Nanoparticles for Targeted Release of Fibrinolytic Drugs.

1. NanoMed 20065th International Workshop on Biomedical Applications of NanotechnologyLogenhaus, Berlin (Germany)16-17 February 2006 Nanoparticles for Targeted Release of Fibrinolytic Drugs. Federica Chiellini, Anna Maria Piras , Cristina Bartoli, Chiara Fiumi, Vincenzina Caruso, Cesare Errico, Matteo Gazzarri, Bruno Fiorentino‘, Robert Anderson*, Marta Muckova°, Chaim Eidelman^, Roberto Solaro, Emo Chiellini. Department of Chemistry and Industrial Chemistry, University of Pisa, Pisa (Italy). ‘Kedrion spa, Castelvecchio Pascoli – Lucca (Italy) *Polymer Laboratories ltd, Church Stratton (United Kingdom) °Vulm a.s. Modra (Slovakia) ^ Novetide ltd, Haifa bay (Israel)

2. TATLYS “A New Biocompatible Nanoparticle Delivery System for Targeted Release of Fibrinolytic Drugs” G5RD-CT-2000-00294 •Kedrion SpA (I) • University of Pisa (I) • Polymer Laboratories Ltd (UK) • Novetide Ltd (IL) •Vulm a.s. (SK) • Polish Academy of Science (PL) • Czech Academy of Science (CZ) • Slovak Academy of Science (SK)

3. Acute Myocardial Infarction (AMI) Formation of Intracoronary Thrombus (95 % of cases) Protease Vascular injury Fibrinogen Prothrombin Thrombin activation Fibrin (monomer) Polymerization Soft clot Fibrin Clot Factor XIIIa t-PA Plasminogen Plasmin Hard Clot Clot Lyses u-PA

4. Available Treatments for AMI  Percutaneous Transluminal Coronary Angioplastic (PTCA)  Fibrinolysis Standard Treatment  Thrombolytic drugs: - Urokinase (Uk); - Recombinant Tissue Plasminogen Activator (rt-PA) Side effects: Fatal Hemorrhage Risks Improvement of Fibrinolytic Therapy: Targeted Selective Activation of Plasminogen in Thrombus

5. Development of the Project • Selection of the Target and Design of Specific Ligands • Design, Synthesis and Biological Characterization of Polymeric Materials • Biofunctionalization of Polymeric Materials • Formulation of Drug Loaded and Targeted Nanoparticles • Biological Evaluation of Polymeric Nanoparticles • Evaluation of Biological Activity of Loaded Drug • Assessment of Nanoparticles Stability

6. Fibrin as Targeting Site Selection of Sequences Exposed on the Surface of Fibrinbut not on Fibrinogen Molecule Selected Epitope on g Chain

7.  Monoclonal Antibody and Fab Fragment for the selected fibrin epitope  Molecular Modeling of fibrin Epitope  Identification of oligopeptides as specific ligands Selection and Design of Targeting Moieties H-Arg-Glu-Lys-NH2 H-Lys-Asp-Arg-NH2 H-Lys-Asp-Lys-NH2 H-Lys-Glu-Arg-NH2 H-Lys-Glu-Lys-NH2 Ac-Arg-Asp-Lys-Asp-Arg-OH H-Arg-Asp-Lys-Asp-Arg-OH H-Arg-Asp-Lys-Asp-Arg-NH2 Design of fibrin-specific ligands for drug targeting I. Massarelli1, A.M. Bianucci2, F. Chiellini1,C. Eidelman3, E. Chiellini1

8. Hemiester of Alternating Copolymer of Maleic Anhydride/n-Butyl vinyl ether (VAM41) C6H6 AIBN 60 °C Mw: 300kDa DCM Mw: 30-40kDa THF 75°C DMAP

9. Modified VAM41 Polymeric Material

10. Biofunctionalized VAM41 Polymeric Material Fab-SH: Monoclonal Antibody Fragment against Fibrin Epitope Oligopeptides-SH

11. Nanoparticle Stabilizers b-cyclodextrin

12. In Vitro Evaluation of Polymeric Materials Cytotoxicity  ISO 10993 (EN 30993) Part 10995: Test for Cytotoxicity– In vitro methods. Cell Line: balb/3T3 clone A31 mouse embryo fibroblasts (ATCC CCL-163)  Polymers dissolved in cell growth media (DMEM)  Cell viability analyzed by mean of : √ Tetrazolium salts (cell metabolism) √ LDH release (cell membrane integrity) Determination of IC50

13. In VitroCytotoxicity of Bioerodible Polymers (1) Cytotoxicity was evaluated by WST-1 cell proliferation reagent (Roche) In vitro toxicity correlates with polymeric materials molecular weight

14. In VitroCytotoxicity of Bioerodible Polymers (2) Cytotoxicity (%)  Cytotoxicity was evaluated by mean of LDH release  Good correlation between results obtained with different methods

15. In VivoCytotoxicity of Bioerodible Polymers Acute Toxicity in mice after single i.v. dosepolymers and stabilisers Acute Oral Toxicity in mice

16. Mutagenicity  GIG-bCD and bioerodible VAM 41 polymers (Mw 300-100 kDa), up to a maximum concentration of 5 mg/plate were not mutagenic in any of the Salmonella typhimurium TA 100, TA 102, TA98 and TA 102 strains  VAM41-F and G polymers (Mw 40 kDa) up to a maximum concentration 2000 mg/plate were not mutagenic in any of the Salmonella typhimurium TA 100, TA 102, TA98 and TA 102 strains  Lack of mutagenic potential was defined as no substantial increasesin the number of revertant colonies observed in any of theSalmonella typhimurium strains.

