Clifford W Fong
email: cwfong@internode.on.net

Molecular mechanisms of cellular transport and resistance of platinum anticancer drugs

Keywords: Platinum drugs, anti-cancer, cellular uptake, resistance, reversal of resistance

The acquired resistance and reversal of resistance of Pt drugs has been examined using an extensive review of the literature and molecular orbital computations. The cellular uptake of Pt drugs is fastest for neutral species, with high lipophilicity, since their desolvation penalties for crossing the cell membrane are lowest. Pt resistant cell lines generally have lower levels of drug uptake, indicating this is a dominant first order cause of cellular resistance. Resistance can be caused by complexation of charged Pt species to phosphatidylserine (PS) headgroups, or by Pt complexation to the inner terminii of the trans-membrane pore of the hCtr1 transporter. Drugs that are known to reverse induced Pt resistance can decomplex the Pt-PS or Pt-hCtr1 complexes, restoring normal PS or hCtr1 functions.

Objectives

Resistance to Pt drugs: to examine mechanisms involved in acquired resistance, particularly the cellular accumulation mechanisms: (a) passive diffusion, active transport including copper transporters, organic cation transporters; (b) active efflux and intracellular reductive processes Reversal of platinum resistance: to develop predictive criteria for drugs that can reverse acquired resistance.

Introduction

Chemotherapy, surgery and radiation are the main treatments for cancer. Platinum chemotherapeutics are the most widely prescribed drugs in modern oncology (administered to about 50 % of all cancer patients), either alone or in combination with other anti-cancer drugs and/or radiation therapy. Platinum drugs are used for the treatment of a broad spectrum of specific cancers, including testicular, ovarian, bladder, head and neck, esophageal, small and non-small cell lung, breast, cervical, stomach and prostate cancers, as well as Hodgkin's and non-Hodgkin's lymphomas, neuroblastoma, sarcomas, multiple myeloma, melanoma, and mesothelioma. One of the greatest successes in chemotherapeutics was the advent of cisplatin treatment of metastatic testicular cancer where the survival rate in 1970 was about 5% of young men, whereas now greater than 80% of such cases are cured. [START_REF] Abu-Surrah | Platinum Group Antitumor Chemistry: Design and development of New Anticancer Drugs Complementary to Cisplatin[END_REF] The development of new platinum (II) and (IV) drugs has been extensively reviewed, [START_REF] Abu-Surrah | Platinum Group Antitumor Chemistry: Design and development of New Anticancer Drugs Complementary to Cisplatin[END_REF][START_REF] Spreckelmeyer | Cellular Transport Mechanisms of Cytotoxic Metallodrugs: An Overview beyond Cisplatin[END_REF][START_REF] Wilson | Synthesis, Characterization, and Cytotoxicity of Platinum(IV) Carbamate Complexes[END_REF][START_REF] Kelland | The resurgence of platinum-based cancer chemotherapy[END_REF][START_REF] Graf | Redox activation of metal-based prodrugs as a strategy for drug delivery[END_REF].

Over 3000 Pt antineoplastic drugs have been examined in vitro, but to date, only cisplatin, carboplatin and oxaliplatin have received world wide approval. [START_REF] Boulikis | Designing platinum compounds in cancer: structures and mechanism[END_REF]] Some Pt(IV) prodrugs, which have better cell permeability characteristics, have been examined [START_REF] Wilson | Synthesis, Characterization, and Cytotoxicity of Platinum(IV) Carbamate Complexes[END_REF][START_REF] Graf | Redox activation of metal-based prodrugs as a strategy for drug delivery[END_REF]]. These neutral prodrugs revert to the active Pt(II) species in the cytosol. None of these drugs have passed clinical trials.

The major limiting factor which affects the therapeutical efficacy of Pt anti-tumour drugs (and other drugs used in combination) is acquired resistance. Causes of resistance are changes in cellular uptake (probably the dominant factor), cellular efflux, increased detoxification, inhibition of apoptosis and increased DNA repair. Combinatorial therapies have increased the efficacy of Pt drugs. Searches for more effective drugs which do not induce resistance have not been successful. Alternatively, searches for drugs that can sensitize Pt resistant cancer cells to antitumour agents have not yet led to clinical success.

The greatest challenge in chemotherapeutics is drug resistance. Numerous mechanisms of Pt drug resistance have been found, including changes in cellular uptake, drug efflux, increased detoxification, inhibition of apoptosis and increased DNA repair. It is rare to find a single mechanism of resistance within a tumour, usually several mechanisms coexist to create a complex multifaceted disease, which confounds cancer treatment strategies. Combinatorial therapies have been used to help overcome resistance, but resistance still remains the principal clinical obstacle to curing cancer. Although clinical remission can be obtained after cytoreductive surgery and first-line chemotherapy, relapse is common. Second-line treatment of recurrent cancers is difficult and requires complex management strategies. For example, in the treatment of ovarian cancer, one of the most difficult to treat diseases because the cancer has most likely metastasized at diagnosis, the widely used definition of "sensitive" and "resistant" disease is whether recurrence occurs within six months of first-line chemotherapy. Refractory ovarian cancer occurs in about 20% of cases where the first line treatment has little or no effect. The majority of patients with recurrent disease eventually develop Pt resistance disease. [START_REF] Markman | Second-line treatment of ovarian cancer[END_REF][START_REF] Lederman | Optimal treatment for relapsing ovarian cancer[END_REF] Finding ways to reverse resistance to Pt (and other) chemotherapeutics is the major challenge to improving clinical outcomes in anti-tumour therapies. There are some isolated examples of such reversals, but there is no comprehensive investigative structure for examining such reversals.

Review of literature findings 1. Resistance to Pt drugs

Resistance to drugs is based on very complex genetic and epigenetic changes in gene expression and alterations in protein localization. Pleiotropic changes occur in almost every mechanism supporting cell survival, including cell growth-promoting pathways, apoptosis, developmental pathways, DNA damage repair, and endocytosis. [START_REF] Shen | Cisplatin Resistance: A Cellular Self-Defense Mechanism Resulting from Multiple Epigenetic and Genetic Changes[END_REF][START_REF] Florea | Cisplatin as an Anti-Tumor Drug: Cellular Mechanisms of Activity, Drug Resistance and Induced Side Effects[END_REF][START_REF] Siddik | Mechanism of Cisplatin Resistance[END_REF][START_REF] Stordal | Understanding Cisplatin Resistance Using Cellular Models[END_REF]] Acquired resistance to administered Pt drugs is commonly observed in the clinic.

There are multiple mechanisms of cellular resistance to Pt drugs including:

1. Decreased cellular accumulation: (a) decreased uptake owing to degradation or delocalisation of channels or transporters, eg Ctr1, Ctr2, OCAT, AQP2, AQP9, FBP or MRP1, MRP2 (b) increased efflux by the copper-transporting P-type adenosine triphosphate ATP7A and ATP7B, or active exocytosis (c) biophysical functional and structural changes to organelles or cell membrane potential (d) defective Ctr2 regulated endocytosis 2. Decreased rate of Pt-DNA adduct formation as a result of (a) decreased cellular accumulation, (b) reduction of the concentration of Pt(II) + or Pt(II) ++ species which bind to DNA by reducing agents such as the thiols glutathione, the cysteine-rich metallothionein, cysteine and (c) various self-defence mechanism such as DNA repair systems 3. Epigenetic changes in resistance, transcription factors affecting platinum sensitivity, micro RNA involvement, epithelial to mesenchymal transition etc. These changes are highly interactive and multicausal.

Decreased cellular accumulation, mainly from reduced uptake not increased efflux, is a dominant rate determining factor resulting in cisplatin resistance. There may also be numerous epigenetic and genetic changes leading to the loss of cell-surface binding sites and/or transporters for cisplatin, and decreased fluid phase endocytosis.

Reduction in Pt concentrations of about 20-70% are observed in cancer cell lines resistant to cisplatin. [START_REF] Shen | Cisplatin Resistance: A Cellular Self-Defense Mechanism Resulting from Multiple Epigenetic and Genetic Changes[END_REF][START_REF] Florea | Cisplatin as an Anti-Tumor Drug: Cellular Mechanisms of Activity, Drug Resistance and Induced Side Effects[END_REF][START_REF] Siddik | Mechanism of Cisplatin Resistance[END_REF][START_REF] Howell | Copper Transporters and the Cellular Pharmacology of the Platinum-Containing Cancer Drugs[END_REF][START_REF] Spreckelmeyer | Cellular Transport Mechanisms of Cytotoxic Metallodrugs: An Overview beyond Cisplatin[END_REF][START_REF] Basu | Cellular Responses to Cisplatin-Induced DNA Damage[END_REF][START_REF] Gately | Cellular accumulation of the anticancer agent cisplatin: a review[END_REF][START_REF] Kelland | The resurgence of platinum-based cancer chemotherapy[END_REF][START_REF] Galuzzi | Molecular mechanisms of cisplatin resistance[END_REF][START_REF] Hall | The role of cellular accumulation in determining sensitivity to platinum-based chemotherapy[END_REF] It is generally accepted that Pt drugs enter the cell via two pathways: (a) passive diffusion and (b) facilitated and active uptake by a number of membrane transporters and channels (collectively the transportome). Decreased influx could result from defective transporters or channels, or functional/structural changes in organelles or membrane potential. Active efflux could result from increased export, secretion, or exocytosis of the platinum. It is possible that a defective passive diffusion process may be involved in reduced uptake of Pt. It is noted that there is conflicting and contradictory evidence in the literature on cisplatin resistance, and in particular on the role of human organic cation transporters on cellular uptake. It may well be that individual resistance pathways are active in specific tissues, for example the large difference in response of testicular cancer and glioblastoma. There are many examples in the literature of variations in resistance found in similar cells lines: (a) in five human ovarian carcinoma cell lines, it was found that the accumulation of cisplatin varied from reduced accumulation having a partial role in intrinsic resistance, to a major role in another cell line where reduced uptake (not increased efflux) was dominant. None of the cell lines showed saturation at 2 hours exposure, and resistant cells showed the lowest accumulation. [Mistry 1992] (b) the presence of extracellular ATP during treatment with cisplatin leads to additive cytotoxicity in a sensitive human ovarian carcinoma cell line but not in cisplatin-resistant cell line. [START_REF] Rotte | Effect of extracellular ATP on cisplatininduced cytotoxicity in human ovarian carcinoma cells[END_REF]] Using the same cell line, it was found that cisplatin triggered redistribution of copper efflux transporters ATP7A and ATP7B from the perinuclear region to more peripherally located sites in the cytosol. Cisplatin exposure had no effect on protein localisation in the resistant cell line and the cisplatin-resistant cells were found to have significantly less lysosomes than the sensitive cells. [START_REF] Kalayda | Altered localisation of the copper efflux transporters ATP7A and ATP7B associated with cisplatin resistance in human ovarian carcinoma cells[END_REF]] (c) the copper transporter Ctr1 has been shown to be degraded by exposing human ovarian cancer cells (and most other cells, but not all) to cisplatin, and the greatest effect is during the early phase. The down regulation is thought to be due to decreased endocytosis. The proteasome inhibitors bortezomib and lactacystin were found to prevent the cisplatin-induced down-regulation of Ctr1. [START_REF] Howell | Copper Transporters and the Cellular Pharmacology of the Platinum-Containing Cancer Drugs[END_REF] While the role of hOCT in Pt uptake is contradictory, the major side effects of nephrotoxicity and ototoxicity of cisplatin are decreased after inhibition of OCT2. [START_REF] Ciarimboli | Organic cation transporter 2 mediates cisplatininduced oto-and nephrotoxicity and is a target for protective interventions[END_REF], 2004[START_REF] Koepsell | Polyspecific organic cation transporters: their functions and interactions with drugs[END_REF] Comparison of accumulation of cisplatin, carboplatin and oxaliplatin in MCM-7 breast cancer cells after 24 hours showed oxaliplatin had the highest uptake, closely followed by cisplatin and both had significantly higher uptakes than carboplatin. The intracellular concentrations were proportional to the concentrations in the culture medium. [Ghezzi 2004] These data reflect the clinical half lives of the drugs.

