In vivo anti-tumor activity of the organometallic ruthenium(ii)-arene complex [Ru(η6-p-cymene)Cl2(pta)] (RAPTA-C) in human ovarian and colorectal carcinomas

Based on the clinical success of platinum-based anti-cancer drugs such as cisplatin, carboplatin and oxaliplatin, a variety of other metal-based anti-cancer compounds are being investigated. In particular, a number of ruthenium-based compounds have been identified which exhibit unique biochemical properties and reduced toxicity profiles compared to the clinically used platinum-based drugs. We have developed a series of organometallic ruthenium(II)-arene complexes that were shown to exert antimetastatic activity with relatively minor activity on primary tumor growth. Here, we show that the prototype compound, [Ru(h-p-cymene)Cl2(pta)], where pta 1⁄4 1,3,5-triaza-7-phosphaadamantane (RAPTA-C), reduces the growth of primary tumors in preclinical models for ovarian and colorectal carcinomas. When administered daily at relatively low doses (0.2 mg kg ), RAPTA-C was shown to significantly reduce the growth of the A2780 ovarian carcinoma transplanted onto the chicken chorioallantoic membrane model. Similar activity was observed in LS174T colorectal carcinoma in athymic mice, albeit at a higher dose. In both models, a clear inhibition of microvessel density was observed, confirming the previously discovered anti-angiogenic mechanism of RAPTA-C. Biodistribution studies with radiolabeled (Ru) RAPTA-C indicate that the compound is rapidly cleared from the organs and the bloodstream through excretion by the kidneys. As such, RAPTA-C is a promising compound for translation to clinical evaluation.


Introduction
Cisplatin, carboplatin and oxaliplatin are widely used in the clinical treatment of a broad spectrum of cancers. 1 Despite the success of these platinum-based drugs, their use is limited due to signicant toxicity and both intrinsic and acquired drug resistance. 2 In light of these limitations, a large focus in current research has been directed towards the development of compounds that are based on metals other than platinum. 2,3In recent years, the prominence of ruthenium-based complexes has signicantly grown as they have been shown to possess antimetastatic properties and overcome both of these main limitations of platinum-based drugs in a number of animal models. 4,5Two ruthenium(III)-based compounds (Chart 1), namely KP1019 (indazolium trans-[tetrachlorobis(1H-indazole)ruthenate(III)]) and NAMI-A (imidazolium trans-[tetrachloro(dimethylsulfoxide)(1H-imidazole)ruthenate(III)]), have undergone phase I clinical evaluation.
Ruthenium(III) complexes, however, are prone to ligand exchange reactions in aqueous media/physiological buffer which hamper, to some extent, the rational design of such new compounds with relevant medicinal properties.For this reason, ruthenium(II)-arene compounds have attracted considerable attention in recent years following encouraging in vivo data on two prototypical compounds (Chart 2), [Ru(h 6 -p-cymene)Cl(en)], where en ¼ ethylenediamine (termed RAED-C) 6 and [Ru(h 6p-cymene)Cl 2 (pta)], where pta ¼ 1,3,5-triaza-7-phosphaadamantane (termed RAPTA-C). 7RAED-C was shown to have a moderate effect on reducing the growth of primary tumors whereas RAPTA-C has shown moderate effects on solid tumor metastases.Recently, these differences have been tentatively attributed to differences in the preferential binding site of each molecule to chromatin, with RAED-C binding to DNA sites and RAPTA-C to the histone core. 8he mechanisms by which ruthenium-based drugs exert their anticancer effects remain to be fully elucidated although, as mentioned above, chromatin binding appears to be an important mechanism.It has been proposed for ruthenium(III) complexes that their reduced toxicity to normal tissue may be attributed to the so-called "activation by reduction" mechanism, i.e. ruthenium(III) complexes act as prodrugs that can be reduced to active ruthenium(II) species in the hypoxic microenvironment of a tumor.Similar to KP1019 and NAMI-A, which are both based on a ruthenium(III) ion, the ruthenium(II) complex RAPTA-C also appears to be well tolerated in vivo showing considerably reduced side-effects compared to platinum-based drugs (as judged by the high doses that may be tolerated by animals).
RAPTA-C was recently shown to also exert a strong antiangiogenic effect, 9 similar to that of sorafenib, a clinically used anti-angiogenic small molecule drug. 10Moreover, there are similarities shared by RAPTA-C and NAMI-A, both of which show limited direct cytotoxic effects on cancer cells in vitro while exhibiting anti-metastatic behaviour in vivo.NAMI-A also has an anti-angiogenic component to its mechanism as demonstrated by its ability to induce apoptosis in a spontaneously transformed human endothelial cell line (ECV304) through the inhibition of MEK/ERK signalling. 11APTA-C presents particular interest when viewed as an antiangiogenic agent, as it has been shown to reduce the growth of lung metastases in CBA mice bearing the MCa breast carcinoma, 12 while many clinically used VEGF-targeted therapies have been shown to induce pro-metastatic phenotypes in treated tumors, such as the VEGF targeted tyrosine kinase inhibitor sunitinib. 13Consequently, RAPTA-C is an attractive compound for further pre-clinical development.Therefore, we decided to evaluate the effects of RAPTA-C in more detail on primary tumors of human carcinomas.The results presented here demonstrate that RAPTA-C possesses anticancer activity, in conjunction with an intrinsic anti-angiogenic mechanism on primary tumors.

