1. 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, Prague 6, Czech Republic

2. 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/D by Hsieh, Ming-Ju; Chen, Mu-Kuan; Yu, Ya-Yen; Sheu, Gwo-Tarng; Chiou, Hui-Ling/ Phytomedicine: International Journal of Phytotherapy & Phytopharmacology

3. 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

4. Multidrug resistance (MDR)
Decrease of drug concentration inside the cells by various mechanisms:
Drug efflux: cytotoxic drugs are pumped out from the cell by transmembrane proteins (e.g. P-glycoprotein, P-gp)
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
M ~ 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“

5. P-Glycoprotein efflux
P-gp efflux
Extracellular space
Intracellular space
Cytoplasmatic membrane
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

6. Inhibition of P-glycoprotein
Kabanov, Batrakova et al., USA – micellar systems based on Pluronic (Poloxamer)
E.V.Batrakova et al. Journal of Controlled Release 130 (2008)
Pluronic F127 (100:65:100) ~ Poloxamer 407
MH ~ 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 ~ g/mol (PPO ~ 1800 Da, 10% PEO block)
L … „liquid“
P … „paste“
F … „flake“
HYDROPHOBICITY
HLB
HLB … „Hydrophilic-lipophilic balance“

7. Amphiphilic block polymer-drug carriers: pHPMA
HYDROPHOBIC PART B = PPG
poly(HPMA-co-Ma-Acap-NHNH-BOC)
poly(propylene glycol)
Amphiphilic block polymer-drug carriers:
pHPMA
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
DEGRADABLE BOND
HYDROPHILIC PART A = pHPMA copolymer
DRUG = Dox
X … -H (diblock A-B)
or hydrophilic part A (triblock A-B-A)

8. Drug effluxed by P-glycoprotein through the cell membrane.
Polypropylene glycol
Low-molecular-weight drug (dox)
pH-sensitive hydrazone bond
Drug effluxed by P-glycoprotein through the cell membrane.
Drug efflux inhibited by polymer conjugate based on block copolymers pHPMA and PPG.
Copolymer based on
N-(2-hydroxypropyl methacrylamide)

9. SYNTHESIS ABIC-TT ABIN CTA-TT
Reversible Addition-Fragmentation chain Transfer Polymerization
DIBLOCK
TRIBLOCK

10. MOLECULAR WEIGHT - GPC Diblock precursor PPG HPMA copolymer PrecursorMn
Mw/Mn
pHPMA copolymer
8 450
1.2
PPG (Aldrich)
4 000
~1.0
Diblock
-NHNH2
12 950
Triblock
19 200
Triblock precursor
PPG
HPMA copolymer
Conjugate
wt % of Dox
Diblock
-NHN=Dox 8
Triblock 10
TSK 3000 Super SW;
80% MeOH/20% acetic buffer

11. TRIBLOCK POLYMER PRECURSOR DIBLOCK POLYMER PRECURSOR
MOLECULAR WEIGHT - FFF LS
dRI 1 2
TRIBLOCK POLYMER PRECURSOR
Peak Mn
Mw/Mn 1
cca
1.8 2
1.5
52% of all injected mass
DIBLOCK POLYMER PRECURSOR
Peak Mn
Mw/Mn
1+2
cca - 3
1.3
over 82% of all injected mass
regenerated cellulose membrane (10 kDa); water/NaN3

12. Light scattering measurements(DLS)
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°.
Precursor
RH /nm
pHPMA copolymer
4.1
PPG -
Diblock
-NHNH2
20.0
Triblock
14.6
Conjugate
RH /nm
Diblock
-NHN=Dox
18.8
Triblock
13.2

13. Stability of prepared particles
DIBLOCK POLYMER PRECURSOR, PBS, pH 7.4

14. Drug release degradation Diblock Polymer conjugate with Dox Triblock
Phosphate buffers:
mimicking environment inside cells - pH 5.0
mimicking environment of bloodstream- pH 7.4
37°C
The amount of released Dox determined by GPC, UV (488 nm)
degradation
Diblock
Polymer conjugate
with Dox
Triblock

15. IC 50 NB3 (sensitive) related to Dox NB3/Dox (MDR) related to Dox
In vitro tests (IC 50) on neuroblastoma cells (sensitive cells: NB3; MDR cells: NB3/Dox) Control: Dox … low-molecular-weight Doxorubicin Linear-Dox … Doxorubicin linked by hydrazone bond to a linear pHPMA copolymer
NB3 (sensitive)
related to Dox
NB3/Dox (MDR) related to Dox
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) .

16. In vitro study of inhibition of P-gp: Calcein assay
Calcein fluorescence intensity
Diblock polymer precursor
Polypropylene glycol
Calcein fluorescence intensity
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.

17. 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 bond
pH 5.0 (intracellular environment) – 80% of released Dox/24h
pH 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

18. Thank you for your kind attention 
Acknowledgement
Ministry of Education, Youth and Sports of the Czech Republic (grant No. EE ) 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
Thank you for your kind attention 