CHAPTER 1 INTRODUCTION AND OBJECTIVES

Transcription

1 CHAPTER 1 INTRODUCTION AND OBJECTIVES 23

2 24

3 : Contents 1.1 Introduction Objectives of the thesis Organization of the thesis References

4 26

5 1.1 Introduction Improving the therapeutic index of drugs is the major impetus for the innovation in therapeutic areas such as cancer, inflammatory, and other infective diseases. Today, vast majority of clinically used drugs are of low molecular weight compounds (typically under 500 g mol -1 ) that exhibit very short half-life in blood and high overall clearance rate from the body. These small molecules usually interact through a multiple but monovalent binding with a receptor, diffuse rapidly into healthy tissues and distribute evenly within the body. Therefore only a very small amount of the drug reach the target site and this create therapy associated with side effects, especially by those drugs that exhibit only a narrow therapeutic index like anticancer and immunosuppressive agents. Usual side effects associated with these drugs are nephrotoxicity, bone marrow toxicity, neurotoxicity, cardiotoxicity, mucositis and gastrointestinal toxicity. These side effects are dose limiting and therefore prevent the effective treatment of the diseases (van Rijt & Sadler 2009). Delivery of very high dose of traditional chemotherapeutic agents especially at tumor sites without causing systemic damage and side effects is still a formidable task. The interface of polymer chemistry and biomedical science has given rise to a new field called polymer therapeutics for the treatment of diseases. It encompasses polymer-protein conjugates, drug-polymer conjugates and supramolecular drug delivery systems. The search for new drug delivery concepts and new modes of action are the major driving force in polymer therapeutics (Duncan et al. 2006). Macromolecular carriers selected for 27

6 synthesis of polymer-drug conjugates should be water-soluble, non-toxic and non-immunogenic as well as degraded and/or eliminated from the organism. In conventional polymeric drug delivery system, the bioactive molecule is entrapped inside the polymer matrix and solubilizes the drug moiety without any covalent conjugation. But such non-covalent interactions can result the leakage of the drug moiety under biological conditions which can alter the pharmacokinetics of drug delivery. Polymer therapeutics has several advantages like precise release of the drug under suitable biological conditions, improved solubility of the drug in water, higher stability of the unstable peptide/protein based drugs and reduction in toxicity of the drug due to its bound state (Haag 2004). Many macromolecular drug delivery systems are under investigation to improve therapeutic potential of a drug (Cho et al. 2008). In nanoparticulate drug delivery systems, the drugs are physically incorporated into the nanoparticles as emulsions, liposomes, and non-covalent micellar carrier systems. In drug polymer conjugates, a drug molecule is covalently linked to polymers such as proteins, polysaccharides or synthetic polymers (Packhaeuser et al. 2004). Coupling a drugs to macromolecular carriers received important impetus from 1975 onwards with the development of monoclonal antibodies (Kohler & Milstein 1975). The concept of a drug delivery system based on synthetic polymers was first proposed by Helmut Ringsdorf in 1975 (Ringsdorf 1975). This model (Figure 1.1) consists of five components: macromolecular polymeric backbone, drug molecule, spacer, targeting group, and a solubilizing agent. Spacer arm is the linker that connects the drug with the macromolecule and it 28

7 should be capable of releasing the drug under specific conditions prevalent at the point of its release. The targeting moieties like antibodies/peptide/sugar should have high affinity or recognition towards the disease related antigens or overexpressed cellular receptors. To follow the course of action of the carrier, an imaging probe like FITC can be incorporated. A solubilizing group like PEG can also be incorporated to the nanocarrier to improve the aqueous solubility of the whole system, if necessary. Figure 1.1 Ringsdorf s model for polymeric drug delivery systems In recent years significant advancement have been made in the field of nano sized drug delivery systems (Janib et al. 2010). These include drug loaded liposomes, micelles, polymeric nanoparticles and dendrimers as diagnostic and therapeutic tools against diseases like cancer, infectious or neurodegenerative disorders (Zhang et al. 2007). In anticancer therapeutics, the nanotechnology based formulations can help to enhance the efficiency of the free form of drugs by the preferential targeting of the desired organ/tissue/cell/cellular compartment. This can improve the pharmacokinetics and reduce the systemic toxicity of the drug (Salata 2004). The linear poly(ethylene glycol) (PEG), poly(lactide-co-glycolide) (PLGA), polysaccharide based, and amino acid based polymers have been used to deliver bioactive compound through the systemic circulation (Soppimath et al. 2001). Advanced polymeric architecture such as 29

