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Cancer: Cancer statistics & types of cancer
Cancer is one of the major leading causes of death worldwide. Around 10.9 million people worldwide are diagnosed with cancer and 6.7 million people die of cancer each year (1), (2) World Health Organisation (WHO) projected that death from cancer will continue to rise with estimation of 11.5 million deaths in 2030. There are hundreds of different types of cancer. According to GLOBOCAN, in 2008, the most commonly diagnosed cancers worldwide are lung (1.61 million, 12.7% of the total), breast (1.38 million, 10.9%) and colorectal cancers (1.23 million, 9.7%) (3). Incidence of cancer varies markedly with nationality and race. For example, Australian is more prone to skin cancer, Japan is a high incidence area for stomach cancer and cases of colon cancer are highly prominent in the USA (4). Cancer incident rate also varies between male and female. Lung cancer is the leading cause of death in men with a reported annual mortality of 16% of an estimated 6.6 million men diagnosed with cancer in 2007. Among women, breast cancer is one of the most frequently diagnosed cases where one out of four women worldwide is diagnosed with breast cancer. Factors that predispose to cancer include genetic abnormality, tobacco, alcohol, obesity, dietary factors, and environmental and occupational risks (5).
Development of Cancer Cells in Human Body
Cancer, also called malignancy, is characterized by an abnormal growth of cells. Somatic cells undergo a process known as cell cycle to produce a genetically identical copy that is essential for generation of new cells and replacement of old cells that dies off. A non-dividing cell will remain in a resting state (G0), i.e. a cell that has reached an end stage of development and will no longer divide. It will only enter the cell cycle when stimulated by growth factors. This cycle involves four distinct phases which are termed G1, S, G2, and M. G1 and G2 represents the gap periods where cell growth and proteins synthesis occurs to prepare the cell for S phase (DNA replication occurs) and M phase (mitosis occurs) respectively (6). These stages are regulated by cell division cycle protein called cyclin dependent kinases. Cells will moves through the various stages of the cell cycle depending on the kinases that are being activated and will be terminated once kinase is degraded. For instance, cyclin-D-Cdk4, cyclin-D-Cdk6, and cyclin-E-Cdk2 are involved in the transition from G1 to S phase of the cell cycle, whereas cyclin-ACdk2 is essential for the progression from S phase (7). There are certain check stages in the cycle to ensure smooth division especially prior to DNA replication. If DNA synthesis goes wrong, checking proteins will either stop replication to allow time for cell to repair the DNA or if damage is too extensive, cell apoptosis (programmed cell death) will occur. One such protein is p53 which is a transcription factor described as an anti-tumour gene that binds to DNA and activates p21 which is responsible for blocking cyclin dependent kinase and thus prevent progression through G1 phase. (8), (9). Mutation or failures of these antitumour genes will results in genomic instability, increase in DNA damage, uncontrolled cell proliferation, and, eventually, formation of tumour (10). According to Reynolds & Schecker.(1995), p53 gene is the most common mutated gene in human cancer including breast, colon, liver and lung cancer. Studies have shown that agents such as cisplatin and nitrogen mustard can stop progression at either G1/S or G2/M checkpoints. (9)
Currently, the four standard way of treating cancer are surgery, chemotherapy, radiotherapy, and biologic therapy. Chemotherapy is the use of drugs to kill or destroy any cancer cells that may spread from the tumour primary cells to other part of the body where secondary cancer may developed. It has effects on the entire body unlike treatments such as surgery and radiotherapy that only act locally. The types of drugs available include alkylating agents, antimetabolites, anthracyclines, plant alkaloids, and topoisomerase inhibitors. Chemotherapy can also be given in a form of drug combination that differs in their mechanism of action and toxicities. For example, the treatment regimen involving cyclophosphamide, adriamycin, and fluorouracil (CAF) is effective at increasing response rates in postmenopausal women with metastatic breast cancer and reducing death by 20%. (11). The choice of drugs used for treatment depends on the type and stage of cancer. For example, cisplatin is approved by the FDA to be used as a single agent for treatment of testicular, bladder, lung, stomach and ovarian cancer. (12) However, the drawbacks of chemotherapy is that this treatment acts by targeting rapidly dividing cells which include cancer cells and some normal cells like hair follicle cells and those lining the skin, mouth, and digestive system. This cause side effects like hair loss, poor appetite, mouth or lip sore, and stomach upset. Large doses are also generally required to obtain desired therapeutic outcomes which at the same time lead to severe toxicity and multiple drug resistance by cancer cells. Therefore, there is a need of on going research in looking into the area of discovering new drugs, new delivery techniques, or to target cancer cells more specifically to reduce side effects.