Results and discussion
The effect of RAPTA-C on tumor growth inhibition was investigated in a human A2780 ovarian carcinoma implanted on the chicken chorioallantoic membrane (CAM) model.A2780 cells were inoculated on embryo development day (EDD) 7 and monitored for 11 days.Established and vascularized tumors were detected 3 days post implantation (EDD 10).Treatment with RAPTA-C was performed by i.v.injections for ve consecutive days (Fig. 1A).Tumors grew to an average size of approximately 160 mm 3 by EDD 18 when le untreated (Fig. 1C).Tumor growth was efficiently inhibited following treatment with RAPTA-C and on the nal experiment day, the growth of tumors was inhibited by about 75% (**p ¼ 0.0007) at a dose of 0.2 mg kg À1 per day (Fig. 1B).An inhibition of ca.45% was observed at a dose of 0.05 and 0.02 mg per kg per day, however, neither were statistically signicant.It is noteworthy that READ-C (10 mg kg À1 , i.p.) tested on tumor derived from the same cell line in athymic mice showed tumor growth inhibition of about 50% at 10-fold higher dose as compared to that used in our study, which resulted in the inhibition of tumor growth by 75%. 6The effect of RAPTA-C was, in fact, comparable to that of cisplatin in this study. 6he effect of RAPTA-C treatment on the process of angiogenesis was assessed by measuring the vessel density using immunohistochemical staining of the CAM tissue sections with the endothelial cell marker CD31. 14Staining of tumor sections for the nuclear proliferation marker Ki-67 was performed to determine intra-tumor cell viability. 15Tumors were resected on the last day of the experiment (EDD 18).The control samples contained viable tumor tissue with many blood vessels whereas the treated tissues contained large areas of non-proliferating tumor cells (Fig. 1D and F, **p ¼ 0.0099).Additionally, quan-tication of the microvessel density in the sparse viable tissue remaining revealed signicantly reduced microvessel density (0.2 mg kg À1 per day), as compared to the control tumors (Fig. 1E).These results conrm the in vivo antitumor activity of RAPTA-C was achieved in parallel to an anti-angiogenic effect.
RAPTA-C was also evaluated in Swiss athymic mice inoculated on the right ank with human LS174T colorectal adenocarcinoma cells.When tumor size reached approximately 5-6 mm in diameter (day 5 aer inoculation), the mice were randomized into groups and treated once daily with RAPTA-C (i.p. 10, 40 or 100 mg kg À1 ) for 11 days or with PBS only (containing 0.9% DMSO) as a control.Spectroscopic characterization of RAPTA-C in DMSO solutions with or without albumin is presented in Fig. S1 (ESI †), showing that the compound is stable under the treatment conditions, as expected from previous studies. 16Tumor growth was monitored during treatment and, aer this period, mice were sacriced and tumors were resected.Tumor growth of RAPTA-C treated tumors (i.p. 100 mg kg À1 per day) was inhibited (**p ¼ 0.008) by approximately 50% compared to the control group (Fig. 2A) on the last experiment day.Lower doses (10 and 40 mg kg À1 per day) did not lead to signicant tumor growth inhibition.We did not observe toxicity during the course of treatment.The average body weight of animals in the treatment groups did not change signicantly over the course of treatment (Fig. 2B).
KP1019 has been evaluated in various colorectal tumor models and was found to have a strong activity in a chemoresistant colon carcinoma model (MAC15A) and was subsequently tested in an autochthonous colorectal tumor model in rats, which more accurately reects both the histological appearance and malignant progression of human disease. 17In this model, KP1019 was highly effective, resulting in approximately 90% tumor growth inhibition.It was more effective that cisplatin, which was completely ineffective, and 5-uorouracil treatment.5-Fluorouracil therapy, which is a standard treatment for colorectal carcinoma and is the most effective single agent therapy, inhibited tumor growth by 60% as compared to the control tumors. 18,19Treatment of CBA mice with MCa mammary carcinoma with KP1019 administered at 40 mg per kg per day from day 6 to day 11 post tumor implantation (6 daily administrations) resulted in a reduction of primary tumor weight by 67% relative to the tumor mass in the control animals. 20reated tumor tissue sections were stained for endothelial cells (with CD31 staining in brown) and smooth muscle actin (with anti-SMA in blue), indicating the presence of mature blood vessels (Fig. 2C and D).Tumor size measurements appeared to be an underestimation of the RAPTA-C activity, as treated tumors showed large areas of necrosis.In addition, around the remaining blood vessels small patches of Ki-67 negative tumor cells were observed, indicating that that tumor cells are in a quiescent state (Fig. S2, ESI †).Quiescence describes reversible growth or proliferation arrest and is considered to represent a state induced by diverse anti-mitogenic signals leading to cell division arrest. 21Interestingly, assessment of microvessel density revealed a signicant decrease in the number of blood vessels in treated tumors (Fig. 2C, for 100 mg kg À1 per day), indicating a strong antiangiogenic effect.SMA staining revealed that RAPTA-C treatment resulted in normalized, more mature blood vessels that were covered by pericytes.While control tumors were characterized by numerous, newly generated and immature blood vessels lacking pericytes (CD31+/SMA À ), we noticed that treatment with RAPTA-C resulted in the disappearance of this newly formed angiogenic vasculature and led to an increased number of mature vessels covered by pericytes (CD31+/SMA+, Fig. 2D).This observation further conrms the angiostatic activity of RAPTA-C.
