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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