For over 25 years, platinum complexes have been used as antitumour agent in cancer treatment. (13). This was started by an accidental discovery when Barnett Rosenberg (a biophysicist) examined the effect of electric fields on the growth of Escherichia coli cells where platinum anti-proliferative effect was uncovered. (13), (14). The filamentous growth (indicate stop of cell growth) of the bacteria observed was not due to the electric field but rather is the product of electrolysis of the platinum electrode (Fig 1). Two active complexes were identified, cisplatin, (Cis-dichloro-diammine-platinum (II), Fig 2) was found to be more active than the trans isomer.
Figure 1. A scanning electron microphotograph of gram negative Escherichia coli: (a) in normal growth medium; (b) in growth medium containing a few parts per million of cis-diamminedichloroplatinum(II). The elongated E. coli cell was due to inhibition of cell division but not cell growth. (15)
Figure 2. Structural formula of Cisplatin
Further test and clinical trials was carried out and cisplatin was granted approval by the US Food and Drug Administration (FDA) in 1978 as treatment in cancer therapy. (16), (17). Since the establishment of cisplatin antitumour activity in 1970, cisplatin is now widely use in treatment of testicular, ovarian, breast, lung, and head and neck cancer. (18) (19).
Mechanical Action of Cisplatin
Cisplatin is given intravenously as a sterile saline solution. The compound remains intact in the bloodstream due to high chloride concentration and which then enters the cell through passive diffusion or active uptake. In the cell, where chloride concentration is low, cisplatin is activated intracellularly by the hydrolysis of the chloride leaving groups forming monoaqua ([Pt(NH3)2Cl(H2O)]+) and diaqua species ([Pt(NH3)2(H2O)2]2+). These highly electrophilic (positively charged) species can covalently bind to DNA, and forms DNA adducts which is a structurally distorted double helix that are either partially unwind or the formation of kink on the DNA. (20) Generally, cisplatin could react with all DNA nucleobases (adenine, guanine, thymine, and cytosine) which have potential site for platinum coordination and prevent DNA replication and transcription that subsequently triggers cell apoptosis. However, the highly nucleophilic N7 position of guanine residues of the DNA strands are considered to be more preferred under physiological conditions. (21) (17) (20) (22). Although nearly 90% of platinum will bind to DNA, it can also target mitochondria DNA (resulting in cell death due to mitochondria damage) and other sulphur containing enzyme (metallothionein and glutathione) which is involved in cell resistance (Fig 3). (23) (13). Cisplatin can also interact with cellââ‚¬â„¢s RNA but the extent of cell damaged will not be significant. Studies have indicate that cisplatin do not affect RNA synthesis as it does in DNA synthesis and only a small portion of RNA were destroyed when a toxic dose of cisplatin was given.
Figure 3. The cellular uptake of cisplatin. Cisplatin enter the cell either through passive diffusion or active uptake which then undergoes hydrolysis due to lower chloride concentration and reacts with nucleus DNA, mitochondria DNA, RNA, or sulphur containing enzymes. (24)
Problem with Cisplatin Therapy: Dose limiting side effects and development of drug resistance.
Several randomisation trials and meta-analyses show that cisplatin can prolong survivor in testicular, ovarian and non-small cell lung cancer especially if cancer are diagnosed in an early stage. Activity of cisplatin can also be increased by combination therapy with drug such as 5-fluorouracil that reduce cisplatin-intrastrand DNA lesion. (20). Unfortunately, cisplatin produced several dose limiting side effects such as nausea and vomiting, nephrotoxicity, neurotoxicity, ototoxicity, and gastrointestinal toxicity which limit its use. (21) (13) (25). Emesis is the most common side effects of cisplatin that are currently overcome by introducing 5-hydroxytryptamine-3 (5-HT3) receptor antagonists (granisetron or ondansetron). Cisplatin induced nephrotoxicity is reduced by hydration with normal saline or if necessarily frusemide is given to maintain good diuresis. (23).