The low toxicity of RAPTA-C when applied at quite high doses is remarkable and therefore the accumulation of RAPTA-C was studied in order to rationalize, at least in part, the absence of toxicity.The biodistribution of radioactively labelled 103 Ru-RAPTA-C in healthy Balb/c mice was determined (Fig. 3).The procedure used to prepare 103 Ru-RAPTA-C is provided in the Experimental section.Two hours aer injection, it was found that the level of ruthenium in the urine was ca.28-fold higher than in liver, spleen, lungs and heart, indicative of fast and efficient renal excretion.Ruthenium was not found to specically accumulate in any of the tested organs.The slightly higher levels in the kidneys (approx.2-fold) and intestines (approx.3-fold) may be expected only 2 hours aer injection.These data parallel a previous pharmacokinetic study in which RAPTA-C was injected i.p. in healthy Swiss CD-1 mice. 7In this previous study, the content of ruthenium in the blood, plasma, and in selected organs (liver, kidney, spleen, and lung) was quantied by atomic absorption spectroscopy.A half-life (T 1/2 ) of about 12 hours was estimated that varied slightly depending on the dose and administration schedule.Two hours aer RAPTA-C administration, the concentration of RAPTA-C in kidneys was 2fold higher than in the spleen, while the majority of ruthenium was eliminated in urine within 1 hour of administration.The previous and current data show that the rate of clearance of  RAPTA-C is faster than that of NAMI-A. 22A 106 Ru analogue of RAED-C revealed that the highest levels of the compound were associated with the liver and kidney. 23
Neutron activation of natural RuCl 3 $3H 2 O gave the 103 RuCl 3 $3H 2 O. Irradiations were performed with the spallation neutron source SINQ at the PSI.Irradiation of natural ruthenium with a neutron ux of 10 14 n cm À2 s À1 gave the following radionucleides: 97 Ru (T 1/2 ¼ 2.9 days), 103 Ru (T 1/2 ¼ 39.3 days) and 105 Ru (T 1/2 ¼ 4.44 h).The thermal cross section of 102 Ru (s ¼ 1.27 barn) and the relative long half-life of 103 Ru compared to the other radionucleides produced allowed for the production of 103 Ru with high radionuclidic purity with 97 Ru or 105 Ru activities undetectable aer 1 week of decay (Fig. S3, ESI †).Neutron irradiation of the chlorides gave negligible 36 Cl and 38 Cl activity.
Example: 15 mg natural ruthenium (39.9 mg RuCl 3 $3H 2 O) were irradiated for 16.2 days with the 10 14 n cm À2 s À1 ux giving a 103 Ru activity of 109.8MBq from the 124.7 MBq expected. 103Ru activity was calculated according to the equation given in the ESI.† The radiosynthesis of 103 Ru-RAPTA-C was monitored with a Cyclone phosphor imager (Perkin-Elmer).
Cell culture, preparation and implantation on the CAM model A2780 human ovarian carcinoma cells (ECACC, Salisbury, UK) were maintained in RPMI-1640 cell culture medium supplemented with GlutaMAX™ (Gibco, Carlsbad, USA), 10% heatinactivated bovine calf serum (Sigma-Aldrich, St. Louis, USA) and 1% antibiotics (Sigma-Aldrich).Fertilized chicken eggs were incubated in a hatching incubator (relative humidity 65%, 37 C), as previously described. 24A2780 cells (10 6 ) were prepared in serum-free RPMI-1640 as a spheroid in a 25 ml hanging drop and 3 h later were transplanted on the surface of the in ovo CAM on EDD 8 25 (Fig. 1A).Vascularized three-dimensional tumors were visible 3 days aer tumor cell implantation, on EDD 11, when treatment was initiated.Tumors were treated daily by i.v.injection of freshly prepared RAPTA-C into a main blood vessel of the CAM.Aer preliminary experiments, we observed that the most efficient tumor growth inhibition was obtained with RAPTA-C aer 5 intravenous injections, in the tested dose range, on 5 consecutive days.Therefore, we used this treatment schedule in the current study.RAPTA-C was dissolved in DMSO to afford a stock solution, which was subsequently diluted to the desired concentrations in 0.9% NaCl.Controls were treated with vehicle (DMSO in 0.9% NaCl).The concentration of drug was administered each day for 5 consecutive days and doses were calculated assuming an average embryo weight of 8.23 grams (i.e. the average embryo weight during the course of the 5 days of treatment).Tumors were monitors for a total of 11 days aer cell implantation, at which time embryos were sacriced and tumors were resected and xed in zinc-xative for additional analysis.N ¼ 5 in all treatment groups; N > 5 in the control group.