8 hyperbranched molecules was also developed as carriers to deliver therapeutic agents (Svenson & Tomalia 2012). The holy grail of nanomedicine is the design and synthesis of a novel advanced polymer macromolecule as nanocarriers that can effectively transport multiple drugs, deliver them at the target site in a controlled manner and finally dismantle itself and clear from the system (Garcia-Bennett et al. 2011). This is the most challenging task for the polymer based nanocarrier synthesis as it required a thorough understanding of the structural diversity requirements, physical and chemical properties of the polymer. Still now not enough polymer architectures are designed and developed as a carrier after a critical evaluation of their physical and chemical parameters. Nanocarrier construction represents a subtle equilibrium between organic chemistry, macromolecular synthesis, physico-chemistry and pharmaceutical chemistry. The successful design of a biodegradable nanocarrier is governed by the nature of the monomer selected for the synthesis and the way it is synthesized. In poly alkyl ether based polymer carriers, the available functionalities to load the drug and the other molecules are very limited. Multifunctional poly alkyl ether based carrier can be developed either as dendrimers or as hyperbranched polymers. These polymers showed low polydispersity, multi-functionality and well defined nanoscale globular structure. But their multi-step solution phase synthesis is a laborious, time consuming and required extensive purification procedures to isolate the pure target molecule. The high production cost limits their large scale use as drug delivery vehicles. These multi-functional carriers also lack chemical differentiation of functional groups (Tomalia et al. 1985) and 30

9 therefore incorporation of different molecules like drug, targeting sequence, biomarkers and solubilizing moiety could be achieved only in a random manner. Considering all these facts, we propose the design and synthesis of a novel multifunctional, biodegradable poly alkyl ether based dendritic nanocarrier below 2000 Da by the block polymerization of ethyl 2-(hydroxymethyl) acrylate. Polymer supported organic synthesis technique was used to develop the multifunctional carrier molecule. This technique can help to drive each synthetic step to completion by using excess reagents and the intermediate purification was carried out by just washing with suitable solvents. After each generation, the functional groups present in the partially grown nanocarrier were utilized to incorporate drug molecule. This allows the loading of enough drug molecules in the carrier. After the fourth generation, receptor specific ligands, biomarker and the solubilizing moiety, if necessary can be incorporated in an orderly manner. This technique helps to achieve improved specificity, enhanced potency and prolonged delivery for the treatment of cancer. This nanocarrier could impart high solubility with reduced toxicity of anticancer drugs to normal tissues and provide improved stability to targeting peptide ligands. The proposed cost effective biodegradable nanocarrier can overcome several limitations of the existing multifunctional dendrimers/hyperbranched carriers, like chemical differentiation of functional groups and the polydispersity to less than 1.2. This nanocarrier could have potential application in the field of chemotherapy of cancer, gene and protein delivery. 31

10 1.2 Objectives of the thesis The main objective of the thesis is to develop a novel poly alkyl ether based multifunctional dendritic nanocarrier that can deliver anticancer drug specifically to the tumour cells. This thesis discusses about the: 1. Design and development of a novel monomer for the synthesis of nanocarrier and its characterization. 2. Design and synthesis of a novel surface functionalized polymer support for the solid phase organic synthesis of the nanocarrier and its characterization. 3. Synthesis of the multifunctional poly alkyl ether based nanocarrier by the block polymerization technique and its characterization. 4. In silico design and synthesis of peptide ligands to specifically target the overexpressed epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor (VEGFR) on tumor cells and in vitro functional evaluation of the peptide ligands. 5. Incorporation of drug molecule (doxorubicin or methotrexate) after each generation of the carrier molecule, incorporation of the targeting peptide ligands and the biomarker to the surface functional sites of the dendrimer and the structural and functional characterization of the nanocarrier. 6. In vitro and in vivo biological evaluation of multifunctional dendritic nanocarrier system for future drug delivery applications. 32

