Final+Report

=**Recent Developments in Cisplatin Chemotherapy Treatment**=

Lauren Reuther

 * Background of Cisplatin**

Various characteristics specific to inorganic compounds place them at the frontline of medicinal research into chemotherapy drugs. The increased coordination numbers that can be achieved by inorganic compounds lead to the possibility of attaching various substituents and/or ligands, possibly increasing specificity of drug target and decreasing the reactivity of the drug with biomolecules in the human body. Due to increased coordination numbers only achieved by inorganic compounds, much more molecular geometry is available to these compounds in comparison with organic molecules. Different oxidation/reduction states for an inorganic element are also feasible, which may be used to drug toxicity to the human body. The thermodynamic and kinetic characteristics and intrinsic properties also offer diverse reactivity for inorganic molecules alone[3]. Discovery of platinum-based compounds, specifically cisplatin, as anticancer agents in the 1960s set in motion the trend of research into the field of inorganic medicinal chemistry for drugs that can be used for the treatment of cancer[1,2,7,8]. The effect of platinum and other group VIIIb transition metals on cell division was first identified by a biophysicist, Barnett Rosenburg, and his research group. They determined that these transition metals inhibited cell division, but did not very much affect the growth process of the cell. It was found that these cells would, instead of dividing, form long filaments and grow to approximately 300 times the normal length of the cell[16]. This revolutionized the field of medicinal inorganic chemistry research because it proved that platinum and other transition metals could be used to stop the division of unwanted, damaged cells in the human body. Of course, carcinoma cells would be the main target of research in this field because it affects the lives of so many people across the world and a cure for cancer would save millions, even billions of lives. A chemotherapy drug using platinum as an agent to stop cell division was highly desirable. This was when the greatest use of cisplatin, a platinum based molecule, was discovered. Cis-diammine-dichloroplatinum (II) is the proper nomenclature for the chemotherapy drug that is commonly known as cisplatin. Cisplatin, which has been coined the “penicillin” of chemotherapy drugs, was first synthesized in 1845 by Michel Peyrone, over 150 years ago! At this time, it was unknown what great benefits the generation of this molecule would have for the medicinal field. It was later discovered that only the cis-isomer of the compound is active against carcinoma cells. A method for solely producing the cis- isomer and a method for identifying and separating the two isomers were then developed, which made production of this drug much easier and more efficient[1]. Since its discovery, cisplatin has proven to be one of the most effective chemotherapy drugs in that has passed clinical trials. It has been confirmed to be especially beneficial for patients with testicular cancer with approximately 90% of the patients that had been treated with cisplatin cured. Also, with an early diagnosis in many other types of cancer a cure rate of almost 100% has been seen which highly surpasses the success rates of other chemotherapy drugs[1,3].


 * How Cisplatin Works**

Cisplatin is injected into the body and enters the bloodstream with chloride ligands attached, neutralizing the charge of the molecule and essentially inactivating it. The bloodstream contains a high concentration of chloride ions, therefore preventing the replacement of the chloride ligands by water molecules. The inactive cisplatin then diffuses through the cell membrane of cancerous cells via the copper transporter Ctr1. The cytosol of a eukaryotic cell has a much lower concentration of chloride ions than in the bloodstream of the human body, and the displacement of the chloride ligands on the cisplatin molecule by water molecules takes place. The process of water molecules replacing ligands on the cisplatin molecule is called hydrolysis or aquation. This process gives the cisplatin molecule a positive charge, hence activating the compound. This positively charged molecule can then bind to the negatively charged DNA bases, creating an adduct in the DNA nucleotide sequence. Due to an improperly formed DNA, there is an interference with the transcription and replication of cellular DNA. Because of the damaged DNA, the cell will then activate the apoptosis pathway, which leads to programmed cell death[1,9,13].