Expression of the human Cu transporter 1 (hCtr1) is thought to result in increased sensitivity to cisplatin, whereas expression of the human homeostatic Cu(I) proteins exporting ATPase, ATP7A and ATP7B, is believed to be involved in the resistance to cisplatin, either by sequestering drug away from its targets (ATP7A), or by exporting the drug from the cell (ATP7B). There is a linkage between the Cu chaperone protein ATOX1 and ATP7A and ATP7B, which are specific Cu(I) ATPases.

However, despite much evidence that Cu transporters are involved in uptake of Pt drugs, there is evidence to the contrary. For example, a comparison of the uptake rates of copper with cisplatin (and several analogs) into human embryonic kidney 293 cells overexpressing wild-type or mutant hCtr1, mouse embryonic fibroblasts that do or do not express Ctr1, and human ovarian tumor cells that are sensitive or resistant to cisplatin led to the conclusion that hCtr1 is not the major entry pathway for Pt drugs. The major pathway is not saturable nor protein mediated. [START_REF] Ivy | A Re-Evaluation of the Role of hCTR1, the Human High-Affinity Copper Transporter, in Platinum-Drug Entry into Human Cells[END_REF][START_REF] Basu | Cellular Responses to Cisplatin-Induced DNA Damage[END_REF][START_REF] Spreckelmeyer | Cellular Transport Mechanisms of Cytotoxic Metallodrugs: An Overview beyond Cisplatin[END_REF]] When examining the role of Ctr2 in cisplatin sensitivity, it was found that the siRNA-mediated down-regulation of Ctr2 increased cisplatin uptake. This result is difficult to explain through a mechanism that involves direct transport of the drug by the transporter. [START_REF] Gupta | Human copper transporters: mechanism, role in human diseases and therapeutic potential[END_REF] It has been found that the uptake of carboplatin in parental human hepatoma cells is time-, temperature-, and energy-dependent and that the rate of uptake is reduced in human hepatoma cisplatin-resistant cells. The efflux of carboplatin in cisplatinresistant cells was comparable to efflux in the parental cisplatin-sensitive cells, implying impaired uptake has a major role in reduced accumulation. [START_REF] Shen | Cisplatin Resistance: A Cellular Self-Defense Mechanism Resulting from Multiple Epigenetic and Genetic Changes[END_REF]] Although there was evidence that cisplatin, carboplatin and oxaliplatin were transported by Ctr1 in murine embryonic fibroblasts at low concentrations, oxaliplatin is not dependent on Ctr1 at higher concentrations. Also the loss of Ctr1 function reduced cisplatin and carboplatin to 36%, indicating another uptake mechanism was operating. [START_REF] Holzer | The internalization and degradation of human copper transporter following cisplatin exposure[END_REF]] This mechanism may involve the OCT transporter, as it has been shown that oxaliplatin uptake (but not cisplatin and carboplatin) and cytotoxicity is substantially increased by OCT1 and OCT2 in colon cancer cell lines. The transported species is thought to be the single charged chloro (possibly DACHPt + Cl(H 2 O) or hydroxyl aquated species (possibly DACHPt + OH(H 2 O). [Zhang 2006] However other studies showed that oxaliplatin is the major species transported in human colon cancer cells, and the hydrolysed species were only minor components that were transported. [Luo] It is also known that cations that are not transported by OCTs can act as inhibitors of the OCTs. [START_REF] Koepsell | Polyspecific organic cation transporters: their functions and interactions with drugs[END_REF] The exposure of mouse embryo fibroblasts and ovarian carcinoma cells to low concentration of cisplatin (2 µM) triggered almost instant disappearance of Ctr1 that was interpreted as protein degradation. However, Ctr1 reappeared when higher concentrations of cisplatin were used (10 and 200 µM). [Larson 2009] These Pt drugs also trigger proteasomal degradation of the Ctr transporter, and with time, limits its own uptake, a process that can be prevented by proteasomal inhibitors like bortezomib and lactacystin. [START_REF] Jandial | Enhanced delivery of cisplatin to intraperitoneal ovarian carcinomas mediated by the effects of bortezomib on the human copper transporter 1[END_REF][START_REF] Al-Eisawi | Carboplatin and oxaliplatin in sequenced combination with bortezomib in ovarian tumour models[END_REF] This result implies a Pt-Ctr1 interaction, which may be prevented by a similar inhibitor-Ctr1 interaction. The 3D X-ray structure of hCtr1 has shown that hCtr1 creates a pore that stretches across the membrane bilayer, and aligned with and sealing both ends of the pore, extracellular and intracellular domains of hCtr1 appear to provide additional metal binding sites. [START_REF] Feo | Three-dimensional structure of the human copper transporter hCTR1[END_REF] These domains could provide extra-and intra-cellular sites where Pt species could bind to the hCtr1 which might explain the contradictory time and concentration Pt uptake and accumulation studies discussed above if kinetic and binding equilibria factors are operating.

From the literature studies the consensus view is that reduced cellular accumulation ie uptake not efflux, is a dominant rate determining feature of acquired Pt resistance, but may be cell line dependent, time and energy dependent.

The experimental evidence that influx is a first order rate determining factor that determines resistance logically follows from first principles: the Pt drug has to first enter the cell by leaving the blood plasma and crossing the cell membrane to reach its DNA target, and if efflux mechanisms are operating, then being effluxed out of the cell back into the blood plasma. Accumulation certainly has a passive diffusion transport element and the role of active transporters, particularly for charged Pt species, is contradictory both for uptake and efflux. There appears to be a time dependent interaction between Pt drugs and active transporters, which affects uptake. Cellular uptake of drugs can be a competitive blend of passive diffusion and active transport by Ctr1 and OCAT transporters, which can be altered by drug induced changes to the cell membrane and/or transporters (time dependent) as well as pre-organizational factors specific to the drugs as they prepare to enter the cell membrane from a aquated environment. Such considerations also apply to drug efflux from the cytosolic environment. There have not been any studies of the role of desolvation, and the energy penalties involved, for neutral and charged Pt species with respect to uptake or efflux mechanisms.

Reversal of resistance to Pt drugs

Since Pt drug resistance is clearly a multi-faceted phenomenon, with pleiotropic changes occurring in almost every mechanism supporting cell survival, studies to find effective means of reversing this induced phenomenon are very complex. It appears from the literature that there are many ways of achieving reversal, and although separate studies have focussed on particular epigenetic changes and reversal outcomes, it may well be that multiple interacting factors are actually involved, and different cell lines probably have different resistance factors. A few examples of reversal of cisplatin resistance illustrate the multi-factorial aspects of reversing cisplatin resistance:

1. Reversal of cisplatin resistance in human ovarian cancer cell lines by a c-jun antisense oligodeoxynucleotide (ISIS 10582): evidence for the role of transcription factor overexpression in determining resistant phenotype. [START_REF] Pan | Reversal of cisplatin resistance in human ovarian cancer cell lines by a cjun antisense oligodeoxynucleotide (ISIS 10582): evidence for the role of transcription factor overexpression in determining resistant phenotype[END_REF]] 2. The combination of glycyrrhizin and lamivudine can reverse the cisplatin resistance in hepatocellular carcinoma cells through inhibition of multidrug resistance-associated proteins. [START_REF] Wakamatsu | The combination of glycyrrhizin and lamivudine can reverse the cisplatin resistance in hepatocellular carcinoma cells through inhibition of multidrug resistance-associated proteins[END_REF]] 3. A comparative study of the reversal of several cisplatin-resistant ovarian cancer cell lines found the following order of effectiveness: colchicine> genistein> triethylenetetramine (TETA)> rapamycin ≥ buthionine sulfoximine (BSO), which was correlated with the cellular accumulation of cisplatin. [START_REF] Yellepeddi | Comparative evaluation of small-molecule chemosensitizers in reversal of cisplatin resistance in ovarian cancer cells[END_REF]] 4. A BH3 mimetic, gossypol, was found to reverse cisplatin resistance in head and neck cancer cells [START_REF] Bauer | Reversal of cisplatin resistance with a BH3 mimetic, (-)-gossypol, in head and neck cancer cells: role of wild-type p53 and Bcl-xL[END_REF]] 5. Reversal of cisplatin resistance in human non-small lung cancer cells by the 1,4-Benzothiazepine derivative JTV-519 also increased cellular accumulation.