11 1.3 Organization of the thesis Chapters of this thesis are organized as follows provides brief introduction about the current strategies in chemotherapy, its drawbacks and the need for a novel target specific multifunctional dendritic nanocarrier for anticancer drug delivery. This chapter also gives a brief description about the objectives and organization of the thesis. Chapter 2 provides detailed review of various nanosized drug delivery systems with special emphasis on current clinical practice, new approaches and developments in the pipeline in anticancer nanotherapeutics. Chapter 3 describes the design and development of the monomer ethyl 2- (hydroxymethyl) acrylate by Morita-Baylis-Hillman reaction and the advantages of microwave energy on this reaction when it was carried out in PEG 200-DABCO the green reaction medium. Chapter 4 describes the synthesis and characterization of novel core-shell amino functionalised tetraethyleneglycol diacrylate (TTEGDA) cross linked polystyrene (PS) support for the solid phase organic synthesis of nanocarrier. This chapter also evaluates the utility of the new support over commercial supports for the solid phase synthesis of polypeptides. Chapter 5 discusses in silico design, synthesis and functional evaluation of peptide ligands to target the overexpressed EGFR and VEGFR in tumor cells and the in vitro functional evaluation of the peptide ligands. Chapter 6 describes solid phase organic synthesis of poly alkyl ether based multifunctional dendritic nanocarrier and its structural and functional characterization. It also discusses the incorporation of anticancer drug molecule, 33

12 peptide ligands to target EGFR and VEGFR in tumor cells and in vitro functional evaluation of peptide-drug conjugates. Chapter 7 discusses the in vitro and in vivo toxicity and in vivo biological evaluation of the new target specific drug delivery system and its biodistribution studies. Summary and conclusion provides brief summary and conclusion of whole thesis work. 34

13 1.4 References Cho, K., Wang, X., Nie, S. and Shin, D. M. (2008) Therapeutic nanoparticles for drug delivery in cancer. Clinical cancer research, 14, Duncan, R., Ringsdorf, H. and Satchi-Fainaro, R. (2006) Polymer therapeuticspolymers as drugs, drug and protein conjugates and gene delivery systems: Past, present and future opportunities*. Journal of drug targeting, 14, Garcia-Bennett, A., Nees, M. and Fadeel, B. (2011) In search of the Holy Grail: folate-targeted nanoparticles for cancer therapy. Biochemical pharmacology, 81, Haag, R. (2004) Supramolecular Drugs-Delivery Systems Based on Polymeric Core-Shell Architectures. Angewandte Chemie International Edition, 43, Janib, S. M., Moses, A. S. and MacKay, J. A. (2010) Imaging and drug delivery using theranostic nanoparticles. Advanced drug delivery reviews, 62, Kohler, G. and Milstein, C. (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature, 256, Packhaeuser, C. B., Schnieders, J., Oster, C. G. and Kissel, T. (2004) In situ forming parenteral drug delivery systems: an overview. European Journal of Pharmaceutics and Biopharmaceutics, 58, Ringsdorf, H. (1975) Structure and properties of pharmacologically active polymers. In: Journal of Polymer Science: Polymer Symposia, Vol. 51, pp Wiley Online Library. Salata, O. V. (2004) Applications of nanoparticles in biology and medicine. Journal of nanobiotechnology, 2, 3. Soppimath, K. S., Aminabhavi, T. M., Kulkarni, A. R. and Rudzinski, W. E. (2001) Biodegradable polymeric nanoparticles as drug delivery devices. Journal of controlled release, 70, Svenson, S. and Tomalia, D. A. (2012) Dendrimers in biomedical applicationsreflections on the field. Advanced drug delivery reviews, 64, Tomalia, D. A., Baker, H., Dewald, J., Hall, M., Kallos, G., Martin, S., Roeck, J., Ryder, J. and Smith, P. (1985) A new class of polymers: starburst-dendritic macromolecules. Polymer Journal, 17, van Rijt, S. H. and Sadler, P. J. (2009) Current applications and future potential for bioinorganic chemistry in the development of anticancer drugs. Drug Discovery Today, 14, Zhang, L., Gu, F. X., Chan, J. M., Wang, A. Z., Langer, R. S. and Farokhzad, O. C. (2007) Nanoparticles in medicine: therapeutic applications and developments. Clinical Pharmacology & Therapeutics, 83,

14 36