 * Side Effects of Cisplatin**

Although cisplatin has proven to be one of the most effective chemotherapy drugs, there are a few drawbacks to the current version being dispensed to patients. One of the major problems associated with the use of cisplatin is it high toxicity[19]. The mechanism of this drug causes it to target and attach to the DNA molecules in cells and, according to the cisplatin paradigm, generates lesions in the amino acid sequence. These lesions in the cellular DNA ultimately lead to cell death, which is the pathway this drug follows to effectively destroy cancer cells. Because there is not yet a mechanism developed to target the cisplatin directly to cancerous cells, it also attacks healthy, non-carcinogenic cells in the body[3]. Side effects that greatly diminish a patient’s health are commonly seen with the use of cisplatin as a chemotherapy agent due to its high toxicity. Neuro-, hepato-, and nephrotoxicity all have been seen as side effects of this drug. Neurotoxicity is a disorder that occurs when a neurotoxin, in this case cisplatin, causes damage to nervous tissue. This tissue is very important in the human body because it transmits neurological signals throughout the brain and human body. Hepatotoxicity occurs very easily because the liver is the organ responsible for filtering and metabolizing harmful substances from the body. Cisplatin can easily be taken through the blood stream to the liver where it would cause damage to the hepatocytes. Nephrotoxicity is the result of chemical damage to the kidneys, which are also a key organ in the excretion pathway of toxic chemicals[2]. All three of these disorders have a very large impact on a patient’s body whom has been subjected to cisplatin treatment, which is why the toxicity of this drug must be regulated and targeted to cancerous cells only. One very interesting study done on the role of the enzyme gamma-glutamyl transpeptidase (GGT) on the toxicity and resistance levels of cisplatin was performed. An increase in the concentration of GGT in cells other than renal proximal tubular cells causes an increase in the resistance of the cells to cisplatin. On the other hand, an increase in GGT in renal proximal tubular cells causes an increased sensitivity to the chemotherapy drug[9]. This increased sensitivity in renal cells is believed to be one of the main causes of the side effect of nephrotoxicity with exposure to cisplatin. Another major drawback to the current version of cisplatin is the severe tumor resistance[19]. When cisplatin is administered to a patient there are various times in which he or she has an inherent immunity to the drug. Also, it has been seen that an acquired resistance to the drug is also possible. These immunities cause the effectiveness of the drug to majorly decrease [3]. Toxic levels of cisplatin would need to be administered to the body in order to have an effect on the tumor cells when a sense of immunity is acquired by the patient’s body. Because cisplatin cannot be used for patients that have an inherent or gained immunity for the drug, another version or addition to the treatment must be created to reduce this immunity. There are some hypotheses as to the reason in which the body becomes immune to cisplatin treatment. One cause for the decreased activity of cislatin is believed to be due to non-specific bonding to biomolecules, such as proteins and phospholipids, in the organs in which cisplatin circulates. It is believed that cisplatin that had been administered to the body and entered the blood stream may be bound by the thiol group on the cysteine residues of various proteins. This would attach and deactivate the compound, leading to a decrease in the active amount of cisplatin in the body[1,17,20]. The major blood-bound protein that is believed to be responsible for this unwanted binding is albumin. This cisplatin-albumin complex has been found in the urine of patients receiving treatment with cisplatin. A mechanism or change in the compound must be made in order for cisplatin to be more active with a lesser dosage. There are also many hypotheses as to attack the toxicity problem of this molecule. Because cisplatin readily distributes throughout the entirety of the human body and lacks specificity to entering cancer cells, changes must be made in order to reduce its toxicity. There are many different strategies that may be employed in order to achieve this goal. Improving plasma stability so that it no longer reacts with biomolecules, targeting the drug specifically to be taken in by cancer cells in tumors, and increasing the desire of the molecule to bind to DNA in tumor cells are theories that can be utilized to decrease cisplatin’s toxicity to non-cancerous cells in the human body[20]. Also, the efficiency at which platinum-based anticancer compounds are absorbed into cancer cells is very low[13]. Because of the low molecular weight of cisplatin, it is readily absorbed into the blood stream and spends very little time inside a tumor(Cisplatin encapculated). This causes a decrease in the effectiveness of the drug to kill cells within tumors and also allows for more toxicity to non-cancerous cells.