[Takashi 2001] 6. Reversal of acquired cisplatin resistance in rat ovarian cancer cells by nicotinamide, an inhibitor of poly(ADP-ribose) polymerase (PARP), was found in vitro and in vivo [START_REF] Chen | Reversal of acquired cisplatin resistance by nicotinamide in vitro and in vivo[END_REF]] 7. In a study of human small cell lung cancer, bladder cancer, and ovarian germ cell tumours, resistance mechanisms identified in vitro include a complex and wide array of related and unrelated pathways such as alterations in cellular drug transport, enhanced DNA repair dependent and independent of signal transduction pathways, and enhanced intracellular detoxification by glutathione and metallothionein systems. Reversal of resistance involved the following factors: reversal of cellular accumulation defects by dipyridamole; inhibition of DNA repair by hydroxyurea, pentoxifylline, and novobiocin; inhibition of the glutathione system by ethacrynic acid and buthionine sulfoximine; and inhibition of signal transduction pathways by cyclosporine, tamoxifen, and calcium channelblocking agents [START_REF] Gosland | Insights into Mechanisms of Cisplatin Resistance and Potential for Its Clinical Reversal[END_REF]] 8. Reversal of cisplatin resistance with ligustrazine and cyclosporin was found in a cisplatin-induced human epithelial ovarian cancer resistant cell line. [START_REF] Chen | Mechanism of drug resistance and reversal with ligustrazine and cyclosporin A in cisplatin-induced human epithelial ovarian cancer resistant cell line 3ao/cddp[END_REF]] 9. SGI-110 (Decitabine + 2-deoxyguanosine] was found to be a hypomethylating agent displaying epigenetic silencing MLH1and ZlC1 in resistant ovarian cancer lines. [START_REF] Munck | The DNA hypomethylating agent SGI-110 reverses the platinum resistance of ovarian cancer models[END_REF]] 10. Reversal of cisplatin resistance in NSLC MDR phenotype by small interfering RNA (siRNA) has been observed. The mechanism appears to involve activation of anti-apoptotic cellular defence: ie DNA damage caused by the chemotherapeutic drugs induces the release of an enzyme that activates the caspases enzymes that cleave cellular proteins [START_REF] Ganesh | Combination of siRNA-directed Gene Silencing With Cisplatin Reverses Drug Resistance in Human Non-small Cell Lung Cancer[END_REF]] 11. Resistance reversal in ovarian cancer cell lines by ATP7 silencing in vivo has been observed. ATP7B siRNA incorporated into neutral nanoliposomes was delivered into nude mice bearing tumours. This combination in conjunction with cisplatin was effective in reducing tumour growth. [START_REF] Mangala | Therapeutic targeting of ATP7B in ovarian carcinoma[END_REF] It is clear from these studies that there are multiple interacting factors involved in the reversal of platinum resistance, which probably means that any mechanistic conclusions from any one study cannot be universal or useful in themselves in seeking methods of overcoming platinum induced resistance. However by examining the biophysical properties of reversing agents, it may be possible to identify those first order dominant rate determining properties which underlie resistance, and hence be useful in identifying new drugs that can overcome resistance.

The ionization energy (IE) is a measure of the ease that a molecule can be oxidized (or lose an electron) and the electron affinity (EA) is a measure of the ease that a molecule can be reduced (or gain an electron). The gap between IE and EA is a measure of the intrinsic stability (or conversely reactivity) of a molecule. In a multifactor and interacting phenomenon like acquired resistance to Pt species, where a series of oxidative and reducing reactions can occur, IE and EA may be useful measures of the ability of a drug to reverse resistance. Theoretical parameters like IE and EA are directly related to the biochemical redox reactions and their oxidation and reduction potentials. Linear relationships have been found between experimental reduction potentials and EAs in platinum and related metal complexes. [START_REF] Wilson | Synthesis, Characterization, and Cytotoxicity of Platinum(IV) Carbamate Complexes[END_REF][START_REF] Bateni | Prediction of reduction potentials from calculated electron affinities for metal-salen compounds[END_REF]] Additionally linear relationships between reduction potentials and cytotoxicity (as measured by IC 50 values) in human ovarian cancer and human colon carcinoma cells have been observed. [START_REF] Choi | Reduction and anti-cancer activity of Pt(IV) complexes[END_REF], Gaviglio, Platts 2011[START_REF] Hall | Quantitative measurement of the reduction of platinum(IV) complexes using X-ray absorption near-edge spectroscopy (XANES)[END_REF]] While these relationships involved reduction potentials of Pt(IV) prodrug complexes, it is known that these drugs exert their cytotoxicity within the cytosol as Pt(II) species. [START_REF] Wilson | Synthesis, Characterization, and Cytotoxicity of Platinum(IV) Carbamate Complexes[END_REF][START_REF] Hall | Quantitative measurement of the reduction of platinum(IV) complexes using X-ray absorption near-edge spectroscopy (XANES)[END_REF][START_REF] Pil | Cisplatin and related drugs[END_REF][START_REF] Galanski | Drug delivery in oncology: from basic research to cancer therapy[END_REF] Homeostatis of the human body is critical to life. In particular, the homeostatis of the intracellular environment is disrupted by cancer, and introducing reactive Pt species into the diseased cells induces apoptosis, which is mediated by the activation of various signal transduction pathways. Cancer cells are known to differ distinctly in their redox metabolism from healthy tissues. Thus, enhanced levels of intracellular reactive oxygen species (ROS) are often observed in tumor cells and the specific area of the solid tumor is characterized by high metabolic activity, hypoxia, and, in general, reductive conditions. The reductive microenvironment of hypoxic tumours results from insufficient formation of new blood vessels during extraordinary rapid cell growth. Metal complexes with their multiple accessible oxidation states can interact with the cellular redox balance with ease. Intracellular interactions of Pt species include reactive oxygen species (such as O 2

•-, 1 O 2 , H 2 O 2 and, •OH), DNA (formation of Pt-DNA adducts etc), tumour necrosis factors, mitachondria, the tumour suppressor protein p53, calcium signalling, caspaces, multidrug resistant proteins. ROS species have been shown to be directly involved in the regulation of diverse signal transduction pathways important for cell proliferation, differentiation, and cell death. [START_REF] Kovacic | Mechanisms of Anti-Cancer Agents: Emphasis on Oxidative Stress and Electron Transfer[END_REF][START_REF] Jungwith | Anticancer Activity of Metal Complexes: Involvement of Redox Processes[END_REF] Normal cells survive oxidative stress by activating coordinated steps to get rid of the stressors and to avoid destructive damage by various transcription factors which activate of an array of diverse genes involved in metabolism, detoxification, export of xenobiotics, as well as in the repair of the induced cellular damages. As anticancer metal drugs are redox-active substances interfering with the cellular redox status and supporting ROS generation by different mechanism, such protective response mechanisms are almost generally activated as a consequence of cell exposure. These responses can counteract the cancer cell-damaging effect of drugs so causing therapy failure, or resistance to the drugs.

Cisplatin treatment was found to deplete cellular NADPH pools resulting in altered mitochondrial redox status, which then causes hydroxyl radical generation. This effect can directly affect energy intensive processes involved in active efflux transport processes. Living organisms maintain a complex oxidant-antioxidant homeostasis system with diverse ROS generating and degrading systems in different compartments of the cell. There are several regulatory levels for maintenance of redox balance in the cell involving enzymatic (such as superoxide dimutases, catalase, thioredoxin reductases), glutathione reductases, and glutathione peroxidises, as well as nonenzymatic antioxidants (such as glutathione, thioredoxin, and several vitamins). Intracellular redox homeostasis is severely affected by cisplatin due to the disruption of the thioredoxin and glutathione reductase systems, which is related to acquired cisplatin resistance. [START_REF] Jungwith | Anticancer Activity of Metal Complexes: Involvement of Redox Processes[END_REF]] However these cytosolic redox processes are still secondary to the actual accumulation of the Pt drugs in the cell after passing through the cell membrane.

A critical intracellular homeostasis redox environment is maintained by thiol molecules (eg glutathione), which can form thiyl radicals. Resistance is thought to be partially based on the formation of Pt-glutathione chelate conjugates, which leads to depletion of intracellular glutathione pools, disturbance of the cellular redox homeostasis, and formation of ROS. The Pt-glutathione complex is effluxed from the cell in an ATP-dependent manner by the glutathione transporter family, termed the GS-X pumps. It was initially thought that cells that are resistant to cisplatin have elevated levels of glutathione, but there is evidence to the contrary. Pt can also interfere with calcium homeostasis extra-and intra-cellularly. [START_REF] Florea | Cisplatin as an Anti-Tumor Drug: Cellular Mechanisms of Activity, Drug Resistance and Induced Side Effects[END_REF][START_REF] Jungwith | Anticancer Activity of Metal Complexes: Involvement of Redox Processes[END_REF][START_REF] Basu | Cellular Responses to Cisplatin-Induced DNA Damage[END_REF] DNA oxidation occurs most readily at guanine residues due to the high oxidation potential of this base relative to cytosine, thymine, and adenine, which explains why the primary interaction of Pt with DNA to form Pt-DNA adducts is at the guanine site. More damage to RNA is caused by ROS than DNA as well.

The accessible redox potential window in normal biological cellular systems ranges from around -0.4 to 0.8V (versus NHE), with the strongest reducing agent being the NADP/NADPH couple (-0.38V NHE). The strongest oxidizing agent is oxygen (0.815V NHE pH 7) which requires enzyme catalysis as oxygen is chemically inert. However ROS and thiyl free radicals for example have very high potentials, eg hydroxyl radical 2.31V, cysteine thiyl radical 0.92V, but their concentrations are usually very low & react where they are formed. Oxidizing radicals like GS • can react with GS -to form strongly reducing GSSG • -radicals with redox potentials of -1.50 V. Glutathione has a half cell potential of -0.26V (NHE) at pH 7.0 [Mills] with a calculated EA of 1.7 eV in water at pH 7 (this study).

