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Biomedical applications of polymer-based pharmaceuticals Biomedical Engineering – Group XII Almeida, Diogo1; Balthazar, Lucas2; Ribeiro, Filipe3 1 [email protected]; [email protected]; [email protected] Abstract The medical use of polymer-based pharmaceuticals is a new hope to healthcare. Polymerdrug conjugates and polymer-protein conjugates gave nanomedicine a whole new meaning, as their application is showing new kinds of treatment for diseases such as cancer and hepatitis C. Judging from the current research, their role will be even more significant in the near future. In this report we will explore natural and synthetic polymers, giving an extra relevance to polymer-drug conjugates, namely its therapeutic benefits. Keywords Polymer-drug conjugate, polymer-protein conjugate, chitosan, hyaluronic acid, dextran, gellan, HPMA, PGA Contents 1. Introduction 2. State of the art 3. Methods and Results 3.1. Polymer-protein conjugates 3.2. Polymer-drug conjugates 3.2.1. Mechanism of action of polymer-drug conjugates 3.2.2. HPMA and PGA as polymers: Brief description 3.2.3. Clinical Results 4. Polymer-drug conjugates: conclusions, challenges and opportunities 5. Natural Polymers 5.1. Chitosan 5.2. Hyaluronic acid 5.3. Dextran 5.4. Gellan 6. Conclusion 7. Acknowledgements 8. References BIOMEDICAL APPLICATIONS OF POLYMER-BASED PHARMACEUTICALS 2008 1. Introduction Over the past decades, research at the level of molecular biology has unveiled the molecular basis for many diseases. New important technologies and concepts, such as recombinant DNA and gene therapy, have provided tools for the creation of pharmaceuticals and methods designed to specifically address such diseases. However, progress towards the application of these medicines outside of the laboratory has been considerably slow, principally due to the lack of effective drug delivery systems, that is, mechanisms that allow the release of the drug into the appropriate body compartment, for the appropriate amount of time, without seriously disrupting the rest of the organism functionality. There has been a growing number of different approaches to counter this issue, each with its own particular applications, given the pathophysiology of the disease. Polymers, and associated nanomedicine technologies, constitute a relatively new and promising approach that has already been proven effective in a wide range of applications. The current work covers mainly two concepts subordinated to the named polymer therapeutics: polymer-protein conjugates (Figure 1) and polymer-drug conjugates. Although both include the binding of polymers to pharmacological substances, polymer-protein and polymerdrug conjugation are effectively different, principally in terms of their final applications and the purpose behind conjugation. It should also be noted that although our work will focus mainly in polymer-drug and polymer-protein conjugation, synthetic and natural polymers are used widely as components of new medical devices, for example, as rate-controlling coatings, of hydrogels or materials for the topical administration of drugs, and as constructs for tissue engineering. Besides the use of polymers as carriers for drug-delivery systems, certain natural polymers such as dextran, gellan, chitosan and hyaluronic acid have very interesting properties, which makes them particularly useful in a diverse spectrum of applications (including protein/drug conjugation). As such, it is also our intention to explore these compounds, in a tentative to find out why this is so. Figure 1: Schematic representation showing the families of polymer constructs called polymer therapeutics.1 Biomedical Engineering 2 BIOMEDICAL APPLICATIONS OF POLYMER-BASED PHARMACEUTICALS 2008 2. State of the art The use of polymers in the medical field is not a novelty - natural polymers have been used as components of herbal remedies for centuries. When it comes to synthetic polymers, however, the situation is very different. Because polymer science is a relatively recent area of research, synthetic water-soluble polymers as macromolecular drugs or as part of drug delivery systems related to inoculation can be considered a modern achievement. Hermann Staudinger, Morawetz, Lehn and Ringsdorf are important names associated to the birth of the polymer science field, which occurred in the 1920s. In 1953, Staudinger received the first Nobel Prize related to this new area of research, about the same time Watson and Crick revealed their theory on the structure of DNA. Such discoveries paved the way for more advanced water-soluble synthetic