Cell implantation in athymic mice
Experiments in female 7-week old Swiss female athymic mice purchased from Charles River (Orleans, France) were carried out according to a protocol approved by the Committee for Animal Experiments for the Canton Vaud, Switzerland (license 2736).Mice were injected subcutaneously in the right ank with Chart 3 Radio synthesis of 103 Ru-RAPTA-C.0.5 Â 10 6 LS174T cells (purchased from Cell Line Service GmbH Eppelheim, Germany), previously cultured in DMEM supplemented with 10% heat-inactivated fetal calf serum and 1% antibiotics).When tumors reached a size of 5-6 mm in diameter, mice received 10, 40 or 100 mg kg À1 of freshly prepared RAPTA-C in 100 ml solution via i.p. injections.This treatment was repeated daily for 11 consecutive days.Tumors dimensions and body weight were measured daily.Tumor volumes (mm 3 ) were calculated as follows: volume ¼ (largest diameter in mm) Â (perpendicular diameter in mm) 2 Â 0.5.Mice were euthanized when the CTRL tumor size reached 1000 mm 3 .N ¼ 9, 4, 6 and 3 mice for CTRL, 10 mg kg À1 , 40 mg kg À1 and 100 mg kg À1 treatment groups, respectively.

Biodistribution studies
The in vivo experiments were approved by the local veterinarian department and conducted in accordance with the Swiss law of animal protection.Three 6-8-week-old female, Balb/c mice were purchased from Charles River Laboratories (Sulzfeld, Germany). 103Ru-RAPTA-C (1.9 MBq, 2.6 mmol, 100 mL per mouse) was injected into a lateral tail vein.The animals were sacriced 2 h aer drug administration.Selected tissues and organs were collected, weighed, and counted for radioactivity using a gspectrometer running with InterWinner soware.The results were listed as a percentage of the injected dose per gram of tissue mass (% ID per g).Counts of a dened volume of the original injection solution counted at the same time were used as a reference.

