Medical Dictionary © Farlex 2012

• „the ability of cancer cells to become simultaneously resistant to different drugs, limits the efficacy of chemotherapy“Psoralen reverses docetaxel-induced multidrug resistance in A549/D16 ... by Hsieh, Ming-Ju; Chen, Mu-Kuan; Yu, Ya-Yen; Sheu, Gwo-Tarng; Chiou, Hui-Ling/ Phytomedicine: International Journal of Phytotherapy & Phytopharmacology

MULTIDRUG RESISTANCE (MDR)• Protective reaction of cancer cells, which is caused by long-term exposure of

cytotoxic drugs on the cells• Negative effects of chemotherapy selection for survival of cancer cells

overexpressing P-gp (resistant cells) from an initially heterogeneous population for P-gp expression a new clonal population of cancer cells resistant to most chemotherapeutic agents MDR

http://www.angioworld.com

MULTIDRUG RESISTANCE (MDR)• Decrease of drug concentration inside the cells by various mechanisms:

o Drug efflux: cytotoxic drugs are pumped out from the cell by transmembrane proteins (e.g. P-glycoprotein, P-gp)

• M ~ 170 000 g/mol, 1280 aminoacids• ATP-dependent efflux pump for

xenobiotic compounds with wide substrate specificity• A member of a large family of „ATP-

binding cassette transporters“

Structure of P-gp

Extracellular space

Intracellular space

Cytoplasmatic membrane

P-glycoprotein (P-gp)„protein in the cell membrane that transports drugs out of the cell conferring resistance of that cell to that drug“Farlex Partner Medical Dictionary © Farlex 2012

P-GLYCOPROTEIN EFFLUX

A,B-exogenous compound, e.g. lipophilic drug; C-endogenous compound

• Healthy cells – xenobiotic transporter (toxins etc.) from intracellular space to extracellular matrix

X• Cancer cells (MDR) - expressed high levels of P-gp - P-gp contributes to the cell resistant to the drug effect Inhibition of Pgp = improve penetration of drugs inside the cells more effective cancer therapy

P-gp efflux

Extracellular space

Intracellular space

Cytoplasmatic membrane

Kabanov, Batrakova et al., USA – micellar systems based on Pluronic (Poloxamer)

INHIBITION OF P-GLYCOPROTEIN

E.V.Batrakova et al. Journal of Controlled Release 130 (2008) 98-106

* Pluronic F127 (100:65:100) ~ Poloxamer 407

MH ~ 12 600 g/mol (PPO ~ 4000 Da, 70% PEO block)

* Pluronic P85 (26:40:26)

MH ~ 4600 g/mol (PPO ~ 2300 Da, 50% PEO block)

* Pluronic L61 (2:30:2) ~ Poloxamer 181

MH ~ 2 000 g/mol (PPO ~ 1800 Da, 10% PEO block)

L … „liquid“

P … „paste“

F … „flake“

HYD

ROPH

OBI

CITY

HLB

HLB … „Hydrophilic-lipophilic balance“

• Amphiphilic block polymer-drug carriers:o pHPMAo PPG responsible for P-gp

inhibition• micelle or nanoparticle increase of

MW passive targeting by Enhanced Permeability and Retention effect)

• Covalent binding of Dox by degradable hydrazone pH-sensitive bond drug release inside the cell

DRUG = Dox

DEGRADABLE BOND

HYDROPHILIC PART A = pHPMA copolymer

HYDROPHOBIC PART B = PPG

poly(HPMA-co-Ma-Acap-NHNH-BOC)

poly(propylene glycol)

X … -H (diblock A-B) or hydrophilic part A (triblock A-B-A)

P-GLYCOPROTEIN

POLYPROPYLENE GLYCOL

LOW-MOLECULAR-WEIGHT DRUG (DOX)pH-SENSITIVE HYDRAZONE BOND

A. DRUG EFFLUXED BY P-GLYCOPROTEIN THROUGH THE CELL MEMBRANE.

B. DRUG EFFLUX INHIBITED BY POLYMER CONJUGATE BASED ON BLOCK COPOLYMERS PHPMA AND PPG.

COPOLYMER BASED ON N-(2-HYDROXYPROPYL METHACRYLAMIDE)