polymers, which were starting to make an important contribution to healthcare. The first polymer-drug conjugates appeared around 1955, being mescaline-Nvinylpyrolidine conjugate one of the first. This conjugate had a drug attached by non-degradable or enzymatically degradable side chains, quite like many polymer-drug conjugates tested today. In the following years, many others biologically active polymeric drugs followed, as their clinical importance started to increase. In the 1960s, divinylether-maleic anhydride copolymer (pyran copolymer) was clinically tested as an anticancer agent, but the early trials failed due to its severe toxicity, later discovered to be caused by minor differences in the polymer molecular weight (and thus in the chain length). About ten years later Frank Davis and Abraham Abuchowski were able to foresee the potential of conjugating poly(ethylene glycol) (PEG) to proteins, causing the birth of a technique called PEGylation. PEGylation consists in the covalent bond of poly(ethylene glycol) polymer chains to another molecule, usually a drug or a protein with therapeutic effects (Figure 2). This covalent attachment is able to disguise the drug or the therapeutic protein from the immune system of the host, reducing its stimulation (immunogenicity) and increasing the size of the agent in solution, which on its turn increases the conjugate’s circulatory time. Figure 2: Schematic of PEG polymer-protein conjugation. 2 Biomedical Engineering 3 BIOMEDICAL APPLICATIONS OF POLYMER-BASED PHARMACEUTICALS 2008 In the early 1990s, the clinical use of the first PEGylated proteins was approved. At this time, PEGylation became a process widely known and it was crucial for the development of localized therapeutics. As it increases the molecular weight of a molecule, this process improves drug solubility and stability, extending its circulating time, therefore reducing dosage frequency with potentially reduced toxicity, and boosting protection from proteolytic degradation. By the time the first PEGylated medicines were approved, a new phenomenon was discovered in Japan. It was the passive tumour targeting phenomenon now known as the EPR effect (Enhanced Permeability and Retention effect), which consists in the passive accumulation of substances, usually liposomes or macromolecular drugs, in tumour tissue (because of the lack of effective tumour lymphatic drainage), and in the hyperpermeability of the blood vessels towards the same kind of molecules referred above (due to the angiogenic tumour vasculature, which has a discontinuous endothelium). Also in the 1990s, many interesting polymeric drugs were created. Those synthetic products were designed to treat different pathologies, such as multiple sclerosis (treated with synthetic random copolymer of L-alanine, L-lysine, L-glutamic acid and L-tyrosine administered subcutaneously) and HIV-1 (treated with dextrin-2-sulfate given orally). In 1994, the first synthetic polymer-drug conjugate designed to treat cancer was clinically tested. It consisted on an HPMA (N-(2-hydroxypropyl)methacrylamide) copolymer conjugate of doxorubicin. Targeted release of anticancer agents can also be made using block copolymer micelles, which have the ability to entrap the drug or to covalently link to it. By that time, HPMA copolymer micelles were clinically tested for the first time. In the 2000s, two polymer-protein conjugates, PEG-interferon-α (an antiviral drug intended to treat chronic hepatitis C and hepatitis B) and PEG-GCSF (PEG granulocyte colony-stimulating factor) were placed in the market, and five years later the first therapeutic nanoparticleI (albuminentrapped paclitaxel) was approved as a treatment for metastatic breast cancer. All the above achievements and researches were the core elements that led to the development of polymer-based pharmaceuticals, namely polymeric drugs, polymer-drug conjugates and polymer-protein conjugates. The clinical trials of these new technologies eventually lead to the resolution of many other unexpected challenges that quickly appeared, such as the manufacturing of the polymers at an industrial scale and the quick and total solubilization of the pharmaceuticals for safe inoculation. The optimization of these clinical tests (in terms of dosage and frequency) is still being evaluated today for a large variety of products.3, 4 I A nanoparticle is a small particle (usually of 20-500 nm dimensions) produced from natural or synthetic polymers, which are used, as mentioned before, to capture drugs for drug targeting and controlled release. Biomedical Engineering 4 BIOMEDICAL APPLICATIONS OF POLYMER-BASED