 * Recent Advances in Cisplatin Anticancer Targeting and Toxicity Research**

It is possible to employ the well-understood enhanced permeability and retention (EPR) effect that occurs in cancerous cells as an uptake mechanism to over express chemotherapeutic drugs inside cells. The EPR effect is an increased absorption of biological macromolecules with a decreased excretion of these molecules. This effect has been used in chemotherapy drugs such as Doxil© because the active compound is soluble in water and has a high lypophilicity, which cisplatin does not[20]. A modification in the method used for non-platinum based compounds must be made in order to be effective with cisplatin. Nucleic acid aptamers are single-stranded oligonucleotides and are recognized by a cell through a selection process known as SELEX, system evolution of ligands by exponential enrichment. Apatmers are recognized by the cell in a similar way as antibodies, which has previously been used as a means of cancer cell targeting for chemotherapeutics, and have very similar binding affinities and specificities. They are also considered nonimmunogenic meaning they will not cause an immune response, which may and frequently does occur with the use of antibodies. One study used aptamer conjugated, cisplatin-encapsulating liposomes as a drug delivery system. Liposomes are tiny vesicles generally composed of phospholipids, which is the same material that composes the cell membrane(Wikipedia). Through experimentation, it was shown that this method proved to exhibit high specificity to cancerous cells and an efficient drug delivery system. In addition, a complementary DNA (cDNA) strand of the aptamer was shown to function as an antidote to disrupt this delivery system[4,21]. Here, reversible drug delivery system has been created that also maintains high specificity and efficiency. Because the liposomes are composed of the same material as the cell membrane, they are easily taken into the cell through endocytosis. With further experimentation, this may contribute to a great new way to deliver chemotherapy drugs without having the harmful side effects of current drugs. Because conventional liposomal encapsulations, which are normally composed of phosphatidylcholine (PC) and cholesterol (CH), of platinum-based drugs showed to have low drug loading levels, a more proficient encapsulation method must be created. Also the uptake of PC and CH into the cell through the cell membrane is not the most efficient process. One study instead utilized phosphatidylethanolamine (PE) to comprise the liposome membrane because this phospholipid has been proven to be taken through the cell membrane much more easily. With this new method using PE as the phospholipid, the encapsulation percentage of cisplatin was found to be 70%. Also, it had been seen that when cells were subjected to incubation with the PE liposomes, almost all cells had effective intake of liposomes[11]. This new method utilizing PE instead of the traditionally used PC and CH has proved to be a much more effective and efficient way of encapsulating small molecules inside liposomes. To reduce the toxicity of platinum-based chemotherapy drugs, such as cisplatin, ligand-receptor-mediated delivery systems have been created and the toxicity effects have been evaluated. The iron storage protein, ferritin, naturally forms a hollow protein cage that contains eight hydrophilic channels that lead through the protein shell to the center. Previously, it has been shown that various inorganic nanoparticles were able to be incorporated into the hollow cage of ferritin. It is also essential to understand that ferritin is recognized by the membrane-specific receptors on the cell membranes specific to cancer cells, especially neoplastic and brain tumor cells, and is easily shuttled through the cell membrane[19]. Using a molecule that already has a tag to be targeted to cancer cells without having to add an antibody or other like methods would greatly decrease the amount of time it takes to produce mass amounts of this drug system. This would be a wonderful way in which targeting cisplatin to cancer cells is possible. In a study performed by Zhen Yang, et. al., two methods of incorporating cisplatin into the hollow center of apoferritin, which is a derivative of ferritin, have proven possible. The apoferritin was dissociated into its subunits in very acidic conditions with cisplatin in the solution mixture. The protein was then reassembled at pH 7.5, trapping cisplatin in the center. PAGE and SDS gels were run to ensure the apoferritin protein cage was intact and stable once the cisplatin was incorporated, and a cytotoxicity assay was performed in rats. This assay did prove toxicity of this cisplatin deliver system to cancer cells in rats. Apoferritin has been proven to be specific to cancer cells expressing ferritin receptors. It is believed that further testing with this method will show decreased cisplatin toxicity to non-cancerous cells and be an effective way of targeting chemotherapy drugs to cancer cells only[19]. The use of pH-responsive nanoparticles has also been shown possible for the encapsulation and controlled release of cisplatin. The cores of these nanoparticles are composed of poly[2-(N,N-diethylamino)ethyl methacrylate] (PDEA). The nanoparticles are dissolved in pH below 6, and taken into the cell and transferred to lysosomes. Because the pH of lysosomes is very acidic, they will readily release cisplatin to the cell initiating apoptosis. This was the first developed efficient method of fast-releasing cisplatin encapsulation nanoparticles[18]. An efficient method for the fast release of cisplatin in the body is also highly desirable. Another type of encapsulation of cisplatin that has been produced is water-in-oil (w/o) nanoemulsions and has been used to target bladder cancer cells. These nanoemulsions must be stabilized by emulsifiers, and in this experiment both a