DIBLOCKTRIBLOCK

ABIN CTA-TT

ABIC-TT

SYNTHESIS

Reversible Addition-Fragmentation chain Transfer Polymerization

MOLECULAR WEIGHT - GPC

PRECURSOR

Mn Mw/Mn

PHPMA COPOLYMER

8 450 1.2

PPG (Aldrich) 4 000 ~1.0

DIBLOCK-NHNH2

12 950 1.2

TRIBLOCK-NHNH2

19 200 1.2CONJUGATE wt % of Dox

DIBLOCK-NHN=DOX 8

TRIBLOCK-NHN=DOX 10

Diblock precursor PPGHPMA copolymer

TSK 3000 Super SW; 80% MeOH/20% acetic buffer

Triblock precursor PPG

HPMA copolymer

MOLECULAR WEIGHT - FFF

LSdRI 1 2

over 82% of all injected mass

52% of all injected mass

regenerated cellulose membrane (10 kDa); water/NaN3

Peak Mn Mw/Mn

1+2 cca 40 000 -

3 1 110 000

1.3

DIBLOCK POLYMER PRECURSOR

Peak Mn Mw/Mn

1 cca 24 000 1.8

2 1 016 000

1.5

TRIBLOCK POLYMER PRECURSOR

LIGHT SCATTERING MEASUREMENTS(DLS)PRECURSOR

RH /nm

PHPMA COPOLYMER

4.1

PPG -

DIBLOCK-NHNH2

20.0

TRIBLOCK-NHNH2

14.6CONJUGATE

RH /nm

DIBLOCK-NHN=DOX

18.8

TRIBLOCK-NHN=DOX

13.2

Measurements were performed at 37°C in PBS buffer (pH 7.4) on a ZEN 3600 (Zetasizer Nano instruments, Malvern, UK) at scattering angle Ө 173°.

STABILITY OF PREPARED PARTICLES

DIBLOCK POLYMER PRECURSOR, PBS, pH 7.4

DIBLOCKPOLYMER

CONJUGATE WITH DOX

TRIBLOCKPOLYMER

CONJUGATE WITH DOX

DRUG RELEASE• Phosphate buffers:o mimicking environment

inside cells - pH 5.0o mimicking environment

of bloodstream- pH 7.4• 37°C

• The amount of released Dox determined by GPC, UV (488 nm)

69

yx

OCH3

CN

NH

N

O

O

CH 3

OH NH 2

H

OH

O

O

OH

OH

CH 2

OH

O

NH

C

C

CH 3

CH 2C

CH 3

C O

NH

OH

CH 3

C

CH 3

CH 3 CH 2 CH 2 CH 2 C

O

NH

CH 3

O

CH 3

NH

O

X

DEGRADATION

In vitro tests (IC 50) on neuroblastoma cells (sensitive cells: NB3; MDR cells: NB3/Dox)

Control:Dox … low-molecular-weight DoxorubicinLinear-Dox … Doxorubicin linked by hydrazone bond to a linear pHPMA copolymer

IC 50

NB3 (sensitive)related to Dox

NB3/Dox (MDR) related to Dox

NB3 (sensitive)related to linear- Dox

NB3/Dox (MDR) related to linear-Dox

Control(Diblock: 0 μg/ml)

0.0049 0.311 0.045 3.206

Diblock(250 μg/ml)

0.0051 0.036 0.043 0.365

There was no cytotoxicity proved in case of diblock or triblock precursors (without Dox) .

IN VITRO STUDY OF INHIBITION OF P-GP: CALCEIN ASSAY

Incubation of NB3/Dox cells (MDR cells resistant to Dox) with inhibitors (diblock polymer precursor and original polypropylene glycol) for 0.5, 2, 4, 6 and 16 h;

There was no inhibition observed in case of NB3 sensitive cells.

Diblock polymer precursor Polypropylene glycol

Calc

ein

fluor

esce

nce

inte

nsity

Calc

ein

fluor

esce

nce

inte

nsity

+ CONCLUSION

Synthesis of di- and triblock copolymers, containing PPG as potential P-gp inhibitor and Dox as an anticancer drug (8 – 10 wt.%)

Physico-chemical characterization of final copolymers: GPC, FFF, LS

Degradation of hydrazone bondpH 5.0 (intracellular environment) – 80% of released Dox/24hpH 7.4 (bloodstream) – 10% of released Dox/24h

Biological evaluation (in vitro) on parental sensitive NB3 and Dox-resistant NB3/Dox cells proved P-gp inhibition by diblock block precursors (IC 50; Calcein assay)

PROMISED AMPHIPHILIC BLOCK COPOLYMERS USEFUL AS MDR INHIBITORS, AS WELL AS DRUG DELIVERY SYSTEMS

• Ministry of Education, Youth and Sports of the Czech Republic (grant No. EE2.3.30.0029) for financial support

• Department of Biomedical Polymers and Biolab, IMC AS CR, v.v.i., Prague• LS - Peter Černoch, IMC AS CR, v.v.i., Prague• ITC - Sergey Filippov, Anna Bogomolova, IMC AS CR, v.v.i., Prague• FFF – Richard Laga, Bedřich Porsch, Zuzana Mašínová, IMC AS CR, v.v.i., Prague

ACKNOWLEDGEMENT

Thank you for your kind attention