PHARMACEUTICALS 2008 3. Methods and Results 3.1 Polymer-protein conjugates Polymer-protein conjugation can be seen as an approach to increase the efficiency of protein, peptide and antibody based drugs, given the vast range of these medicines that are being created as a result from genomics and proteomics research, associated with new technologies such as recombinant DNA and monoclonal antibodies. Their limitations often include a short plasma half-life, poor stability, and, especially in the case of proteins, immunogenicity.3 Research done in the 1970’s foresaw the potential of binding the polymer PEG (polyethylene glycol) to proteins and since then a great progress was achieved. Nowadays, the advantages of this technique have become evident. It is used in a wide variety of products, including enzymes, cytokines and monoclonal antibody fragments. PEGylation has been proven to provide among other things, increased protein solubility and stability, reduce receptor-mediated protein uptake by cells of the reticuloendothelial systemII, reduce protein immunogenicity, prevent the rapid renal clearance of small proteins, and prolonging plasma half-lifeIII, thus requiring less frequent dosing, which is of great patient benefit.1 The first polymer-protein conjugate to enter the market was PEG-adenosine deaminase in 1990. Since then, others have followed (Table A). PEG-L-asparaginase, for instance, is used as a treatment for acute lymphoblastic leukaemia, with the advantage of reduced hypersensitivity reactions when compared to the native enzyme. Other PEGylated protein compounds includes PEG-G-CSF (G-CFS stands for a recombinant methionyl human granulocyte colony-stimulating factorIV), which is used to prevent cancer chemotherapy induced neutropaeniaV, with the advantage of requiring less frequent dosing when compared to free G-CSF. In addition to proteins, PEG has also been used to produce a number of polymer-cytokines conjugates. Two PEGinterferon-α conjugates, which have shown better activity in vivo compared to IFN-α, have been approved as treatments for hepatitis C. Despite the possibility of using other polymers for polymer-protein conjugation, PEG has been more widely used due to a number of reasons. Firstly, it has a number of other uses apart from serving as a polymeric carrier, and is known to be non-toxic and non immunogenic. Secondly, it’s flexible, highly water-soluble chain provides a hydrodynamic radiusVI up to 10 times greater than a globular protein of equivalent molecular weight, and in addition to that, it is high degree of hydration means it effectively has a water shell, which helps to mask the protein to which it is II The reticuloendothelial system is a part of the immune system, which consists in the phagocytic cells, primarily monocytes and macrophages. III Plasma half-life can be defined as the time a certain substance takes to lose half its pharmacological or physiological activity. IV Granulocyte colony-stimulating factor is a substance that stimulate the bone marrow to produce granulocytes and stem cells. V Neutropaenia is an abnormal condition characterized by a low number of neutrophils in the blood. VI The hydrodynamic radius is a mathematically defined quantity that arises from the study of the dynamics of polymers moving in a solvent. Here it can be seen as a measure of the easiness a polymer can move in water. Biomedical Engineering 5 BIOMEDICAL APPLICATIONS OF POLYMER-BASED PHARMACEUTICALS 2008 bound. And thirdly, the fact that PEG can be prepared with a single reactive group at one terminal end aids site specific conjugation and prevents protein crosslinkingVII during this process. Table A: Examples of polymer-protein conjugates in clinical use or development. Compound Status Indications PEG-adenosine-deaminase Market Hepatocellular carcinoma PEG-l-asparaginase Market Acute lymphoblastic leukaemia PEG-GCSF Market Prevention of neutropaenia associated with cancer chemotherapy PEG-IFNα2a Market / Phase I/II Hepatitis B and C / Melanoma, chronic myeloid leukaemia and renal-cell carcinoma PEG-IFNα2b Market / Phase I/II Hepatitis C / Melanoma, multiple myeloma and renal-cell carcinoma PEG-arginine deiminase Phase I Hepatocellular carcinoma 3.2 Polymer-drug conjugates Polymer-drug conjugation has been explored so far mainly as a means of targeted drugdelivery for anti-cancer drugs. Most of the anti-cancer polymer-drug conjugates designed rely on the EPR effect for passive targeting. Although extracellular drug-delivery can account for some anti-cancer activity, a main concept behind polymer-drug conjugation is that of lysosomotropic and endosomotropic drug