Calculated electron affinities (EA) have been shown to be highly linearly related to peak reduction potentials from cyclic voltammetry in solution for a series of Pt(IV) carbamates. [START_REF] Wilson | Synthesis, Characterization, and Cytotoxicity of Platinum(IV) Carbamate Complexes[END_REF]] Similar linear relationships have been also observed for a wide range of metal(II) complexes. [START_REF] Bateni | Prediction of reduction potentials from calculated electron affinities for metal-salen compounds[END_REF]] The reduction potentials of Pt(IV) complexes shows that axial Cl ligands are most easily reduced, (-0.06V NHE) and hydroxyl ligands most difficult (-0.71V NHE). Acetato ligands have potentials around -0.41V SHE. The redox potential order for Pt(IV) axial ligands is OH [START_REF] Graf | Redox activation of metal-based prodrugs as a strategy for drug delivery[END_REF]] The oxidation and reduction peak potentials of cisplatin in the presence of 5% human serum at pH 7.4 are 0.28V and -0.05V (NHE). [START_REF] Ye | Electrochemical Determination of Cisplatin in Serum at Graphene Oxide/Multi-walled Carbon Nanotubes Modified Glassy Carbon Electrode[END_REF] A comparative study of several drugs and their ability to reverse resistance in cisplatin-resistant ovarian cancer cells found the following order of effectiveness: colchicine> genistein> triethylenetetramine (TETA)> rapamycin ≥ buthionine sulfoximine (BSO). This order was directly correlated with the cellular accumulation of cisplatin. [START_REF] Yellepeddi | Comparative evaluation of small-molecule chemosensitizers in reversal of cisplatin resistance in ovarian cancer cells[END_REF]] An examination of the EA values for these drugs in water shows that this order is reflected in the reversal effectiveness with the exception of rapamycin. The EA is directly related to the reduction potential, a higher EA indicating a higher reduction potential and increased ease of reduction. The difference for rapamycin is the much larger free energy of solvation for rapamycin and its molecular size compared to the other drugs. As rapamycin has to substantially desolvate before it can be reduced, as well as enter the cell (a larger molecular size increases the energy needed to actively transport rapamycin across the cell membrane), both these factors effectively lower its ability to be reduced. [START_REF] Fong | Permeability of the blood-brain barrier: molecular mechanism of transport of drugs and physiologically important compounds[END_REF] It has been shown that a combination of cisplatin, BSO and temozolomide in vivo has a synergistic effect on malignant glioma, a severe brain tumour, and the cisplatin resistant cells have higher levels of glutathione. The effect is independent of the tumour suppressing protein p53. These observations could be driven by redox reactions with cisplatin, as BSO is a known inhibitor of glutathione production. [START_REF] Rocha | Glutathione depletion sensitizes cisplatin-and temozolomide-resistant glioma cells in vitro and in vivo[END_REF]

-< RC(O)O -< Cl -< CF 3 C(O)O -.

Changes to cell membranes as a basis for reduced accumulation of Pt in resistant cells

The reduced accumulation of Pt drugs in resistant cell lines has also be focussed on changes to the biophysical properties of the cell membrane, including specific membrane components such as transporters, channels, etc. Changes in the biophysical fluidity or viscosity of Pt resistant membranes has been observed using dyes, ESR, NMR and flow techniques which indicate that resistant cells lines show: (a) higher membrane potentials (b) lower polarization values and consequently more fluid membranes (c) ESR probes indicative of more fluid membranes (d) less fluorescent dye packs into resistant cells, indicative of increased fluidity in resistant cells. [Liang 2004[START_REF] Taylor | Pressure tuning infrared spectroscopic study of cisplatin-induced structural changes in a phosphatidylserine model membrane[END_REF]. Changes in cell membrane potential such as depolarization (where the normally negative charge on the inside of the membrane becomes briefly positive, and induces electrical signalling processes, are very important processes to the body. So Pt drug induced changes that reverse the membrane potential will affect diffusion and active transport processes across the membrane. Charged species are known to induce larger changes to the membrane potential than neutral species. [START_REF] Fong | Permeability of the blood-brain barrier: molecular mechanism of transport of drugs and physiologically important compounds[END_REF] Changes in the membrane lipids between sensitive and resistant cells have been detected during cisplatin induced apoptosis by 1 H and 31 P NMR techniques as well as membrane microviscosity fluidity measurements. [START_REF] Huang | NMR studies of the relationship between the changes of membrane lipids and the cisplatin-resistance of A549/DDP cells[END_REF]] Early membrane fluidification, which is independent of cisplatin-DNA adduct formation in human colon cancer cells, is triggered by cisplatin apoptosis via various intracellular processes. Membrane disruption triggers the Fas death receptor pathway. [START_REF] Rebillard | Cisplatin-Induced Apoptosis Involves Membrane Fluidification via Inhibition of NHE1 in Human Colon Cancer Cells[END_REF][START_REF] Rebillard | Cisplatin cytotoxicity: DNA and plasma membrane targets[END_REF][START_REF] Rebillard | Cisplatin-induced apoptosis involves a Fas-ROCK-ezrin-dependent actin remodelling in human colon cancer cells[END_REF] When model membranes were treated with cisplatin, a cisplatin-phosphatidylserine complex was detected in permeabilized and lysed human erythrocytes and tumour cell lines. When glutathione was present, there was a reduction in complex formation. The active species was shown to be the {(H 3 N) 2 Pt(H 2 O) 2 } ++ species. Mass spectral analysis showed the Pt interacted with the carboxylate and amine of the hydrophilic head group, and not the fatty acyl chains. [START_REF] Speelmans | Cisplatin complexes with phosphatidylserine in membranes[END_REF][START_REF] Burger | Interaction of the anti-cancer drug cisplatin with phosphatidylserine in intact and semi-intact cells[END_REF] Solid and solution NMR studies of extracted phosphatidylserine bilayers shows that the serine head group of phosphatidylserine phospholipid binds to the aquated form of cisplatin, followed by another cisplatin-phosphatidylserine complex formation, probably via the oxygen atoms of the phosphate moiety. [START_REF] Jensen | Anticancer cisplatin interactions with bilayers of total lipid extract from pig brain[END_REF][START_REF] Jensen | Cisplatin interaction with phosphatidylserine bilayer studied by solid-state NMR spectroscopy[END_REF] Electrical stimuli are known to cause reversible permeabilization of cell membranes, and the uptake of cisplatin has been observed to increase up to 3 fold, with increasing cytotoxicity in human cervix carcinoma cells. [START_REF] Melvik | Increase in cisdichlorodiammineplatinum(II) cytotoxicity upon reversible electropermeabilization of the plasma membrane in cultured human NHIK 3025 cells[END_REF]] Cisplatin dose dependency with electrical changes to a model phosphatidylcholine membrane have been ascribed to a strong surface interaction between the cisplatin and the polar zwitterionic head group. [START_REF] Maheswari | Interaction of cisplatin with planar model membranes -dose dependent change in electrical characteristics[END_REF]] It is unclear whether neutral cisplatin or its charged hydrolysed products is responsible for bonding with zwitterionic head group of phosphatidyl choline. The interaction does not fluidize the membrane sufficiently to cause breakdown, but is thought to cause small defects in the membrane to allow increased permeability.