sugar ester of sorbitan monooleate (Span 80) and a polyoxyethylene 20 sorbitan monooleate (Tween 80) were utilized to form stable emulsion systems. Also, to prove that the nanoemulsions were not toxic to the cells and it was actually the cisplatin that caused a stop in the cell’s growth, non-cisplatin emulsions were also injected into rats. This study found that encapsulated cisplatin had an increased effect on bladder cancer cells in rats over lone cisplatin. Also, emulsions without cisplatin did not cause apoptosis in cells[10]. Next, antibody-directed enzyme prodrug therapy (ADEPT) has also been exploited as a means of reducing toxicity of cisplatin. In this method, an antibody is bound to an enzyme and administered to the patient. A prodrug containing the antigen for that antibody will then combine, releasing an activated drug. A similar idea for chemotherapy use can be utilized to activate a prodrug administered to the body without having to administer a labeled enzyme. Using the fact that many enzymes are known to be found in increased concentration in tumor tissues, the prodrug, inactive cisplatin, is administered to the body in its inactive form near the tumor. The nearby enzymes then cleave the drug through stereoselective ester hydrolysis. This results in a localized activation of cisplatin in very close proximity to the source of the cancer, the tumor tissues[20]. This method would provide another way in which cisplatin can be targeted to cancer cells without the need for antibodies. Rodney P. Feazell, et. al. had previously developed a method that utilized the more inert platinum(IV) as a prodrug to cisplatin activation, which is the commonly utilized method, and also the usage of carrier molecules as a drug delivery system. Estradiol units were attached to the platinum(IV) compounds and upon reduction of the platinum(IV) to platinum(II) inside the cell, a drug such as cisplatin was released into the cell. They have also developed a new system that utilized soluble single walled carbon nanotubes (SWNT) as a drug carrier. This nanotube is taken into the cell through clathrin-dependent endocytosis. Only once this nanotube is taken into the cell will it release the lethal dose of cisplatin, resulting in apoptosis and cell death[6]. This is very creative and inventive way in which to specifically release cisplatin to cancer cells. Another method of drug targeting to specific cells would be the incorporation of precise peptide sequences that will target receptors on the surface of tumor cells. There are various peptide sequences that have been identified to target cancer cells. The Asn-Gly-Arg (NGR) motif exclusively identified murine breast carcinoma models. Arg-Gly-Asp (RGD) and luteinizing hormone releasing hormone (LHRH) have also been shown to target certain receptors on cancer cells. Doxorubicin (DOX) and 5-fluoro-2′-deoxyuridine (5-FdUrd) conjugates have previously successfully been targeted using the NGR motif(Aminopeptidase N). Margaret W. Ndinguri, et. al. conjugated CNGRC to cisplatin in order to target the drug to CD13-expressing cancerous tissues. Through experimentation, it was shown that attaching this motif to the drug cisplatin caused an increase in the concentration of the drug inside the cells expressing CD13. Also, it was proven that cisplatin was not found inside cells that lacked the CD13-receptor[13]. Due to the results of this experiment, it was proven that attaching a peptide that specifically recognizes receptors on cancer cells is a possible way to target platinum-based chemotherapy drugs. This greatly increases the specificity of the drug, hence decreasing the toxicity to non-cancerous cells. The use of chemotherapy drugs along with radiation therapy has been done in various types of carcinomas. One precise type of cancer that this combination therapy has been performed is localized cervical cancer. The idea of combining therapies came about due to the fact that the patients with larger tumors would need a greatly increased amount of radiation therapy. Many times this would reach toxic levels. Exposing the cancerous cells to a chemotherapy drug sensitized the cells to the radiation. This occurred because adding a toxic drug to the radiated cells prevented repair mechanisms inside the cells from working and stopped the cells in a particular region of the cell cycle that is especially sensitive to radiation. Less radiation was then needed at the cancerous area. Cisplatin, in combination with and without other chemotherapy drugs, was tested in conjugation with varying amounts of radiation therapy in localized, advances cervical cancer tumors. It was found that cisplatin alone and in synchronization with cisplatin, fluorouracil, and hydroxyurea and combined with radiation therapy most effectively eradicated cancerous cells in locally advanced cervical carcinoma[15]. This would be a great method of killing cancerous cells because not very much of either chemotherapy or radiation therapy must be used. On another angle of the downfalls of cancer treatment, a method of drug delivery into the body that would increase a patient’s comfort level throughout chemotherapy is highly desirable. A method of localized, time-controlled release of cisplatin to the body was developed for use in conjugation to radiation therapy. An insertable device composed of poly(ethylene-co-vinyl acetate) (EVAc) that mimicked the device used for vaginal contraceptive delivery was created. Cisplatin crystals were created inside the EVAc, and the device was inserted into the cervixes of women with localized advances cervical carcinoma. This device released cisplatin over a period of time without requiring painful injections[12]. If this idea of inserting a device that contained cisplatin crystals to be released into the body over a period of time was copied and developed for the treatment of other carcinomas, it would greatly increase the comfort of patients with cancer.