delivery, that is, the liberation of the drug inside lysosomes and endosomes, respectively. As of 2006VIII, quite a few polymer-drug conjugates were under clinical evaluation as shown in Table B. Their clinical performance will be discussed below. Mechanism of action of polymer-drug conjugates Polymer-drug conjugates mechanism of action is based on two main aspects: EPRmediated targeting and endocellular drug-delivery through the endocytic pathway. The following image (Figure 3) summarizes the current understanding of the mechanism of action of polymerdrug conjugates. VII A cross-link is a link between two polymers (in this case, proteins). Cross-links are normally formed by chemical reactions initiated by heat, pressure or radiation. VIII The results presented here are all based on 2006 studies. Biomedical Engineering 6 BIOMEDICAL APPLICATIONS OF POLYMER-BASED PHARMACEUTICALS 2008 Figure 3: A. After intravenous administration of the conjugate, the increased leakiness of the tumour angiogenic vasculature would lead to a preferential accumulation of the drug in the tumour interstitium, by the EPR effect (a and b). The addition of cell specific targeting ligands (as in the case of HPMA copolymer-doxorubicin-galactosamine, see below) could increase the targeting effect. B. After arrival in the tumour tissue, the molecule would be internalized either by fluid-phase pinocytosis, non-specific receptor-mediated pinocytosis or ligand-receptor docking. Lysosomal proteases (such as cathepsin B, which is more expressed in tumoural cells) or the decrease in pH inside endosomes/lysosomes would lead to either the cleavage of the polymer-drug linker or the polymer itself (as in the case of PGA-paclitaxel, see below), releasing the drug inside the cell. Such a system of delivery could, in theory, bypass certain resistance mechanisms, namely those associated with membrane efflux pumps as in the case with MRP (multidrug resistant protein) and p-glycoprotein. Note that if the polymeric carrier is non-biodegradable, its size must be limited in order to assure renal elimination and preventing polymer-associated unwanted toxic effects.3 Table B: Examples of polymer-drug conjugates in clinical development.3 Polymer-drug conjugates Compound name Status Indications HPMA copolymer-doxorubicin Phase II Various cancers, particularly lung and breast cancer HPMA copolymer-doxorubicingalactosamine Phase I/II Particularly hepatocellular carcinoma. HPMA copolymer-camptothecin Phase I Various cancers. HPMA copolymer-paclitaxel Phase I Various cancers. HPMA copolymer-carboplatin platinate Phase I/II Various cancers. HPMA copolymer-DACH-platinate Phase I/II Various cancers. PGA-paclitaxel Phase III Various cancers, particularly non-small cell lung cancer; ovarian cancer Biomedical Engineering 7 BIOMEDICAL APPLICATIONS OF POLYMER-BASED PHARMACEUTICALS 2008 Polymer-drug conjugates PGA-camptothecin Phase I/II Various cancers. Dextran-doxorubicin Phase I Various cancers. Modified dextran-camptothecin Phase I Various cancers. PEG-camptothecin Phase II Various cancers. HPMA and PGA as polymers: Brief description HPMA (N-(2-hydroxypropyl)methacrylamide) homopolymer was initially designed for use as a plasma substitute, and doxorubicin is a drug from the family of the anthracyclinesIX. HPMA copolymer-doxorubicin uses a Gly-Phe-Leu-Gly tetrapeptide as a linker, which is degraded by the lysosomal protease chatepsin B after cellular uptake of the conjugate, following the lysosomotropic model. HPMA copolymer-doxorubicin-galactosamine has a similar structure, except for the addition of galactosamine, which is intended to work as a targeting ligand for the asialoglycoprotein receptor present in hepatocytesX, thus increasing hepatocyte-associated drug concentrations. PGA (polyglutamate), unlike HPMA, is a biodegradable polymer. Therefore, PGA polymerdrug conjugates release drug through hydrolysis during systemic circulation, but most of the drug is released inside the cells, after cleavage of the polymer backbone by the protease chatepsin B. Clinical Results Both HPMA-doxorubicin and HPMA-doxorubicin-galactosamine have displayed, in some areas, significative clinical improvement when compared to their non-conjugated counterparts. HPMA-doxorubicin showed a maximum tolerated dose of 320 mg/m2, which represents a five-fold increase compared to free doxorubicin. Maximum dose associated toxicity was typical of the anthracyclines (mucositisXI and neutropaenia), with the notable exception of cardiotoxicity. During phase I trials, anti-cancer activity was observed in patients with non-small-cell-lung cancer, colorectal cancer and