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: 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 ~ 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“

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

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) 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“ HYDROPHOBICITY HLB HLB … „Hydrophilic-lipophilic balance“

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)

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)

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

MOLECULAR WEIGHT - GPC Diblock precursor PPG HPMA copolymer Precursor Mn 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

TRIBLOCK POLYMER PRECURSOR DIBLOCK POLYMER PRECURSOR MOLECULAR WEIGHT - FFF LS dRI 1 2 TRIBLOCK POLYMER PRECURSOR Peak Mn Mw/Mn 1 cca 24 000 1.8 2 1 016 000 1.5 52% of all injected mass DIBLOCK POLYMER PRECURSOR Peak Mn Mw/Mn 1+2 cca 40 000 - 3 1 110 000 1.3 over 82% of all injected mass regenerated cellulose membrane (10 kDa); water/NaN3

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

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

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

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) .

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.

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

Thank you for your kind attention  Acknowledgement 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 Thank you for your kind attention 