Statistical analysis
Values are given as mean values AE standard error of the mean (SEM).Data are represented as averages of independent experiments.Statistical analysis was done using the two-way ANOVA test and t-test using GraphPad Prism soware® (GraphPad Soware Inc., CA).*p indicating p-values lower than 0.05 and **p indicating p-values lower than 0.01 were considered statistically signicant.

Conclusions
7][28][29] Our studies were focused on an organometallic ruthenium(II)-arene complex, i.e.RAPTA-C, that was shown to exhibit both anti-metastatic and anti-angiogenic effects. 9,30Herein, we describe the effect of RAPTA-C on the growth inhibition of primary tumors.In this respect, RAPTA-C treatment was found to efficiently reduce the growth of tumors by approx.75% in one model, with the inhibition of angiogenesis (determined by vessel density assessment) being at least partly responsible for this action.An efficient and general induction of tumor cell death was observed in the tumor tissue, with remaining viable tumor islands completely negative for the proliferation marker Ki-67.These results support the translational development of RAPTA-C into clinical studies.

Fig. 1
Fig. 1 Inhibition of A2780 tumor growth in the CAM model by RAPTA-C.(A) Treatment protocol: RAPTA-C was administered i.v. on treatment days 1-5 (corresponding to EDD 11-15) and tumors were excised on the last day of the experiment.(B) Growth curve of A2780 tumors grafted on the CAM showing tumor volume with respect to treatment day.(C) Images show representative tumors from the vehicle treated (CTRL) and RAPTA-C (0.2 mg kg À1 ) treated CAMs.(D) Representative images of the immunohistochemical staining of the endothelial cell marker CD31 (in brown) showing reduced microvessel density per mm 2 in tumors treated with RAPTA-C normalized to the tumor surface area and provided as a % of the control (E) and Ki-67 positive nuclei (in blue) (D) and quantification of the percentage of the tumor surface area staining positive for Ki-67 (as a % of CTRL) (F).Black bar in the right image of (D) represents 500 mm and is valid for both images.Error bars represent standard error of the mean, SEM; N ¼ 5 in all treatment groups; N > 5 CTRL group; **p < 0.01.

Fig. 2
Fig. 2 RAPTA-C induced inhibition of human LS174T colorectal carcinoma tumor growth in athymic mice.(A) RAPTA-C dose dependent growth curves of LS174T tumors.(B) Relative weight change of treated mice during the experiment.The average weight of mice in the RAPTA-C 100 mg kg À1 treatment group was not significantly different between treatment days 1 and 11 (p ¼ 0.06).(C) Representative images of CD31 positive immunohistochemical sections of CTRL and RAPTA-C (100 mg kg À1 ) treated tumors on the last day of experiment and microvessel density quantification, expressed as the number of vessels per mm 2 (**p ¼ 9.6 Â 10 À7 ).Magnification 4Â, bar in the right image represents 500 mm and is valid for both images.(D) Representative images of SMA and CD31 positive immunohistochemical sections of CTRL and RAPTA-C (100 mg kg À1 ) treated tumors on the last day of experiment and their quantification (*p ¼ 0.0166).Magnification 20Â, bar in the right image represent 100 mm and is valid for both images.Error bars represent SEM; N ¼ 3-9 mice per group.*p < 0.05, **p < 0.01.

Fig. 3
Fig. 3 RAPTA-C biodistribution after injection in Balb/c mice.Mice were injected i.p. with 103 Ru-RAPTA-C (1.9 MBq per mouse) and were sacrificed 2 hours post injection and their organs were resected.The data are presented as % injected dose per gram of tissue (% ID per g).Indicated values are means, error bars represent SEM.