anthracycline-resistant breast cancer. However, during phase II trials, no activity was observed from colorectal cancer patients.3 HPMA-doxorubicin-galactosamine was designed as a treatment for primary liver cancer. A maximum tolerated dose of 160 mg/m2 was observed during phase I/II trials, and maximum tolerated toxicities were again typical of the anthracyclines. Of the 23 patients treated, two displayed partial responses, one displayed a reduction in tumour size, and eleven had stable IX Anthracyclines are a class of drugs widely used in cancer treatment. Their mechanism of action involves the inhibition of topoisomerase II, an enzyme involved in DNA replication. X Hepatocytes are the main type of cells that constitute the liver. XI Mucositis is the painful inflammation of the mucous membranes lining the digestive tract. Biomedical Engineering 8 BIOMEDICAL APPLICATIONS OF POLYMER-BASED PHARMACEUTICALS 2008 disease. However, hepatocyte-associated drug was 12-50-fold higher than what can be achieved with free drug.3 First generation HPMA copolymer-conjugates contained anthracyclines, as these were the most used anti-cancer drug at the time. Camptothecin and paclitaxel, once discovered, became interesting candidates for polymer conjugation given their poor water solubility (and thus the conjugation to a more soluble polymer could help to improve the drug biodistribution), and the hypersensitivity reactions associated with free drug administration, in the case of paclitaxel. However, HPMA copolymer-camptothecin and HPMA copolymer-paclitaxel early clinical trials were disappointing.5 HPMA-camptothecin displayed no anti-tumour activity, whereas HPMApaclitaxel showed no improvement in therms of dose-limiting toxicity compared to free paclitaxel. Both conjugates use a normal ester linkage between the drug and the polymer, and unexpected excessive liberation of drug during systemic circulation and renal clearance could explain some of the observations, namely, the lack of preferential EPR mediated tumour accumulation and severe cystitisXII in the case of HPMA-camptothecin. Partial successes resultant from PGA-paclitaxel phase I/II studies granted eligibility for phase III studies. PGA-paclitaxel was compared with gemcitabineXIII or vinorelbine as a first-line treatment for poor performance status non-small-cell lung cancer (NSCLC) patients.3 A significative improvement in terms of survival was observed when compared with vinorelbine. Such an increase was particularly high in pre-menopausal women treated with PGA-paclitaxel, which could be related to the increased expression of cathepsin B due to greater oestrogen levels.3 A pivotal trial is ongoing in order to compare PGA-paclitaxel with paclitaxel as a first line-treatment for NSCLC in women. 4. Polymer-drug conjugates: conclusions, challenges and opportunities The fact that both HPMA copolymer-doxorubicin conjugates showed a decrease in toxicity when compared to the administration of free drug proves the feasibility of polymer-drug conjugation as a means of decreasing chemotherapy-related general toxicity. On the other hand, HPMA-camptothecin and HPMA-paclitaxel disappointing results make clear the need for careful linkage selection, since the excessive unwanted release of the drug in the bloodstream can seriously hazard anti-cancer activity, either by lowering EPR mediated targeting or increasing general toxicity.3 Most of the conjugates have showed superior in vivo results when compared to their parent drug, and there are some indications that HPMA-copolymer conjugates can circumvent multi-drug resistance (MDR), which would be consistent with lysosomotropic delivery of the drug. It is not yet clear to what extent polymer-drug conjugates display EPR mediated targeting when considering clinical metastatic cancer. Preliminary studies with HPMA copolymerdoxorubicin using rodent models indicated that variations in lysosomal thiol-protease activity XII Cystitis is the inflammation of the urinary bladder. Gemcitabine is a nucleoside analog which replaces cytosine, preventing DNA replication and thus leading to cell apoptosis; vinorelbine acts as an anti-mitotic drug. XIII Biomedical Engineering 9 BIOMEDICAL APPLICATIONS OF POLYMER-BASED PHARMACEUTICALS 2008 (such as cathepsin B) were more important in determining anti-cancer activity than the extent of EPR mediated targeting.3 In addition, the fact that some patients displayed more EPR mediated targeting than others means that certain individuals would benefit more from the administration of conjugates than others, and thus a better understanding of the factors behind EPR-targeting