Phosphatidylcholine is a major constituent of cell membranes and pulmonary surfactant, and is more commonly found in the exoplasmic or outer leaflet of a cell membrane. It is thought to be transported between membranes within the cell by phosphatidylcholine transfer protein (PCTP). Phosphatidylcholine also plays a role in membrane-mediated cell signalling and PCTP activation of other enzymes. It exists as a zwitterion over a wide range of pH in the cell, which makes it amenable to interacting with charged species, such as the small amount of charged Pt species, such as {(H 3 N) 2 Pt(H 2 O)Cl} + (or less likely {(H 3 N) 2 Pt(H 2 O) 2 } ++ ) which might be present in blood plasma. Phosphatidylcholine also has a relatively high dipole moment by virtue of the zwitterionic head group and long hydrophobic tails, which allows a charged interaction with the strong dipole of cisplatin. It has been previously shown that a new model of the permeability of the blood brain barrier indicates that the dipole moment of drugs (and how it interact with the cell membrane potential) is a key component that explains permeability of cell membranes. [START_REF] Fong | Permeability of the blood-brain barrier: molecular mechanism of transport of drugs and physiologically important compounds[END_REF] Phosphatidylserine is an acidic (anionic) phospholipid with three ionizable groups, i.e. the phosphate moiety, the amino group and the carboxyl function. The head group is negatively charged in the cell. It has a high natural propensity to chelate to cellular metal ions eg calcium. This chelating ability is consistent with the observed formation of cisplatin-phosphatidylserine complexes, particularly where the concentrations of the positively charged species {(H 3 N) 2 Pt(H 2 O)Cl} + and {(H 3 N) 2 Pt(H 2 O) 2 } ++ are relatively high in the low Cl -intracellular cystolic environment, whereas in blood plasma the higher Cl -concentration precludes significant concentrations of these charged Pt species . So while these charged Pt species are known to react with DNA to form adducts, they can also react with the inward cystolic facing anionic head groups of phosphatidylserines in the cell membrane. Phosphatidylserine(s) are actively held facing the cytosolic (inner) side of the cell membrane by the enzyme flippase. This is in contrast to normal behaviour of phospholipids in the cell membrane which can freely flip their heads between the two faces of the membrane they comprise. However, when a cell undergoes apoptosis phosphatidylserine is no longer restricted to the cytosolic domain by flippase. Flippases or scramblase can move phosphatidylserine in both directions across the membrane. When the phosphatidylserines naturally flip to the extracellular (outer) surface of the cell, they act as a signal for macrophages to engulf the cells. The process of apoptosis is often accompanied by generation of reactive oxygen species, which bring about rapid oxidation of the fatty acids in phosphatidylserine before this lipid is externalized. Only molecular species of phosphatidylserine with an oxidatively truncated sn-2 acyl group that incorporates terminal γ-hydroxy(or oxo)-α,β-unsaturated acyl moieties are recognized by scavenger receptors in macrophages as a prerequisite for engulfment of apoptotic cells. [Christie, Leventis 2010] Phosphatidylserine externalization is involved in apoptosis which is a controlled type of cell death that is energy-dependent and characterized by cell shrinkage, chromatin condensation, membrane budding, and activation of a family of cysteine proteases called caspases. Caspase activation is thought to be a key step in the genesis of apoptosis, and numerous stimuli activate caspases, including those that activate plasma membrane death receptors and cause mitochondrial dysfunction. Cisplatin induced apoptosis of renal proximal tubular cells and malignant mesothelioma cells involves increased phosphatidylserine externalization and caspace activation. [START_REF] Cummings | Cisplatin-Induced Renal Cell Apoptosis: Caspase 3-Dependent and -Independent Pathways[END_REF][START_REF] Inez | Mechanisms of cisplatin-induced cell death in malignant mesothelioma cells: Role of inhibitor of apoptosis proteins (IAPs) and caspases[END_REF] As noted above, there is contradictory evidence on the importance of active OCT and CTR transporters on cellular uptake, possibly due to varying time, temperature and energy factors and different cell lines. The structural modification of these transporters by Pt could be an explanation accounting for some of the contradictory results. It has been shown in studies of carboplatin and nedaplatin that a maximum of two Pt atoms can bind to each monomer unit of the end terminus of hCtr1, via methionine residues to form ring-opened monofunctional adducts. Nucleophiles in plasma played a pre-activation role with the drugs. [START_REF] Wang | Activation of carboplatin and nedaplatin by the N-terminus of human copper transporter 1 (hCTR1)[END_REF]] A comparison of cisplatin, carboplatin and oxaliplatin with model hCtr1 peptides, similarly found the formation of Pt-hCtr1 adducts at the methionine residues. With cisplatin the removal of all ligands occurred within hours, carboplatin took 48 hours and oxaliplatin maintained its chelating diammine ligand. [START_REF] Crider | Coordination of platinum therapeutic agents to met-rich motifs of human copper transport protein1[END_REF] Copper chelators are known to increase cisplatin accumulation and increase cytotoxicity, observations attributed to changes to Ctr1 processes. [START_REF] Ishida | Enhancing tumor-specific uptake of the anticancer drug cisplatin with a copper chelator[END_REF][START_REF] Holzer | The internalization and degradation of human copper transporter following cisplatin exposure[END_REF] Transport of organic cations mediated by the three OCT subtypes (OCT1, OCT2, and OCT3) is electrogenic, independent of Na + , and reversible with respect to direction, and driven solely by the electrochemical gradient of the transported organic cation. The uptake mediated by OCT is concentration dependant, as opposed to the transport mediated by Ctr1 that is equilibrative. Hence, OCTs may have a stronger activity than Ctr1 with respect to the transport of platinum derivatives. [START_REF] Ciarimboli | Organic cation transporter 2 mediates cisplatininduced oto-and nephrotoxicity and is a target for protective interventions[END_REF]] However it has been shown that oxaliplatin may be transported by OCT1 1nd OCT2, but not cisplatin or carboplatin. [Zhang 2006] It is also possible that charged Pt species that are not transported by OCT might actually inhibit normal OCT function, a process observed for other cationic species. [START_REF] Koepsell | Polyspecific organic cation transporters: their functions and interactions with drugs[END_REF]] If such species were acting as intracellular inhibitors, they might make cells resistant to further Pt drug treatment, similarly to that observed for hCtr transporters where complexes have been observed between Pt and phosphatidylserine.

Results and Discussion

Resistance to Pt drugs

Acquired resistance is clearly a multi-faceted and multi-factor phenomenon of chemotherapeutics. However from the review above, the reduced accumulation (ie uptake) of the drug in tumour cells is clearly the dominant factor, and intracellular processes that contribute to resistance, including efflux, reactions with nucleophilic or free radical cytoplasmic scavengers (such as S containing species) and signalling pathways, are secondary and follow-on factors arising after drug uptake. Resistance to Pt drugs consists of four factors (a) processes before the binding of Pt to DNA, pretarget resistance (b) processes related to Pt-DNA binding, on-target resistance (c) processes related to the lethal signalling pathways elicited by Pt mediated damage to DNA, post-target resistance, and (d) off-target resistance. [START_REF] Galuzzi | Molecular mechanisms of cisplatin resistance[END_REF]] The approach taken in this study focuses on (a) as a dominant primary rate determining step in acquired Pt resistance. That is, processes (b-d) can only occur after the Pt drug in the blood plasma enters the cell by passing through the cell membrane to reach its DNA target.

Uptake can be by passive diffusion or active transport by OCT or CTR transporters. The critical factors in determining uptake are: (a) Molecular properties of the various Pt drug species and how likely they are to passively transport across cell membranes: the charge on the Pt species, the desolvation energy required before entry into the cell membrane, the dipole moment of the Pt species and how this attribute interacts with the membrane dipole, the molecular size, and lipophilicity of the drug. Active transport processes also require a substantial desolvation of the drug before being taken up by a carrier protein or channel, or engulfed as in exocytosis, all of which require substantial cellular energy input. The greater the energy of desolvation of the drug required, the lower the likelihood or uptake rate by energy intensive active transporters of the drug. (b) The likelihood that charged Pt species in the blood plasma can chemically bond to the zwitterionic phosphatidylcholine headgroup of the cell membrane, and so alter the membrane potential of the cell wall, hence altering the electromechanical fluidity of the membrane (c) The potential of charged Pt species to complex to the receptor sites (where Cu is bound) at the terminus of CTR transporters, both from the extra-and intra-cellular side, and hence degrade the transport ability of the CTR (d) The likelihood that charged Pt species in the cytosol can chemically bond to the zwitterionic phosphatidylserine headgroup of the inner leaflet of the cell membrane, and so alter the membrane potential of the cell wall, hence altering the electromechanical fluidity of the membrane.

A new four component model of drug transport across cellular membranes has the following additive components: (1) a negative dependence on the desolvation from water, (2) a positive dependence on the lipophilicity, and smaller dependencies on (3) molecular volume and (4) dipole moment. [START_REF] Fong | Permeability of the blood-brain barrier: molecular mechanism of transport of drugs and physiologically important compounds[END_REF]]. The model was originally developed for drugs crossing the blood brain barrier, but the same principles apply to any cellular membrane system. These data are shown in Table 1 for the various drugs and their hydrolysis products. The outstanding feature in Table 1 is the very large differences in the solvation energies for the neutral parent Pt drugs, or the neutral hydrolysis products of these drugs, and the singly or doubly charged hydrolysis Pt species. Desolvation is effectively the reverse of solvation. While it is not known how complete the desolvation of a drug is required before it can begin to permeate through the hydrophobic flexible lipid bilayer after it negotiates entry past the tightly bound zwitterionic phosphatidyl headgroups and leaves the watery blood plasma environment. It is clear that highly charged species, with their large desolvation penalties, cannot easily enter the hydrophobic environment of the lipid bilayer. The charges on the Pt atoms of the neutral and charged Pt hydrolysis species are shown in Table 1, and are clearly related to the solvation energies. It is also obvious that charged Pt species could complex with the zwitterionic phosphatidyl headgroup, with the likely attachment points being the O of the phosphate group, or less likely the carbonyl group of the acyl group for phosphatidylcholine phospholipids, or the zwitterionic carboxy or phosphate groups of the phosphatidylserine headgroups. Similar complexes have been characterized between the phosphatidylcholine head group and cationic species. [START_REF] Fong | Permeability of the blood-brain barrier: molecular mechanism of transport of drugs and physiologically important compounds[END_REF] The lipophilicity of the various Pt species (calculated from the solvation energies in noctane) are shown, noting that these negative values are largely repulsive forces. The differences between the lipophilicity of the neutral Pt species and the charged Pt species quantifies the magnitude of the effect. The molecular volumes and dipole moments are also shown in Table 1, although these parameters are known to be much smaller factors in affecting cell membrane permeability than desolvation and lipophilicity.

The desolvation energies of the Pt drugs and their hydrolysis products are also very relevant to active transport by Ctr1 or OCAT transporters. Again substantial desolvation is required before the Pt drugs can be actively taken up by the transporter, and then transported across the cell membrane. In the case of hCtr1, a transmembrane pore is formed, Some Pt(IV) drugs are also included in Table 1 as a reference point for the Pt(II) drug species, since these prodrugs are well characterised as being largely passively transported across cell membranes, and have fairly high lipophilicities. [START_REF] Wilson | Synthesis, Characterization, and Cytotoxicity of Platinum(IV) Carbamate Complexes[END_REF][START_REF] Graf | Redox activation of metal-based prodrugs as a strategy for drug delivery[END_REF]] A comparative study of the Pt(IV) drug LA-12 with cisplatin in H1299 cells showed that the uptake of LA-12 was faster than cisplatin, (as well as a higher anti-tumour activity), [START_REF] Kvardova | The new Pt(IV) drug LA-12 shows stronger inhibitory effects Hsp90 function compared to cisplatin[END_REF]] a somewhat surprising result when comparing the desolvation energies, lipophilicity and larger molecular size values in Table 1. However this result is understandable because LA-12 does not hydrolyse in the blood serum as fast as cisplatin, so a larger concentration of LA-12 is available to penetrate the cell membrane. This explanation is consistent with the observed concentration effects seen with the uptake of cisplatin. It is noted that LA-12 has a higher antitumour activity than satraplatin and cisplatin when tested against a panel of 14 cell lines, and no cross resistance with cisplatin.

The molecular properties of several drugs commonly used in conjunction with Pt anticancer drugs in chemotherapy regimes, such as paclitaxel, docetaxel, gemcitabine, folinic acid, 5-fluorouracil are also given in Table 1. It can be seen their properties, particularly their reduction potentials, are very similar to the key Pt drugs, consistent with being in the same "therapeutic window" as the Pt drugs.