Although some of these side effects can still be overcome, certain type of tumour cell like colorectal cancer and non-small cell lung cancer are insensitive to cisplatin treatment (intrinsic resistance). Others such as testicular and ovarian cancer may develop acquired resistance after frequent drug administration. Several resistance mechanism had been identified which includes failure of cell apoptosis, degradation and deactivation of drugs by intracellular thiols (glutathione and metallothionein), ability of cells to tolerate higher DNA damage, and decreasing uptake and increasing efflux of drug from cells. (26). Therefore, there is a need to develop newer platinum drugs to overcome cisplatin resistance and to reduce its side effects.
Development of New Platinum Drugs
Over the years, many cisplatin analogues were investigated in an attempt to improve therapeutics efficacy, reduce toxicity and overcome drug resistance. Currently, only a few out of the many platinum complexes that have entered clinical trials received approval. Studies had shown that over 3000 compounds tested in vitro, 28 platinum based drugs entered clinical trials and only 5 were approved for clinical used. Only oxaliplatin and carboplatin receive worldwide approval; Lobaplatin is sold just in China and nedaplatin are merely approved in Japan. (13) (22).
Carboplatin is shown to bind to DNA strands, similar to cisplatin, forming DNA adduct, but it has been discovered to have reduced toxicity to the peripheral nervous system and the kidney. Reduced nephrotoxicity is mainly due to the more stable cyclobutane ring (Fig 5) compare to the chloride leaving group in cisplatin. This lowered reactivity thus allows it to be administered at a higher dose (maximum of 2000mg/dose) than cisplatin. Carboplatin is now mainly used to treat patients with ovarian cancer that suffer from severe toxicity and used in a variety of combination chemotherapy regimen. (22) (13) (16).
Figure 4. Chemical structure of carboplatin
Oxaliplatin is the first antitumour drug to overcome cisplatin resistence. It can effectively inhibit DNA replication due to the lipophilic 1,2-cyclohexane carrier ligand which enhance cellular uptake. Nedaplatin has similar antitumour activity and show reduced nephrotoxicity when compare to cisplatin. Hence, Nedaplatin could be used to replace cisplatin in patients with renal impairment. (13). Many more drugs are currently in various stages of clinical trials. Satraplatin and Picoplatin are polymer or liposomal based platinum drugs that are in the process of getting marketing approval. Since 1999, triplatin tetranitrate, BBR3464 (a chainlike structure that connects platinum with aliphatic linker), is the last small molecule platinum drugs that entered clinical trial. It has been evident that efforts to discover cisplatin analogue or to design new platinum drug shows little advancement. Hence, researchers have now shift focus to look into the use of delivery vehicles and improve drug delivery system. (27)
Targeted drug delivery in antitumour drugs
The development of cancer therapy had shown little advancement, though many drugs are discovered to be effective. These drugs are often restricted by its dose limiting side effects, risk of drug resistance and several pharmacological deficiencies like short plasma half life, low bioavailability due to poor affinity to cancer cell and poor water solubility. (28) (29) Drug administered orally are exposed to metabolic pathways in the body and while drug given via intravenous route (such as anticancer platinum drug) has reduced specificity that lead to harmful effect to normal tissues. (29) Different measures are included to optimize drug therapy such as multiple drug regimens, monitoring of serum drug level, and slow and careful timing of drug infusion. Alternatively, specific drug delivery system could be exploited where the differences between cancer and normal cells were studied. It has been found that tumour cells exhibit unique pathophysiological patterns that could help to enhance the time for which tumour is expose to the therapeutic drug or to increase drug accumulation at site of action and thus minimize effects on healthy tissues. (30) Generally targeted drug delivery system can be classified either as passive or active targeting.