could provide a foundation for the selection of patients better suited for treatment with polymerdrug conjugates.3 Also, the tumour targeting of many polymer-based pharmaceuticals could be improved by co-administration of agents that enhance vascular permeability, providing another possibility for increased effectiveness of the EPR effect. The possibility of administrating more than one drug, either as two different conjugates, or the conjugation of more than one drug to the same polymer in combinatorial therapies is also something that is worth being explored. An ongoing study is examining a combination of PGApaclitaxel and carboplatinXIV as a first-line therapy for ovarian cancer, and initial results showed a positive response in 80 out of 82 patients.3 Another interesting approach has been the administration of a polymer-drug and a polymer-protein conjugate, in a two-step prodrug therapy. In this sense, HPMA copolymer-Gly-Phe-Leu-Gly-doxorubicin could be administrated followed by an HPMA copolymer-chatepsin B, thus triggering a faster release of doxorubicin, after accumulation in the tumour tissue by the EPR effect. It should be noted that although the results of many researches have been satisfactory, polymer-drug conjugation is a concept that allows a lot more of development. New polymer technologies, such as graft polymers, star polymers, multivalent polymers, dendrimers, and dendronized polymers (Figure 4) constitute new polymeric architectures that could (and this is the current trend at the moment) be applied to the field of polymer conjugation, thus providing new opportunities for clinical development. Figure 4: New polymeric architectures that could provide interesting possibilities for polymer conjugation. Their advantages include a more defined chemical composition, tailored surface multivalency, providing more possibilities for conjugation, and a defined three dimensional architecture.1 XIV Carboplatin is a chemotherapy drug used against some forms of cancer, namely ovarian carcinoma, lung, head and neck cancers. Biomedical Engineering 10 BIOMEDICAL APPLICATIONS OF POLYMER-BASED PHARMACEUTICALS 2008 5. Natural Polymers 5.1 Chitosan Figure 5: Chitosan is derived from chitin by a process named alkaline deacetylation, which consists in treating chitin with 50% of hydroxide for a few hours. 6 Chitosan (poly-D-glucosamine) is a natural polymer derived from chitin (Figure 5). After cellulose, chitin is probably the most abundant polysaccharide in nature, whereas chitosan is a very rare compound that only exists in some kinds of fungi. Comparisons drawn between chitosan and chitin show that the former has a better chemical and biochemical reactivity. In addition to that, chitosan is known to have a series of interesting properties: low toxicity, biocompatibility and biodegradability.6 Chitosan is composed by glucosamine units with a free amino group located in the second carbon. The aminogroup (rare in polysaccharides) is frequently used as the reactive side. There are four polymorphic forms of chitosan, discovered from X-ray diffraction measurements, three in a hydrated form and one anhydrous form. Because of its specific characteristics, explained by the unique physicochemical proprieties of the polymer, chitosan has recently attracted more attention over chitin, although the latter also has its uses in pharmaceutical and biomedical branches as a bioactive agent. Chitosan is known to possess an intrinsic antibacterial activity, even though the exact reason for this isn’t fully known. It is believed that the positive amino group of glucosamine units changes the barrier proprieties of the microbial cell membranes, interacting with their negative charged components, either preventing the nutrients from entering the cell or causing the leakage of intracellular contents.6 Another possible explanation for this is that the penetration of chitosan Biomedical Engineering 11 BIOMEDICAL APPLICATIONS OF POLYMER-BASED PHARMACEUTICALS 2008 inside the cell and subsequent binding to DNA provokes the inhibition of RNA transcription and consequently protein synthesis.7 Depending on molecular size, solubility and partial acetylation, chitosan may also have an anti-tumoural activity, displayed specially by low weight forms of the polymer. Chitooligosaccharide with six sugars, for example, can inhibit the proliferation of CT26 cells inducing their apoptosis, whereas partially acetylated chitosan has been shown to inhibit the growth of sarcoma180 tumour cells in a rodent experiment. In vitro tests with 293 cell lines and HeLa Cells reveal that complexes with different cooper to chitosan