Reversal of resistance to Pt drugs

If resistance to Pt drugs is principally dominated in the first order by the decreased accumulation of the drug in the cell, then reversal of resistance should also be dominated by factors that increase cellular accumulation, ie uptake and efflux. This implies that some factor(s) that induced resistance can be reversed. It is hypothesised that these factors are dominantly related to Pt induced changes to the cell membranes, both extra-and intra-cellularly. Changes to cell membranes might include electromechanical fluidity and related membrane potential changes induced by a reversible interaction between charged Pt species and phosphatidylcholine phospholipid (PC) on the extracellular leaflet side) and phosphatidylserine phospholipid (PS) on the intracellular leaflet side. Other changes to the cell membrane might be the reversal of Pt-Ctr complexes that inhibit Ctr transport uptake of the drug.

A model to explain the literature findings of Pt drug resistance, and the known examples where such resistance has been reversed by certain drugs, is described in the box.

A model for analysing the acquired resistance of cancer cell caused by Pt drugs, and the reversal of the acquired resistance by other drugs complexes with the zwitterionic phosphatidylserine headgroup of the inner leaflet of the cell membrane, so altering the cell membrane potential, and reducing the permeability of drugs entering the cell. This is a second order effect, which can alter drug uptake, so affecting the first order uptake process, and is time and concentration dependent. 5. The known examples of the reversal of Pt resistance in some cell lines by some drugs can then occur if these drugs can complex with the membrane bound Pt species and allow the cell membrane to return to its normal structure and function.

6. Where the active transporters CTr1 and OCAT have been shown to operate in certain cell lines, the uptake of the drug before interacting with the protein transporters is energetically favoured for drugs with smaller desolvation penalties. 7. The known examples of functional degradation of the hCtr1 transporter by Pt species can occur if the charged Pt hydrolysis species can complex to the terminals of trans-membrane pore of the transporters, so degrading the function of the transporter. 8. Where reversal of the degradation of the hCtr1 transporter is known to be caused by certain drugs, this reversal can be attributed to the de-complexing interaction of the reversing drugs with the hCtr1-Pt complex, thus returning the hCtr1 transporter to its normal state and function.

Figure 1 also shows a schematic of how charged Pt species can complex with the intracellular phosphatidylserine headgroup of the inner leaflet of the cell membrane and the intracellular terminus of the hCtr1 transporter. Figure 2 show the likely structure of the Pt complex with the carboxy group of the phosphatidylserine headgroup. The various drugs which are known to reverse Pt resistance some of which are given in section 3 of the review section above, are postulated to be able to complex with charged Pt-PS complexed species so "disengaging" (or "decomplexing" by shifting the equilibrium between the free and complexed species) the charged Pt species from the PS cell membrane components. Similarly, these species could also disengage the Pt from the terminus ends of the hCtr1 transporters. It is also conceivable that charged Pt hydrolysis species could complex with the extracellular phosphatidylcholine headgroup, but this effect should show up in early cellular accumulation, and not have a time dependency which seems common in induced Pt resistance, and the higher extracellular Cl -ion concentration in blood plasma compared to the much lower intracellular Cl -concentration means that the rate of hydrolysis in the blood plasma is lower compared to that in the cytosol. The zwitterionic carboxy group of PS has a strong bidentate chelating ability, stronger than a phosphate O-Pt binding ability (Figure 2). Hence Pt-PS complexation is favoured over Pt-PC complexation.

As noted above in the section 4 of the review section, a cisplatin-phosphatidylserine complex has been detected in permeabilized and lysed human erythrocytes and tumour cell lines. When glutathione (a strong intracellular reducing agent) was present, there was a reduction in complex formation. The active species was shown to be the {(H 3 N) 2 Pt(H 2 O) 2 } ++ species. The serine head group of 1-palmitoyl-2-oleoyl phosphatidylserine bilayers binds to the aquated form of cisplatin, (Figure 2) followed by another cisplatin-phosphatidyl complex formation, probably via the oxygen atoms of the phosphate moiety. (Figure 1)

It has been shown previously that cisplatin complexes with PS model membranes and the rate is fastest at pH 6 and with no Cl -present, and PS can be regenerated by glutathione. The complexation does not occur with other membrane phospholipids. [START_REF] Speelmans | Cisplatin complexes with phosphatidylserine in membranes[END_REF]] However another study showed that the Pt-PS complex only existed in permeabilized human tumour cells, and that the complex could only have a transient lifetime, and was probably not involved in cancer cell cytotoxicity. [START_REF] Burger | Drug transporters of platinum-based anticancer agents and their clinical significance[END_REF]] It should be noted that these studies did not look at Pt resistant cells. In this study, the molecular properties of the complexation of cisplatin with PS have been examined where (H 3 N) 2 Pt ++ (H 2 O) 2 is first complexed with the carboxy group of PS then followed by a second complexation with the O atom of the phosphate group, consistent with the structural studies of these complexes. [START_REF] Speelmans | Cisplatin complexes with phosphatidylserine in membranes[END_REF][START_REF] Jensen | Anticancer cisplatin interactions with bilayers of total lipid extract from pig brain[END_REF], 2010] The first complex is thought to be that shown in Figure 2, designated {Pt-PS} coo . The second complex adds a (H 3 N) 2 Pt + (H 2 O) to the O of the P-O of the phosphate group as well, designated {2Pt-PS}. The formal molecular charge on the headgroup, the atomic charge (au) on Pt, and dipole moment (D) for these two complexes in water are 1, 2.587, 38.6D and 1, 2.813 and 50.8D respectively, compared to the uncomplexed PS values of which has a formal charge -1 on the headgroup, and dipole of 46.5D. These data suggest that the first complexation reduces the overall dipole compared to the free PS, while the second complexation increases the dipole moment. It appears that the Pt complexation to the phosphate O atom is most likely to affect the dipole potential of the membrane, particularly the lipophilic bilayer component.

Previously it has been shown that cationic species can interact with phospholipids, affecting the dipole of the phospholipid, and so altering the membrane potential of the cell membrane, which induces electromechanical changes in the cell membrane, thereby affecting membrane permeability. [START_REF] Fong | Permeability of the blood-brain barrier: molecular mechanism of transport of drugs and physiologically important compounds[END_REF]] Another resistance mechanism that could come into play is a steric repulsive effect where other free charged Pt species in the cytosol are repelled by the complexed Pt groups on the intracellular membrane surface. Alternatively, the formation of Pt-PS complexes on the inner leaflet of the cell membrane may act to deactivate the action of flippase or scramblase that trigger the flip of the direction of PS to the extracellular side, so triggering macrophages that usually engulf apoptotic cells.

The EAs for the {Pt-PS} coo {2Pt-PS}complexes are 1.6 and 1.9 eV respectively, similar to gluthathione (1.7 eV) which is known to reverse the complexation of Pt to PS. It can be predicted that drugs with suitable structures which have EAs in this region could reverse the Pt complexation to PS, and so potentially reverse Pt resistance in cancer cells. These drugs would need to be able to penetrate the cell membrane first, so consideration of their desolvation and lipophilicity properties would also need to be considered.

Similar arguments apply to induced Pt resistance where the transporter is a Ctr1 transporter. It has been shown in studies of carboplatin and nedaplatin that a maximum of two Pt atoms can bind to each monomer unit of the end terminus of hCtr1, via methionine residues to form ring-opened monofunctional adducts. [START_REF] Wang | Activation of carboplatin and nedaplatin by the N-terminus of human copper transporter 1 (hCTR1)[END_REF]] It is known that the hCtr1 structure has a trans-membrane pore with receptor sites (for Cu or other suitable ions) at the terminii. [START_REF] Feo | Three-dimensional structure of the human copper transporter hCTR1[END_REF] Copper chelators, such as glutathione, are known to increase cisplatin accumulation and increase cytotoxicity, observations attributed to changes to Ctr1 processes in Pt resistant cells. [START_REF] Ishida | Enhancing tumor-specific uptake of the anticancer drug cisplatin with a copper chelator[END_REF][START_REF] Holzer | The internalization and degradation of human copper transporter following cisplatin exposure[END_REF][START_REF] Spreckelmeyer | Cellular Transport Mechanisms of Cytotoxic Metallodrugs: An Overview beyond Cisplatin[END_REF] The molecular indicators for potentially active drugs that reverse down-regulation of hCtr1 are copper chelators with an EA similar to glutathione (1.7 eV), and with suitable cell membrane permeability properties. For example the copper chelator triethylenetetramine, TETA (EA 1.95 eV) in combination with carboplatin is a known to be effective in treating some patients with high grade Pt resistant epithelial ovarian cancers. [START_REF] Spreckelmeyer | Cellular Transport Mechanisms of Cytotoxic Metallodrugs: An Overview beyond Cisplatin[END_REF][START_REF] Fu | Overcoming Platinum Resistance through the Use of a Copper-Lowering Agent[END_REF] The molecular indicator of the resistance reversing ability of a drug is the reduction potential (as measured by the EA in water) compared to the reactivity of the Pt-PS complexes, and how well such drugs can themselves permeate through the cell membrane to react with the Pt-PS complexes. The reversal of several cisplatinresistant ovarian cancer cell lines found the following order of effectiveness: colchicine> genistein> triethylenetetramine (TETA)> rapamycin ≥ buthionine sulfoximine (BSO), which was correlated with the cellular accumulation of cisplatin. The calculated EAs in water are 2.2, 2.2, 1.95, 3.0 and 0.7 (or 0.2 for the zwitterionic BSO) eV respectively, and the desolvation energies are 20.6, 21.2, 17.9, 49.2 and 31 (or 66.6 for the zwitterionic BSO) kcal/mol respectively. Their lipophilicities are -11.6, -11.3, -5.6, -28.5, and -10.5 (or -22.2 for the zwitterionic BSO) kcal/mol respectively. As expected the large highly polar rapamycin and zwitterionic BSO have low lipophilicities. These EA data appear to reflect the order of effectiveness of the reversing drugs, and the desolvation energies and lipophilicities which are measures of their ability to permeate the cell membrane, indicate that these drugs have roughly comparable abilities to cross the cell membrane and then interact with Pt-PS complexes on the inner leaflet. The exception is rapamycin which is a very large molecular and correspondingly has a much higher desolvation penalty and lower lipophilicity affecting its cellular permeability compared to the other drugs. It is likely rapamycin needs an active transporter to cross the cell membrane. The EA of glutathione is 1.7 eV by comparison. Other resistance reversing drugs examined are JTV cation, lamivudin, SGI-110 anion, and decitabine which have EA values of 1.5, 1.8, 2.5, and 2.0 eV respectively, desolvation energies of 63.9, 26.4, 132.5, and 30.5 kcal/mol respectively, and lipophilicities of -37.9, -11.7, -46.7 and -13.9 kcal/mol respectively. The neutral drugs lamivudin and decitabine are predicted to have the greater reversing effectiveness.