 * Alternative Platinum Based Drugs**

Carboplatin is an analogue of cisplatin, a second generation drug, that in recent years has become more widely used than cisplatin because it has shown to have less cytotoxic side effects and is also effective against cisplatin-resistant tumors[13]. In contrast to cisplatin, carboplatin is a larger molecule. The chloride ligands of cisplatin have been replaced with a dicarboxylate ligand, increasing its size and adding an organic substituent to the molecule. This larger molecule is broken down much slower by the human body due to a much more stable leaving group, which in turn slows the rate of formation of toxic waste products. Carboplatin has been proven to be much less toxic to kidney and liver cells, which is a major improvement over cisplatin. Although the toxicity of cisplatin has been decreased through the development of carboplatin, the effectiveness of the drug has not. The mechanism in which carboplatin targets DNA molecules in cancerous cells is very similar to the mechanism followed by cisplatin[2,5]. Carboplatin also creates identical lesions in DNA as cisplatin[14]. One adverse side-effect of carboplatin is bone-marrow suppression with increasing anemia, but nephrotoxicity and neurotoxicity are not seen[9]. Oxaliplatin, which is the third generation drug of cisplatin, was approved for use as a chemotherapy drug in 2002. The benefit of this drug is that it can be used with high efficiency in cisplatin resistant tumors because it does not have the same mechanism of action as cisplatin. Oxaliplatin forms less crosslinks with molecular DNA, hence requiring a lower concentration for the same effectiveness as cisplatin or carboplatin[14]. Some of these recent advances to reduce the toxicity of cisplatin may possibly be the next big breakthrough in the fight against cancer. If a drug is found that can be targeted to various type of cancer cells, it would decrease the toxicity of the chemotherapy drug being used and also be much easier to give to a larger group of the population. With the modification and exact tuning of some of the above mentioned discoveries, cisplatin just may be that wonder drug to defeat cancer.


