Mecanismos de seguridad

Osmium tosyl (TsDPEN) 2 (p-cymene), 7 (biphenyl) and 8 (m-terphenyl),and iridium complexes 12-14 (Cp*, CpxPh and CpxBip) showed broad activity across the majority of cell lines investigated (Table 4.2), and in particular, the complexes showed promise in cancers of the reproductive organs, specifically of the breast, ovary and prostate (MCF7, A2780 and PC3, respectively). This trend could be linked to the fact that cancers of these tissues are typically hormone-responsive, and the mechanism of

0 30 60 90 0.0 1.0 2.0 ng m etal × 10 6cel ls Log P DPEN-X 0 10 20 0.0 0.2 0.4 0.6 ng m etal × 10 6cel ls Log P BMEN-X

Ruthenium conjugates of the peptide hormone somatostatin have been designed to increase cancer cell selectivity by generation of 1O2 upon light irradiation, utilising the

overexpression of somatostatin receptors in various cancer cell types relative to healthy cells.40 Ruthenocene complexes derived from the anticancer drug Tamoxifen were found to bind to estrogen receptor alpha (up to 85% binding, compared to 39% for hydroxytamoxifen) but did not exhibit potency towards ER-α-negative cells.41

Os and Ir sulfonamide complexes displayed particularly low antiproliferative activities (in some cases, >50 μM) in gastrointestinal cell lines (OE19 and HCT116), suggesting selectivity for cells of particular tissues – an important property in the design of new anti-cancer agents. In addition to specific tumour selectivity, efficacies of the complexes were investigated between cancer cells (A2780 and A549) and two non- cancerous primary cell lines, ovarian (HOF) and lung fibroblasts (MRC5) as a model for healthy cells. Ovarian fibroblasts were chosen for comparison to A2780 ovarian cancer cells, in which the complexes were highly active. In this case, Os complexes 2

(p-cymene), 7 (biphenyl), and 8 (m-terphenyl) displayed good selectivity for cancer cells over healthy cells. Many other osmium complexes have displayed similar positive selectivity towards cancerous cells.18, 42 Iridium compounds 12-14 were not as selective, achieving only ca. 2× selectivity. The selectivity of iridium complexes for cancer cells over normal cells is highly dependent on the nature of the bidentate ligand.33, 43 While cyclometalated iridium bipyridine complexes were found to possess

ca. 11-fold selectivity for cancer cells over healthy cells, Ir(III)-curcumin complexes lack the selectivity found in structurally similar Ru(II)-curcumin derivatives.44 In this study, both Ir(III) and Os(II) sulfonamide complexes were poorly selective between cancerous (A549) and healthy (MRC5) lung cells, largely due to the low activity in the cancerous lung cell line.

Table 4.5. Resistance factors (A2780cis/A2780) calculated for complexes of Os (2, 7-8) and Ir (12-14).

Complex 2 7 8 12 13 14

Metal Os Os Os Ir Ir Ir

Arene p-cym bip m-terp Cp* CpxPh _CpxBip A2780 15.5 ± 0.5 6.5 ± 0.3 4.4 ± 0.3 20.9 ± 0.7 14 ± 2 10.2 ± 0.6 A2780cis 21.6 ± 0.2 25.9 ± 0.7 10.4 ± 0.5 17 ± 2 18 ± 1 6.3 ± 0.8

R.F. [a] _{1.4 4.0 2.4 0.8 1.3 0.6}

[a] _{Resistance factor (R.F.) defined as the ratio of the IC}

50 determined in A2780cis / A2780.

Osmium(II) complexes 2 (p-cymene), 7 (biphenyl), and 8 (m-terphenyl) exhibited less potency towards cisplatin-resistant cells (A2780cis) compared to cisplatin-sensitive cells (resistance factor > 1; Table 4.5). Less cross-resistance was observed with iridium complexes 12-14 and in fact Ir complexes 12 and 14 were more potent in the resistant cell line (resistance factor = 0.8 and 0.6, respectively). This is in agreement with other iridium(III) half-sandwich complexes.10, 30 Importantly, all sulfonamide complexes of osmium and iridium investigated exhibited less cross resistance than cisplatin (resistance factor = 11.2). This is an important finding, since the treatment of drug-resistant tumours is a problem of clinical significance, as cisplatin-resistant cells have been shown to decrease drug transport, modify apoptotic pathways and develop drug detoxification mechanisms to maintain proliferative growth.45