Experimental methods

All calculations were carried out using the Gaussian 09 package on optimised structures. Electrostatic potential at nuclei were calculated using the CHELPG method in Gaussian 09. The atomic charges produced by CHELPG are not strongly dependant on basis set selection. Using the B3LYP level of theory, calculated atomic charges were almost invariant amongst the basis sets 6-31G(d), 6.311(d,p), 6-311+(2d,2p), 6-311G++(3df,3dp). Errors between calculated and experimental dipole moments were 3%. [Martin 2005, Kubelka] All calculations were at the B3LYP/6-311 + G**(6d, 7f) level of theory for all atoms except for Pt where the relativistic ECP SDD Stuttgart-Dresden basis set for transition metals was used. The atomic radii used for neutral Pt(II) and charged/ionic Pt(II) species (4 coordinated square planar configuration) in the CHELPG calculations were 1.75 and 0.74 A respectively, and for neutral 6 coordinated octahedral Pt(IV) complexes, a radius of 1.75 A was used.

(http://www.webelements.com/platinum/atom_sizes.html).

Solvent calculations used optimised gas state geometries. Generally good agreement was found with published X-ray structures for similar Pt complexes. [START_REF] Adams | New classes of bridging and chelating ligand motifs emphasizing: ruthenium(II) molecular squares, ruthenium(II) diphosphino carborane complexes, and acceptor PCP complexes of platinum(II), iridium(I/III), and ruthenium[END_REF][START_REF] Bruck | The crystal structures and absolute configurations of the anti-tumour complexes Pt(oxalato)(1R,2R-cyclohexanediamine) and Pt(malonato)(1R,2R-cyclohexanediamine)[END_REF][START_REF] Beagley | The crystal and molecular structure of cis-diammine-1,1cyclobutanedicarboxoplatinum(II) [cis-Pt(NH 3 ) 2 CBDCA]. Dynamic puckering of the cyclobutane ring[END_REF][START_REF] Beagley | The crystal and molecular structure of cis-diammine-1,1cyclobutanedicarboxoplatinum(II) [cis-Pt(NH 3 ) 2 CBDCA]. Dynamic puckering of the cyclobutane ring[END_REF][START_REF] Neidle | The structure of the anti-tumour complex cis-(diammino)(1,1-cyclobutanedicarboxylato)-Pt(II)[END_REF][START_REF] Dodoff | A DFT/ECP-Small Basis Set Modelling of Cisplatin: Molecular Structure and Vibrational Spectrum[END_REF][START_REF] Tyagi | Structural aspects of the anti-cancer drug oxaliplatin: A combined theoretical and experimental study[END_REF][START_REF] Galanski | Synthesis, crystal structure and cytotoxicity of new oxaliplatin analogues indicating that improvement of anticancer activity is still possible[END_REF][START_REF] Lippert | Crystal structures of trans-dichloroammine(1methylcytosine-N3)platinum(II) hemihydrate, [PtCl2(NH3)(C5H7N3)].1/2H2O, and trans-diamminebis(1-methylcytosine-N3)platinum(II) dinitrate. Evidence for the unexpected lability of ammonia in a cis-diammineplatinum(II) complex[END_REF], Senguptal 2009[START_REF] Varbanov | Theoretical Investigations and Density Functional Theory Based Quantitative Structure-Activity Relationships Model for Novel Cytotoxic Platinum(IV) Complexes[END_REF], Perez 2000] Some examples were studied using optimised structures in water, and although small differences were observed, these did not change the various trends (eg. solvation energies for neutral versus charged Pt species) which are central to the results from this study. It is noted that very high accuracy for each calculation is not the object of this computational study, rather the method relies on comparative differences between different species and various trends amongst different classes of species.

Ionization energies (IE) and electron affinities (EA) were calculated as vertical values in water, where the optimised configuration in water was used to calculate the excited states. Some adiabatic IE and EA values were calculated to see if any solvent effects were operating in the excited states, by re-optimising the excited state structures in water. There were small differences indicating some solvent effects in the excited state. To test the accuracy of the computational method, Me 2 Pt(PMe 3 ) 3 and Me 2 Pt(NMe 2 CH 2 CH 2 NMe 3 ) had calculated gas IE of 7.3 and 7.2eV which can be compared with the NIST literature experimental values 7.2eV and 7.0eV, indicating the computational model gives accurate results.

The accuracy of the chosen solvation energy model has been tested by comparing the differences in solvation energies in water between cis-and trans-(H 2 O) 2 PtCl 2 which is -1.0 kcal/mol compared to the experimental value of -0.1 kcal/mol [START_REF] Hush | A quantum chemical computational study of the relative stabilities of cis-and trans-platinum dichloride in aqueous solution[END_REF], and within the mean unsigned error of 0.6 -1.0 kcal/mol in the solvation free energies of tested neutrals, and 4 kcal/mol on average for ions for the SMD solvent method [START_REF] Marenich | Universal Solvation Model Based on Solute Electron Density and on a Continuum Model of the Solvent Defined by the Bulk Dielectric Constant and Atomic Surface Tensions[END_REF]. It has been found that the B3LYP / 6.31G+* combination gives reasonably accurate PCM and SMD solvation energies for some highly polar polyfunctional molecules, which are not further improved using higher level basis sets [START_REF] Rayne | Accuracy of computational solvation free energies for neutral and ionic compounds: Dependence on level of theory and solvent model[END_REF]]. Adding diffuse functions to the 6-311 + G** basis set had no significant effect on the solvation energies with a difference of less than 1% observed, which is within the literature error range for the IEFPCM/SMD solvent model.

Conclusions

The consensus of literature studies indicate that decreased cellular accumulation, mainly from reduced uptake, not increased efflux, is a dominant rate determining factor resulting in induced cisplatin resistance. It is shown in this study that the time dependant resistance to Pt therapy is likely to be a dominated of structural modifications of the cell membrane by: (a) complexing of charged hydrolysed Pt species in the cytosol with the phosphatidylserine (PS) headgroup of the intracellular leaflet, giving a Pt-PS complex or (b) complexing of a charged Pt species in the cytosol with the intracellular end terminus of the hCTr1 transporter (Pt-hCtr1). Both these complexes are thought to electrodynamically alter the cell membrane potential and the electromechanical properties of the lipid bilayer, and/or alternately, electrostericly hinder the approach of further Pt species from the cytosol. The overall effect is that drug species trying to cross the cell membrane from the extracellular side are prevented from entering the membrane.

It is shown that the reversal of induced Pt resistance by some drugs is likely to be due to a de-complexation of the Pt moiety of the Pt-PS or Pt-hCtr1 complex by these drugs, thereby reverting the cell membrane to its normal function. Molecular reactivity criteria that can predict the efficacy of de-complexing ability of potential reversing drugs have been identified.

Figure 1 .Figure 2 .

 12 Figure 1. Schematic representation of the extracellular zwitterionic head group of the phosphatidylcholine component of the cell membrane, and the complexation of 2 charged Pt species to the intracellular zwitterionic head group of the phosphatidylserine component of the inner leaflet of the membrane bilayer via the carboxy and phosphate groups. Lower part of the figure schematic shows the transmembrane pore of the hCtr1 transporter with a complexed Pt + species attached to the intracellular terminus.

  ) 2 Pt + CBDCH(OH) has one arm of the bidentate cyclobutane-1,1-dicarboxylate ligand free with the free carboxylate as an acid and a hydroxyl ligand in its place. Oxaliplatin is (cis-[(1R,2R)-1,2-cyclohexanediamine-N,N*]oxalato(2-)-O,O*] platinum; DACH is 1,2 cyclohexanediamine. DACHPtOxalateH(OH) has one arm of the bidentate oxalate ligand free with the free carboxalate as an acid and a hydroxyl ligand in its place. DACHPt + Oxalate -(OH 2 ) ) has one arm of the bidentate oxalate ligand free with the free carboxalate as an anion and a water ligand in its place. Nedaplatin is diammine[(hydroxy-κO)acetato(2-)-κO]platinum, (NH 3 )Pt(HydroxyAcetato -)(OH) has one arm of the bidentate hydroxyacetate ligand free with the free carboxalate as an anion and a hydroxyl ligand in its place. Lobaplatin is 1,2-diammino-methy lcyclobutane-platinum (II) lactate or BAMCBPtLactate, and BAMCBPt(OH)LactateH has one arm of the bidentate BAMCB ligand free with the free carboxalate as an acid and a hydroxyl ligand in its place. Heptaplatin is4a,5b)]]-[2-(1-Methylethyl)-1,3-dioxolane-4,5-dimethanamine-N,N'][propanedioato(2-)-O,O']-platinum or MDDMAPtMalonate, and MDDMAPt + MalonateH(OH) has one arm of the MDDMAP ligand free with the free carboxalate as an acid and a hydroxyl ligand in its place Picoplatin is (2-methylpyridine)ammineplatinumdichloride