Active Targeting: Via surfaced-attached specific ligand
Active targeting is where the drug is being specifically delivered to the target cells using a targeted ligand like peptides, small molecule and antibodies which have high affinity towards highly concentrated receptors, enzymes or DNA. (31) (32) For example, in certain type of cancer (breast and prostate cancer), estrogen receptors are found to be overexpress. Hence, platinum drugs are bind to either polyaromatic ligands or estradiol derivatives, which have high affinity for this receptor, to improve activity in tumours. However, study has shown that there is no difference in activity between cells with overexpression of estrogen receptors and those that are without it. (33)
Passive Targeting: Via Enhanced Permeability and Retention (EPR) Effect
In passive targeting, drug and its carrier non-specifically accumulate via the EPR effects in the tumour interstitial space, leading to a higher drug concentration in the tumour cells and this subsequently enables an increased to the maximum tolerated dose and therapeutic window of the anticancer drug, with minimum effect to the surrounding healthy tissues. EPR effects are based on the differences between tumour and normal cells. The differences are as follow:
Increase leaky vasculature : increase permeability of cancer tissue
Normal endothelium cells have tight junctions and no gaps between cells, thus have low permeability for large or hydrophilic molecules (Fig 5). Drugs that are small and lipophilic will permeate through the cells by diffusion while macromolecules can only exit the circulation by a mechanism known as transcytosis. In cancer sites, new blood vessels are developed (angiogenesis) to provide sufficient nutrients and oxygen to sustain rapid tumour growth. Usually, these newly formed blood vessels are irregular in shape, dilated and defective. This leads to the formation of leaky vasculature around the tumour. The pores surrounding the vessels walls have size range of 200 nm to 2 ÎÂ¼m, with an approximate pore size of 400nm. (34) Macromolecules and nanoparticles can extravasate into the tumour but not into the intact tight junction between endothelial cells of the healthy tissues. Particle size range of approximately 10-100nm are shown to readily penetrate and accumulate in tumours (35), (36). Therefore, a high concentration of drugs (10-50 folds higher than normal tissue) can be achieved at tumour sites within a few days. However, the EPR effects based on leaky vasculature is only applicable to drugs with high molecular weight that do not undergoes rapid diffusion from the blood circulation and which subsequently cleared by the kidney. A study has shown that the EPR effects are also influenced by tumour size where smaller sized tumour will produced greater effect, which most probably due to greater vessels density. (37)
Figure 5. The Enhance Permeability and Enhancement Effect. (a) Normal tissue vasculature: blood vessels are lines by tight endothelial cells. (b) Tumour tissue vasculature: new blood vessels formed (angiogenesis) has a leaky vasculature and is hyperpermeable. Higher concentrations of drugs are preferentially accumulated in the tumour tissue. (29)
Lack of lymphatic drainage: increase drug accumulation in tumour bed
Lymphatic vessels which are widely distributed in the body are more permeable to solutes and fluids compare to blood vessels. The lymphatic pathway helps to return interstitial fluids to the blood circulation. It is also known that higher molecular weight compounds (>40kDa) are found to have higher resident time and are mostly cleared through the lymph. (34) (37) ,. (38) In tumour cells, there is a lack of lymphatic drainage and when in combination with the leaky vasculature, it will lead to the accumulation and retention of the compounds in tumours. Lack of lymphatic drainage will also cause an increase in interstitial fluid pressure in solid tumours which subsequently limit the extravasations of compounds. (32)
Drug Delivery Vehicles
Over the years, targeted drug delivery has been intensively looked into in the area of cancer therapy to deliver the desired dosage of drug to the tumour sites. Traditional chemotherapy agents are prevented from reaching the tumour cells due to the complex tumour vasculature and the existence of p-glycoprotein. There are also many limitations which arise to improve the efficacy of the anticancer drugs which includes poor solubility, being highly cytotoxic to the normal tissue and having narrow therapeutic window. Therefore, many nanotechnology based materials such as nanoparticles, micelles, liposomes, carbon nanotubes, quantunm dots and dendrimers are currently under development in efforts to discover a better treatment for cancer. (39). Out of the many nanomaterials available, several are listed below and they have shown great potential to increase the effectiveness of anticancer agents with much reduced toxicity.