ratios can be used as potential anti-tumour agents. Chitosan also has an important antioxidant activity. Antioxidants delay or inhibit the oxidation of some cell substrates, helping the body to fight against the damage caused by reactive oxygen species (ROS) and degenerative diseases. Free radicals of oxygen, produced for example by ultraviolet light, are very unstable and react easily with the cell or tissue. Low molecular weight partly deacetylated chitosan is known to be a natural antioxidant, and although the exact reason for this isn’t yet known, it is thought to be due to the amino group and hydroxyl groups in C-2, C-3 and C-6 positions, which react with free radicals to form more stable macromolecular radicals. Chitosan-based compounds have also been clinically tested as a polimeric carriers for anticancer drugs, such as camptothecin. Since a simple chitosan-camptothecin conjugation was known to have a series of disadvantages, namely, scarce water-solubility and possible toxic side-effects, aggregates of O-carboxymethylchitosan (OCMSC) were prepared.6 Other uses of chitosan in the area of drug delivery systems includes the production of anti-cancer drug carrying microparticles and as polimeric vectors for gene therapy. 5.2 Hyaluronic Acid Hyaluronic acid (HA) is a natural polymer and a polysaccharide which is characterized by having a high molecular mass and viscoelastic properties. It belongs to a group of substances called glycosaminoglycans and is a linear polysaccharide composed of alternating D-glucuronic acid and N-acetylglucosamine (Figure 6).7 Figure 6: Structure of the disaccharide repeating unit of hyaluronic acid.7 Biomedical Engineering 12 BIOMEDICAL APPLICATIONS OF POLYMER-BASED PHARMACEUTICALS 2008 HA is a component of many different kinds of biological tissues and fluids and is also present in the capsules of some bacteria (e.g. strains of streptococci), but is absent in fungi, plants and insects.7 It has important physiological roles in living organisms: in human body, for example, we can find HA in synovial fluid, umbilical cords, vitreous humor of the eye and skin (mainly the one located in intracellular space). HA is also a key element in the human body due to its role in embryogenesis, signal transduction and cell motility and it is strongly linked with cancer invasiveness and metastasis. The intrinsic biocompatibility of HA makes it excellent for medicine. Some of its applications are: 1. viscosurgery, protecting tissues and providing space during surgeries like in ophthalmological surgery; 2. viscoaugmentation, filling and augmenting tissue spaces, as in vocal and pharyngeal tissues; 3. viscoseparation, separating connective tissue surfaces that are traumatized to prevent adhesions and excessive scar surfaces; 4. viscosupplementation, replacing tissue fluids, as the replacement of synovial fluid in complicated cases of arthritis and to relieve pain; 5. viscoprotection, protecting tissue surfaces from dryness or dangerous environmental agents, and promoting the healing of injured surfaces. HA has an important role in infection scenarios, as it affects the infected cells and causes the reduction of inflammation. The analgesic effects of the substance are widely known at the moment. There is also a great variety of applications of HA in the targeted release of medicines. This polymer has been used to create a cross-linked hydrogel for drug delivery, and nowadays scientists can also directly conjugate HA to drugs or use the polymer to prepare microcapsules for optimized drug delivery capsules. 5.3 Dextran Dextran is a polysaccharide synthesized from sucrose by lactic acid bacteria. It is a component of the human body, being part of the dental plaque, for example. Since the polymer is a natural compost, it has some important advantages, as biocompatibility, biodegradability and low toxicity. Some of dextran’s clinical applications are the following: Decreasing of vascular thrombosis Lubricant agent in some eye drops Increasing blood sugar levels Replacement of blood loss Plasma substitution Volume expansion Rheological improvement Biomedical Engineering 13 BIOMEDICAL APPLICATIONS OF POLYMER-BASED PHARMACEUTICALS 2008 Recent researches in mice indicate that it is possible to use dextran as a carrier in anticancer therapy, which is an important discovery to the future of an efficient anticancer treatment. 