In addition to comparing between different cancer cell types, antiproliferative activities were determined in 3 ovarian cancer cell lines: A2780, SW626 and SK-OV- 3. A2780 are a platinum-sensitive cell line, while SW626 and SK-OV-3 are increasingly platinum-insensitive cell lines (cisplatin: 1.2 µM, 15.7 µM and 16.8 µM, respectively). Similarly, the antiproliferative activities of both osmium sulfonamide (2, 7, 8) and iridium sulfonamide (12, 13, 14) complexes are in agreement with this trend, with complexes exhibiting highest potency in A2780 and lower potency in the

platinum-insensitive cell lines. Taken in combination with the reduced cellular metal accumulation in A2780cis (platinum-resistant) cells, a resistance mechanism is likely common to Pt drug exposure and treatment with Os / Ir complexes.

Comparing antiproliferative activities between parental HCT116 colorectal cancer cells and derived cell lines with genetically-engineered knockouts of key genes (P21

and the tumour-suppressor gene P53, in HCT116-p21-/- cells and HCT116-p53-/- cells, respectively) provides an indication of the gene’s involvement in the mechanism of action. If the gene is involved in the mechanism, the determined IC50 concentration

would be higher (less active) in the knock-out cell line. Conversely, the IC50

determined in a knock-out cell line would not be affected if the gene is not critical to the mechanism of action. For cisplatin, antiproliferative activities are decrease in both the p21 (9.2 µM) and p53 (36.7 µM) knock-out cell lines, relative to the parental line (5.2 µM). This trend is also conserved in both the osmium and iridium sulfonamide series of complexes, which all exhibited the highest relative activity in the parental cell line. In each case, these data suggest that it is likely that p21 and/or p53 are involved in the mechanism of action, reflected in the decreased antiproliferative activities determined in the knock-out cell lines.

Since complexes 2 and 12 have been shown to carry out the catalytic oxidation of 1,4- NADH to NAD+ under biologically-relevant conditions, it is likely that the complexes may interfere with cellular metabolic pathways, which require NADH as a hydride source for the reduction of substrates, for example, the conversion of pyruvate to lactate, which cancer cells utilise to respire (described by the Warburg Effect).46-48 However, perturbation of the NADH/NAD+ _{ratio is also known to induce the}

dependent cell death. While expression of the cyclin-dependent kinase inhibitor (CDK) p21 is known to be up-regulated by p53 in response to anticancer agents, its expression can also be regulated without functional p53.50 A structurally-similar osmium(II) half-sandwich complex [Os(η6-p-cymene)(4-(2-pyridylazo)-N,N- dimethylaniline)I]PF6 generates mitochondrial ROS, leading to the up-regulation of

both p53 and p21, implicating their involvement in the mechanism of action.18 _Since

p53 and p21 appear to also be involved in the mechanism of action of 2 (less activity the knock-out cell lines), which also produces ROS and superoxide in A2780 cancer cells, Os(II) arene complexes may share a common mechanism of action.

Overall, osmium(II) complexes typically exhibit higher antiproliferative activities than their Ir(III) counterparts, though at the expense of slightly increased cross- resistance against platinum-resistant cells. However, the accumulation of iridium in cells treated with Ir complexes 12-17 surpassed the levels of osmium internalised by cells treated with Os complexes 2-10. Since examples of both Os and Ir sulfonamide complexes have been shown to convert NADH to NAD+ under biologically relevant conditions, it may be possible that the mode of action is catalytic in cells. While both osmium and iridium complexes have been shown to be highly stable in biological media, the higher charge of Ir(III) compared to Os(II) may more readily promote catalyst deactivation by nucleophilic components in the cell, such as nucleophilic amino acid side-chains: cysteine (R-SH) serine (R-OH), histidine (R-NHR) and lysine (R-NH2).

While the nature of the metal centre appears to be crucial when comparing biological activities of Os and Ir complexes (both the antiproliferative activity of the complex and the extent of metal accumulation into the cell), exchange of the arene to afford neutral complexes (for example, Os-p-cymene for Ir-Cp*) does mean that specific

conclusions about the role of the metal cannot be made. Even if the directly analogous complexes, [Os(Cp*)(diamine)]- or [Ir(p-cymene)(diamine)]+ complexes could be synthesised, the anionic / cationic nature of the complexes would likely dramatically alter their properties; by increasing water solubility (and therefore would have a lower octanol / water partition coefficient, Log P) which may influence the antiproliferative activity towards cancer cells, cellular accumulation, and ability to carry out the catalytic oxidation of NADH.