 * References**

[|DOI] [1] Alderden, R. A., Hall, M. D., & Hambley, T. W. (2006). The discovery and development of cisplatin. Journal of Chemistry Education, 83, 728. [|DOI] [2] Arnesano, F., & Natile, G. (2009). Mechanistic insight into the cellular uptake and processing of cisplatin 30 years after its approval by FDA. Coordination Chemistry Reviews, 253(15-16), 2070-2081. [|DOI] [3] Bruijnincx, P. C., & Sadler, P. J. (2008). New trends for metal complexes with anticancer activity. Current Opinion in Chemical Biology, 12(2), 197-206. [|DOI] [4] Cao, Z., Tong, R., Mishra, A., Xu, W., Wong, G. C. L., Cheng, J., et al. (2009). Reversible cell-specific drug delivery with aptamer-functionalized liposomes. Angewandte Chemie, 48(35), 6494. [5] Carboplatin information. Retrieved 12/03, 2009, from http://www.carboplatin.org/ [|DOI] [6] Feazell, R. P., Nakayama-Ratchford, N., Dai, H., & Lippard, S. J. (2007). Soluble single-walled carbon nanotubes as longboat delivery systems for platinum(IV) anticancer drug design. Journal of the American Chemical Society, 129(27), 8438. [|DOI] [7] Guo, Z., & Sadler, P. J. Medicinal organic chemistry. Advances in Inorganic Chemistry, 49, 183. [|DOI] [8] Hambley, T. W. (2007). Developing new metal-based therapeutics: Challenges and opportunities. Dalton Transactions,, 4929. DOI [9] Hanigan, M. H., & Devarajan, P. (2003). Cisplatin nephrotoxicity: Molecular mechanisms. Cancer Ther., 1, 47. [|DOI] [10] Hwang, T., Fang, C., Chen, C., & Fang, J. (2009). Permeation enhancer-containing water-in-oil nanoemulsions as carriers for intravesical cisplatin delivery. Pharmaceutical Research, 26(10), 2314. [|DOI] [11] Hwang, T., Lee, W., Hua, S., & Fang, J. (2007). Cisplatin encapsulated in phosphatidylethanolamine liposomes enhances the in vitro cytotoxicity and in vivo intratumor drug accumulation against melanomas. Journal of Dermatological Science, 46(1), 11-20. [|DOI] [12] Keskar, V., Mohanty, P. S., Gemeinhart, E. J., & Gemeinhart, R. A. (2007). Cervical cancer treatment with a locally insertable controlled release delivery system. Journal of Controlled Release, 115(3), 280. [|DOI] [13] Ndinguri, M. W., Solipuram, R., Gambrell, R. P., Aggarwal, S., & Hammer, R. P. (2009). Peptide targeting of platinum anti-cancer drugs. Biconjugate Chemistry, 20(10), 1869. [|DOI] [14] Rabik, C. A., & Dolan, M. E. (2007). Molecular mechanisms of resistance and toxicity associated with platinating agents. Cancer Treat Rev, 33(1), 9. [|DOI] [15] Rose, P. G., Bundy, B. N., Watkins, E. B., Thigpen, J. T., Deppe, G., Maiman, M. A., et al. Concurrent cisplatin-based radiotherapy and chemotherapy for locally advanced cervical cancer. The New England Journal of Medicine, 340, 1144. [|DOI] [16] Rosenberg, B., Van Camp, L., & Krigas, T. (1965). Inhibition of cell division in escherichia coli by electrolysis products from a platinum electrode. Nature, 205, 698. [|DOI] [17] Scanlon, K. J., Kashani-Sabet, M., Tone, T., & Funato, T. (1991). Cisplatin resistance in human cancers. Pharmacology & Therapeutics, 52(3), 385-406. [|DOI] [18] Xu, P., Van Kirk, E. A., Murdoch, W. J., Zhan, Y., Isaak, D. D., Radosz, M., et al. (2006). Anticancer efficacies of cisplatin-releasing pH-responsive nanoparticles. Biomacromolecules, 7(3), 829. [|DOI] [19] Yang, Z., Wang, X., Diao, H., Zhang, J., Li, H., Sun, H., et al. (2007). Encapsulation of platinum anticancer drugs by apoferritin. Chemical Communications, (May 17), 3453. [|DOI] [20] Zutphen, S. v., & Reedijk, J. (2005). Targeting platinum anti-tumour drugs: Overview of strategies employed to reduce systemic toxicity. Coordination Chemistry Reviews, 249(24), 2845-2853. [|DOI] [21] Woo, J., Chiu, G. N. C., Karlsson, G., Wasan, E., Ickenstein, L., Edwards, K., et al. (2008). Use of a passive equilibration methodology to encapsulate cisplatin into preformed thermosensitive liposomes. International Journal of Pharmaceutics, 349(1-2), 38-46.