5.4 Gellan Gellan is a high molecular weight exopolysaccharide (EPS) produced by bacteria like Sphingomonas elodea. Gellan has important characteristics as a natural polymer and a large variety of applications, particularly in the food, pharmaceutical and biomedical fields, as seen in Table C. Table C: Selected list of worldwide-issued and published patents covering the use of gellan gum categorized into application fields.8 Application Patent Title Country / Patent nº Gellan gum beverage and process for making a US5,597,604 gelled beverage Gelatin-free gummy confection using gellan gum US6,586,032 and carrageenan Calcium stable high acyl gellan gum for enhanced CN101001538 colloidal stability in beverages Food containing native gellan gum JP2005253473 Gellan gum tablet film coating Gellan gum based oral controlled release dosage forms-a novel platform technology for gastric retention Liquid aqueous ophthalmic composition containing gellan gum Spray able wound care compositions comprising gellan gum Controlled release compositions comprising gellan gum gels PHB-free gellan gum broth Mutant strain of Sphingomonas elodea which produces nonacetylated gellan gum US2004033261 Genetically purified gellan gum US2006003051 Modified gellan gum and its production JP11341955 US2006177497 NZ217662 NZ523126 WO9922768 US5300429 US2003100078 Cosmetic composition comprising gellan gum and GB2384705 carrageenan Oil-in-water emulsion comprising gellan and a WO0078442 particular surfactant and uses Biomedical Engineering 14 BIOMEDICAL APPLICATIONS OF POLYMER-BASED PHARMACEUTICALS 2008 Composition of toothpaste having improved physical properties and stability, comprising gellan KR20050023598 gum Gellan seamless breakable capsule and process for manufacturing thereof WO2006136198 Process using gellan as a filtrate reducer for waterUS5744428 based drilling fluid Paper coating composition comprising gellan US6290814 gum/starch blend Purification and use of gellan in electrophoresis US2004168920 gels Media and methods for promoting maturation of US20050003415 conifer somatic embryos a) Tissue culture gels, air freshener gels, paper coating, oil recovery, etc. Due to gellan’s stability, biocompatibility and biodegradability, researchers began to investigate the use of the polymer as a drug delivery vehicle. 6. Conclusion Polymer-based pharmaceuticals are starting to be seen as key elements to treat many lethal diseases that affect a great number of individuals, such as cancer or hepatitis C. At the moment, the impact of the researches on polymers applied to nanomedicine is more significant than it ever was, as new properties and applications of the polymer conjugates are being found. Nonetheless, clinical testing is still an obstacle for many developments in the field, as sometimes theory does not apply in its entirety to practice. There is still a need for the cautious design of new conjugates and careful validation of the chemical characterization before clinical trial. The previously revised study shows many applications of some widely known natural polymers and synthetic polymer-based medicines, which are being seen as crucial elements to boost healthcare. Further studies will surely be able to clarify some blurred concepts and if the obstacles found in clinical trials are surpassed, biomedical applications of polymer-based pharmaceuticals may overcome expectations. 7. Acknowledgements The authors would like to show their gratitude towards Prof. Leonilde Moreira, who guided us through the development of this report. Biomedical Engineering 15 BIOMEDICAL APPLICATIONS OF POLYMER-BASED PHARMACEUTICALS 2008 8. References 1 Duncan, R. 2003. The dawning era of polymer therapeutics. Nat Rev Drug Discov 2:347-60. Biotechnology. PEGylation. Accessed in November, 26, 2008. Biotecnology: http://www.biology.iupui.edu/biocourses/Biol540/3DrugCaseStudies2k7.htm 3 Duncan, R. 2006. Polymer conjugates as anticancer nanomedicines. Nat Rev Cancer 6:688-701. 4 Duncan, R., H. Ringsdorf, and R. Satchi-Fainaro. 2006. Polymer therapeutics - polymers as drugs, drug and protein conjugates and gene delivery systems: past, present and future opportunities. J Drug Target 14:337-41. 5 Duncan, R. 2007. Designing polymer conjugates as lysosomotropic nanomedicines. Biochem Soc Trans 35:56-60. 6 J. Vinsova and E. Vavrikova. 2008. Recent Advances in Drugs and Prodrugs Design of Chitosan. Current Pharmaceutical Design, 14: 1311-1326 7 Kogan, G., L. Soltes, R. Stern, and P. Gemeiner. 2007. Hyaluronic acid: a natural biopolymer with a broad range of biomedical and industrial applications. Biotechnol Lett 29:17-25. 8 Arsenio M. Fialho, Leonilde M. Moreira, Ana Teresa Granja, Alma O. Popescu, Karen Hoffmann and Isabel SáCorreia. 2008. Occurrence, production, and applications of gellan: current state and perspectives. Appl Microbiol Biotechnol. 79:889–900. 2 Biomedical Engineering 16