Polymer nanoparticle size is less than 1 ÎÂ¼m in diameter and can be either obtain naturally or synthetically synthesized. Anticancer agent, without being chemically modified can be encapsulated in the polymer which is then delivered to the cancer sites and undergoes a controlled released either via diffusion, swelling followed by diffusion, bulk erosion or depending on the local environment. Up to the year 2007, there are 12 polymer-drug conjugate which have entered phase 1 and 2 clinical trial with most of it aims to target the blood vessels in tumours. Examples of drug that are being incorporated include doxorubicin and paclitaxel. (40).
Micelle is a self-assembling lipid based carrier that has a size range of 20-80 nm in diameter. (41) (42). It is an amphophilic molecule with a hydrophobic core and a hydrophilic shell which is beneficial as a carrier for many chemotherapeutics drug. It is also used due to its high biocompatibility, biodegradability, and being able to isolate the drugs from the surrounding environment. An example of drug using micelle as carrier is NK 105, where paclitaxel is enclosed within a micelle and was tested against pancreatic, colon, and stomach cancer. (40)
Liposomes is used for drug encapsulation by containing a hydrophilic core surround by one or more phospholipid bilayer. Chemotherapeutics drugs are bind to this hydrophobic phopholipid bilayer and hence cause reduction of the side effects such as peripheral neurotoxicity that are commonly related to cisplatin and vincristine. Generally, liposomes used in cancer treatment is 100 nm in diameter as this enable it to extravasate from blood circulation to the leaky vasculature of tumour sites. However, liposomes face some challenges which include rapid clearance, fast release of drug, higher production cost, and the rapid oxidation of certain phospholipids. (40)
Dendrimer is a spherical, three dimensional tree-like branching polymer. It starts from a centre core followed by symmetrical branching shell monomers and for each layer it is termed as generation which is identified with a particular generation number. (Fig 6) As the generation increases, the molecular weight and the number of surface group will also increases exponentially. (43) One of the widely recognize family of dendrimers for studies include the Tomalia-type poly(amidoamine) (PAMAM) dendrimers. (44) The size of PAMAM dendrimers will increase by approximately 1 nm per generation and have a size range of 1.1 to 12.4 for generation 1 to 10. The outer surface group of a full generation dendrimer consists of amine group that can be protonated to formed polycationic charge. (43) A half generation dendrimer (which is used in this study) will have a carboxylate group that forms a polyanionic charge. Anionic dendrimer is shown to have faster rate of uptake compare to other macromolecules and are less toxic than the full generation dendrimers. (45)
Dendrimer: A promising polymeric structure in treatment of cancerous cells
Dendrimers are found to be superior for drug delivery and have demonstrated certain advantages over the other nanocarriers due to their low polydispersity (uniform shape and size), nanoscale architecture, high water solubility, lower toxicity and large surface binding sites. (46) (47) In comparison with linear polymer, the multiple reactive surface groups of dendrimers also allow more drug binding capacities and hence making them ideal drug carriers. Their relatively small size (less than 5nm) enables it to be rapidly cleared by the kidney. Its efficacy had also been proven in various studies for compounds like doxorubicin, DNA, and methotrexate due to the ability to protect drugs from being degraded by protein and peptides. (45) This is shown in some studies which look into in vivo delivery of a same molar concentration of methotrexate and dendrimer bound methotrexate and has shown a much reduced tumour sized (tenfold) for the later. (40).
Aims of the Thesis
Platinum drugs especially cisplatin are being extensively used in the treatment of many cancers despite the various side effects which have been associated with it. Thus, this study aims to develop a more effective anticancer drug with fewer side effects, involving the synthesis of drug with bifunctional platinum centres which could bind to DNA in a different way than cisplatin and by incorporating an aminoalkane linking ligand (diaminobutane) in between the platinum complexes. Charge of a compound is also found to affect the rate and site where platination occurs. Thus, in this present studies, a positively charged compound is synthesis as it is believe that cationic compound have a better selectivity towards tumours cells and show higher vascular permeability compare to a anionic compound. In addition to that, PAMAM dendrimer of generation 6.5 is selected for coupling with this newly synthesise drug. This is due to its ability to protect the platinum drug from degradation and also being able to target the cancerous site more specifically which take into account of EPR effect in tumour sites. The number of drugs bind to a dendrimer will be investigated and eventually a test on anticancer activity will be carried out on certain cancer cell line to test for its efficacy.