  1. Solvated Pt drug in blood plasma requires substantial desolvation before passive diffusion or active transport by protein carriers (CTRL or OCAT transporters) through cell membrane commences. Neutral Pt species have lower desolvation penalties than charged species. The rate of permeation through the membrane also depends on drug lipophilicity, molecular volume and dipole moment. This desolvation step is the first order rate determining step for anti-cancer efficacy. 2. The Pt drug enters the cytosol and begins the apoptotic process, including formation of charged species such as (NH 3 ) 2 Pt + (H 2 O)Cl or (NH 3 ) 2 Pt ++ (H 2 O) 2 from cisplatin for example, which can then form Pt-DNA adducts, triggering apoptosis. 3. The efflux of Pt species from cytoplasm, or other signalling mechanisms that counter apoptosis by Pt drugs, including DNA repair mechanisms etc can occur. These are second order effects, which can change drug efficacy, but can only occur after the Pt drug enters the cell by passing through the cell membrane . 4. Intracellular interactions between charged Pt hydrolysis products in the cytosol, (such as (NH 3 ) 2 Pt + (H 2 O)Cl or (NH 3 ) 2 Pt ++ (H 2 O) 2 from cisplatin) can form

Table 1 . Solvation energies, molecular volumes, dipole moments, atomic charge on Pt, lipophilicity, ionization energies and electron affinities for various Pt drug species and some anti-cancer drugs used in combination with Pt anti-cancer drugs Species in water

 1 All ionization properties are vertical (same optimised geometry in gas and water) unless shown as adiabatic (where the ionized geometry was optimised in the solvent, resulting in small changes to the geometry). Vertical (and adiabatic) ionization energies and electron affinities are in eV. Solvation energies are in kcal/mol. Desolvation energies are essentially the reverse of solvation energies (positive values). The CDS solvation values are the nonelectrostatic components including cavity formation terms. Molecular volumes are in cm 3 /mol. Dipole moments µ are in D. The atomic charges on Pt are in a.u. Lipophilicity is the solvation energy in n-octane, essentially a repulsive effect for polar and charged molecules. Carboplatin =(NH 3 ) 2 Pt CBDC where CBDC is cyclobutane-1,1-dicarboxylate, (NH 3 ) 2 Pt + CBDC -(OH) has one arm of the bidentate cyclobutane-1,1-dicarboxylate ligand free with the free carboxylate as an anion and a hydroxyl ligand in its place, and (NH 3

		Solvatn	CDS Molec	Dipole	Pt	Lipop-	(IE -	EA
		Energy		Volume	µ	Charge	hilicity	EA)
	(NH 3 ) 2 PtCl 2 Cisplatin	-33.1	4.4	76.2	16.8	0.15	-14.3	4.9	1.4
	(Adiabatic)	-29.0	4.6	109.8	17.1	0.134	-11.9	3.5	2.8
	(NH 3 ) 2 PtCl(OH) (NH 3 ) 2 Pt + Cl(OH 2 )	-40 -87.3	4.8 5.3	78.6 85.9	16.3 14.8	0.33 -13.5 2.484 -40.3	5.0 4.6	1.0 2.1
	(Adiabatic)	-91.2	5.5	83.6	15.0	2.363	-33.0	3.3	3.3
	(NH 3 ) 2 Pt ++ (OH 2 ) 2	-222.3	6.4	77.7	5.2	2.726 -101.7	4.5	2.4
	(NH 3 ) 2 Pt CBDC Carboplatin	-33.1	8.6	130.5	18.5	0.073	-13.4	4.9	1.4
	(Adiabatic)	-39.5	9.8	162.1	22.4	0.159	-13.4	3.7	2.4
	(NH 3 ) 2 Pt CBDCH(OH) (NH 3 ) 2 Pt + CBDC -(OH 2 ) (NH 3 ) 2 Pt + CBDCH(OH 2 )	-37.5 -91.1 -90.6	8.7 8.7 8.9	158.1 191.3 150.3	16.8 34.2 14.5	0.141 -13.0 2.598 -28.7 2.514 -36.9	4.2 5.0 4.0	1.6 1.8 2.6
	Carboplatin Dimer	-92.4 10.2	243.5	8.6	0.107 -31.2	2.0	2.1
	DACHPtOxalate Oxaliplatin	-45.8	6.9	144.6	24.6	0.171 -20.1	3.7	2.8
	DACHPtOxalateH(OH)	-47.1	7.2	178.9	17.8	0.426 -17.2	2.4	3.4
	DACHPt + Oxalate -(OH 2 ) DACHPt ++ (OH 2 ) 2	-82.3 -207	7.8 5.9	158.3 123.9	29.5 4.6	2.424 -28.0 2.614 -95.0	4.2 4.5	2.2 2.3
	DACHPt(OH) 2	-46.4	4.5	147.9	15.3	0.298 -16.4	2.4	2.3
	DACHPtCl 2	-39.8	4.2	147.3	20.8	0.142 -20.4	2.4	2.5
	DACHPt(OH)Cl	-48.3	4.3	177.9	20.4	0.231 -19.8	2.4	2.4
	DACHPt + (H 2 O)Cl DACHPt ++ (OH 2 ) 2	-81.6 -212.4	5.0 5.8	157.6 115.4	12.5 2.8	2.242 -38.5 2.682 -97.3	2.1 4.5	2.9 2.3
	DACHPtCBDC	-52.6	7.3	186	21.9	0.287 -22.3	1.3	2.5
	DACHPtCBDCH(OH)	-50.9	8.0	193.8	9.6	0.324 -19.2	1.3	2.3
	DACHPt + CBDC -(OH 2 )	-83.1	8.5	245.1	30.5	2.696 -31.5	1.3	2.8
	(NH 3 ) 2 Pt(HydroxyAcetato)	-46.3	6.5	79.2	17.2	0.205 -15.3	1.3	2.8
	Nedaplatin
	(NH 3 )Pt + (HydroxyAcetato -)(OH 2 ) (NH 3 )Pt(HydroxyAcetato -)(OH)	-98.7 -101.1	8.1 7.4	91.4 96.2	36.1 35.2	2.195 -25.8 0.251 -10.2	1.9 1.8	1.8 1.6
	(NH 3 )Pt(HydroxyAcetatoH)(OH)	-55.3	7.8	105.8	21.4	0.293 -17.0	1.9	2.1
	(NH 3 )(MePyrid)PtCl 2 Picoplatin (NH 3 )(MePyrid)Pt + Cl(OH 2 )	-26.3 -65	5.7 6.6	141.3 131.8	17.8 9.8	0.183 -15.9 2.101 -33.5	4.5 4.5	1.8 2.1
	(NH 3 )(MePyrid)PtCl(OH) (NH 3 )(MePyrid)Pt ++ (OH 2 ) 2	-30.0 -196.5	6.0 7.5	124.1 135.1	15.9 6.9	0.305 -14.2 2.31 -92.5	4.1 4.4	1.8 2.4
	BAMCBPtLactate Lobaplatin	-48.1	6.2	181.2	16.3	0.157 -19.3	2.8	1.6
	BAMCBPt + (OH 2 )Lactate -BAMCBPt(OH)Lactate -	-95.2 -125.1	8.3 7.6	199.6 204.6	34.5 37.8	2.951 0.252 -37.7 -25.4	1.9 2.0	1.7 1.5
	BAMCBPt(OH)LactateH	-56.5	7.9	178.8	17.3	0.277 -20.9	2.9	1.5
	BAMCBPt ++ (OH 2 ) 2	-215.2	6	116.3	8.9	2.734 -98.6	2.1	2.5
	MDDMAPtMalonate						-19.6	3.0	2.6
	Heptaplatin	-47.2	8.8	204.1	17.3	0.236
	MDDMAPt + Malonate -(OH 2 ) MDDMAPtMalonate -(OH)	-100 -134.7	9.4 9.3	236.23 251.4	34.4 45.9	2.66 -32.1 0.202 -43.3	3.1 3.1	2.6 2.5
	MDDMAPtMalonateH(OH)	-60.0	9.6	205.5	16.1	0.186 -20.5	3.0	2.7
	MDDMAPt ++ (OH 2 ) 2	-216.1	7.5	181.3	13	2.482 -95.6	3.1	2.7
	(NH 3 ) 2 PtCl 2 Transplatin	-16.8	4.5	95.9	0	0.112	-9.0	4.6	1.5
	(Adiabatic)	-18.4	4.6	91.0	0	0.052	-9.7	3.3	2.8
	(NH 3 ) 2 PtCl(OH) Trans (NH 3 ) 2 Pt + Cl(OH 2 ) Trans (NH 3 ) 2 Pt ++ (OH 2 ) 2 Trans	-23.2 -77.4 -222.3	4.8 5.4 6.5	90.1 62.4 79.9	2.5 10.2 4.1	0.217 2.973 -35.4 -8.5 2.856 -96.7	4.7 4.6 3.9	1.1 2.2 3.1
	DACHPtCl 4 Tetraplatin Pt(IV)	-31.5	2.8	147	17.5	0.308 -16.2	3.7	3.7
	(H 3 N) 2 PtCl 4 Cis Pt(IV)	-33.6	4.6	135.1	15.6	0.457 -15.0	4.5	3.2
	Satraplatin Pt(IV)	-52	7.7	200.1	23.6	0.553 -19.4	3.7	3.1
	LA-12 Pt(IV)	-46.4	7.9	236.6	23.1	0.475 -19.7	2.7	3.2
	Asplatin Pt(IV)	-45.3	9.3	225.7	18.9	1.014 -18.4	4.1	2.6
	Glutathione	-48.5	4	175.6	7.8		-14.5	4.9	1.7
	Paclitaxel	-60.5 21.4	651.2	17.1		-31.1	3.6	2.0
	Docetaxel	-50.1 18.9	587.3	8.8		-25.8	4.3	1.6
	Doxorubicin Ion	-107.7	12	403.8	28.8		-47.2	3.4	2.8
	Doxorubicin	-45.8	11.8	316.4	7.0		-19.3	2.6	3.0
	Gemcitabine	-30.2	3.4	159.7	7.3		-11.2	4.0	1.6
	Folinic Acid	-61.1	6.9	282.9	17.5		-25.5	3.6	1.8
	Folinic Acid Dianion	-242.6	6.9	276.4	72.3		-92.4	3.8	1.5