Cellular accumulation in other cancer cell lines

The cellular metal accumulation was determined in seven cancer cell lines (Figure 4.6) treated with equipotent concentrations of either Os complex 2 or Ir complex 12. ICP- MS was used to detect xenobiotic elements in cell pellets digested in acidic solution. Despite the lower osmium cellular accumulation compared to iridium across the range of cell lines investigated (30 ng Os × 106 cells for complex 2 compared to 52 ng Ir × 106 cells for complex 12 in A2780), osmium complexes were typically more potent than their iridium counterparts (15.5 ± 0.5 μM for Os complex 2 compared to 20.9 ± 0.7 μM for Ir complex 12 in A2780). Structurally similar osmium half-sandwich compounds have been determined to accumulate in cancer cells at similar concentrations (20-60 ng Os × 106 cells) to complexes 2-10 investigated in this chapter (ranging from 5-30 ng Os × 106 cells, depending on the nature of the arene and sulfonamide ligand),51 while previous iridium arene complexes have been shown to accumulate rapidly in cells,33 similarly to finding using complex 12.

Across multiple cell lines treated equipotent concentrations of either complex 2 or 12, increased metal accumulation in treated cells correlated with increased drug potency (ranging from 2.32-32 ng Os × 106 cells for 2, and 6.2-74 ng Ir × 106 cells for complex

12, with corresponding IC50 values between 10.9-50 μM, and 14.6-50 μM

respectively). Particular cell lines may be more specialised for the internalisation of hydrophobic molecules, or conversely, efflux xenobiotic species more efficiently than other types of cell. Specifically, gastrointestinal (GI) epithelial cells are particularly adapted to circumvent toxin bio-accumulation,52 which may explain the low antiproliferative activities determined in OE19 (esophageal) and HCT116 (colorectal) cell lines. However, the cellular accumulation of anticancer compounds in GI cancers may be increased by functionalisation of molecules with substrates for nutrient transporters.52 Additionally, vitamin B12 is readily absorbed via receptor-mediated

endocytosis.53 B12 conjugates of nanoparticles and peptides have been explored to

increase GI tract drug adsorption.53, 54 A similar strategy could be explored for accumulation of future anticancer drugs, increasing oral bio-availability.

The metal accumulation studies in this thesis were normalised by cell number but did not account for cell volume, which varies greatly between cell lines, so an accurate in- cell concentration cannot be determined. Cell volume may be determined using flow cytometry, a Coulter counter,55 or more recently, by use of a confocal laser scanning microscope combined with 3D reconstruction software.56 Suspended microchannel resonators have been described which measure simultaneously the volume, density and mass of single cells,55 however this work goes beyond the scope of this thesis. Alternatively, single-cell ICP techniques could allow for the evaluation of metal accumulation in cells, providing data concerning the accumulation of metal in a population of cells, rather than whole-pellet digest.

Accumulation of Os and Ir in cisplatin-resistant cells (A2780cis) decreased to approximately ⅓ of the metal accumulation in the platinum-sensitive cell line (30 to 9.5 ng Os × 106 cells). Reduction in total metal accumulation has also been observed

with platinum drugs for various ovarian cancer cell lines, in many cases reduced by the same amount as observed for the sulfonamide complexes (for cisplatin, this is between 36-66% of the accumulation determined in platinum-sensitive cells, depending on the study and cell lines investigated).57-59 Platinum resistance has been shown to result in approximately one order of magnitude fewer platinum-DNA adducts forming.60 _{As well as decreased drug accumulation, resistance has also been}

associated with increased platinum drug efflux.32 More detailed studies of the mechanism(s) of drug uptake and efflux may therefore help to explain the apparent absence of a trend between total accumulation and drug hydrophobicity. For example, the P-glycoprotein efflux pump (a translocating ATPase) has a broad specificity for many hydrophobic drugs, which are known to include many anticancer agents,61 and cells from particular tissues may be better adapted for the accumulation of molecules.