17. Nanoparticles Preparation Co-precipitation Technique - Original Procedure - Avoid use of aggressive organic solvents - Straightforward and Reproducible - Tunable for different type of polymeric materials +

18. Naked Nanoparticles • Nano-sized particles (mean diameter: 125-150 nm) • Monomodal dimension distribution • Fairly reproducible results • Yield: 60%

19. Biofunctionalized Nanoparticles • Targeted Nanoparticles, Pegylated-Targeted Nanoparticles. • Nano-sized particles (mean diameter: 90 - 120 nm) • Monomodal dimension distribution • Fairly reproducible results • Yield: 60%; Encapsulation Efficiency: 70%

20. Z Potential Sample concentration: 0.1 mg/ml, T= 25°C. pH 5-5.2 Sample Polymer Grafted Z Pot Molecule (mV SD) NPs VAM41 - -18.5  1.1 NPs-Fab VAM41-Fab Fab -20.1 1.1 NPs-PEG VM2000 PEG -7.8  0.5 NPs-PEG-NHM VM2000-NHM PEG -7.8  0.8 NPs-PEG-Fab VM2000-Fab PEG, Fab -10.6  1.2

21. Purification Method: Centrifugation Sample Polymer Diameter (nm  SD) Suspension pellet NPs VAM41 78  52 14500  9890 NPs-Fab VAM41-Fab 88  56 827  916 NPs-PEG VM2000 130  16 645  884 NPs-PEG-Fab VM2000-Fab 131  15132  15 NPs-PEG-Peptide VM200-Peptide 95  13 - NPs NPs-PEG-Fab

22. In VitroCytotoxicity of Nanoparticles  Nanoparticles Cytotoxicity was evaluated by WST-1 cell proliferation reagent at a concentration of 5 mg/ml  Cytotoxicity of Nanoparticles resulted fairly low in respect to the polymers itself  Bioerodible pegylated nanoparticles show a lack of in vitro toxicity

23. In VivoCytotoxicity of Nanoparticles Nanoparticles were prepared with pegylated VAM41 bioerodible polymer

24. Loaded Enzymes Model Drug: Porcine Trypsin Serine Protease, Highly Homologous to Urokinase Fibrinolytic Drug: Human Urokinase Serine Protease

25. Encapsulation Efficiency and Enzyme Activity Size Exclusion Chromatography Chromogenic assay (BApNA/S-2444)

26. Trypsin Loaded Nanoparticles Diameter (nm  SD) 139  18 Sample Polymer Loading Activity EE (mg/ml) Maintenance NPs VAM41-G 138 42% 78%

27. Urokinase-Loaded Targeted-Nanoparticles Diameter (nm  SD) 132  17 Sample Polymer Loading Activity EE (UI/ml) Maintenance (UI/mg) NPs VAM41-G 10000 83% 1500 NPs-PEG VM2000 10000 - 1800 NPs-PEG-Fab VM2000-Fab 10000 - 2500

28. Release Kinetics Studies • Dialysis (T: 37 °C) • Regenerated Cellulose Membrane, MWCO 60000 Da • Mean: 0.2% EDTA, PBS 5X pH 7.4 Protein release Activity of the released enzyme

29. Fibrinolytic and Thrombolytic activity In vitro 125I fibrin-labelled human plasma clot lysis assay in vitro UK loaded NPs-PEG Fibrinolytic activity: % of radioactivity of 125I-fibrin released from the clot

30. Fibrinolytic and Thrombolytic activity In vitro 125I fibrin-labelled human plasma clot lysis assay in vitro UK loaded NPs-PEG Thrombolytic activity: % of thrombus weight loss

31. Fibrinolyticand thrombolytic activity correlationIn vitro

32. Time-course of mean blood flow in a carotid artery after i.v. application Activity of urokinase loaded nanoparticleIn vivo Artherial thrombosis in rats

33. Stability programs based on Urokinase shelf life: two years at 5 ± 3 °C. Stability Assessment • Nanoparticle suspension:15 months at room Temperature (21 ± 3 °C) • Lyophilized Nanoparticles : • Accelerated:6 months at 25 ± 2 °C, 60 ± 5% Relative Humidity • Stressed:3 months at 35 ± 2 °C, 75 ± 5% Relative Humidity

34. Stability Studies Stressed Stability studies performed on Urokinase-loaded Nanospheres Batch Purification Lyophilization Analysis NPs-UK Centrifugation Mannitol Activity / NPs Granulometry Sterilisation technique: 0.20 mm filtration

35. Conclusions • Bioerodible polymeric matrices where synthesized with different molecular weight and successfully biofunctionalized with PEG and targeting moieties such as Fab-fragments. • In vitro and in vivo evaluation of polymeric materials showed an increase in biocompatibility of low molecular weight and pegylated matrices • Nanoparticles display an appreciable biocompatibility both in vitro and in vivo

36. Conclusions • Polymeric nanoparticles based on combination of synthetic multifunctional biocompatible polymer and stabilizing agent have been prepared. • Biofunctionalized nanoparticles structured for targeting fibrin g-epitope have been prepared. • Formulation of nanoparticles loaded with proteolytic enzymes has been performed • Controlled Release of the drug has been achieved • In vitro & In vivo studies confirmed prolonged fibrinolytic and thrombolytic activity. • Stability of nanoparticles was undertaken

37. Acknowledgments Dep.t of Chemistry and Ind. Chemistry University of Pisa, Pisa (Italy). Anna Maria Bianucci Ilaria Massarelli Laura Paolini Piero Narducci Kedion SpA Lucca (Italy) Claudio Farina Claudio D’Urso Pierangelo Giovacchini Renato Mariniello