Biological Properties of IRIM, the Iridium(III) Analogue of (Imidazolium (Bisimidazole) Tetrachlororuthenate) (ICR)

Some biological aspects of the new complex imidazolium bisimidazole tetrachloro iridate(III)-IRIM- the iridium(III) analogue of ICR, were considered. More in detail the conformational effects produced by IRIM on DNA and the cytotoxic properties of IRIM on some selected human cell lines were measured. Dialysis experiments and DNA thermal denaturation studies are suggestive of poor binding of IRIM to DNA; formation of interstrand crosslinks is not observed. In any case CD measurements suggest that addition of increasing amounts of IRIM to calf thymus DNA results into significant spectral changes, that are diagnostic of a direct interaction with DNA. A number of experiments carried out on the A2780 human ovarian carcinoma, B16 murine melanoma, MCF7 and TS mammary adenocarcinoma tumor cell lines strongly point out that IRIM does not exhibit significant growth inhibition effects within the concentration range 10-4-10-6 M. It is suggested that the lower biological effects of IRIM compared to ICR are a consequence of the larger kinetic inertness of the iridium(III) center with respect to ruthenium(III).


INTRODUCTION
Following the success of cisplatin as an anticancer agent, new interest raised on the design and development of new metal based antitumor agents 1,2]. Within this frame Keppler and coworkers prepared and characterized a series of antitumor ruthenium(Ill) complexes, of which ICR (imidazolium bisimidazole tetrachloro ruthenate(III)) is probably the most representative [1]. Later on, Alessio and Sava developed NAMI A, a compound structurally related to ICR, that has recently entered phase clinical trials [3]. The biological evaluation of these innovative metal complexes has clearly pointed out that the mechanism of action of ruthenium compounds is substantially different from that of platinum complexes. Generally the direct cytotoxic effects of ruthenium compounds are much lower than those produced by equimolar amounts of cisplatin while the antimetastatic effects are more pronounced [3]. Also the direct DNA damaging effects are of much lower intensity [4]. In this context it is of interest to evaluate the biological effects of isostructural metal complexes, obtained by simple replacement of the central metal ion. The comparative analysis of the pharmacological profiles provides information on the specific role ofthe metal. Ruthenium(Ill) complexes may be easily mimicked by rhodium(Ill) complexes and iridium(Ill) complexes. Some studies already appeared on the rhodium(Ill) analogue of ICR [5]; the iridium(lII) analogue of ICR-IRIMhas been recently synthesized by Mura [6]. The most striking difference between ICR and its rhodium and iridium analogues consists in the increasing kinetic inertness of the metal center. In this study we report on some significant biological properties of IRIM. More specifically the effects of IRIM on DNA stability and conformation in solution and its cytotoxic properties toward a number of human tumor cell lines were investigated.

MATERIALS AND METHODS
IRIM was synthetized as reported [6]. . DNA was treated with IRIM at different mol/bp ratios (r 0.01, 0.1, 1.0)and each sample was incubated for 24 hrs at room temperature. Thermal denaturation experiments were performed in quartz cuvettes with a Perkin Elmer Lamba 20 Bio spectrophotometer equipped with a thermostated cell as described in reference. Samples were continuously heated with a rate of temperature increase of 0.5C/min while monitoring the absorbance changes at 260 nm. The investigated interval of temperature ranged from 50C to 90C. Upon reaching 90C, samples were cooled back to 40C in order to follow the renaturation process. Values for melting temperatures (Tin) and for the melting interval (AT) were determined according to the reported procedures [7]. CD spectra were recorded at increasing IRIM/calf thymus DNA ratio (r= 0.0, 0.01, 0.1, 1.0) after mixing on a Jasco J600 dichrograph operating at room temperature, interfaced with a PC, and analyzed through the standard Jasco software package.
Cytotoxicity measurements were carried out on the A2780 ovarian carcinoma cell line, both sensitive and resistant to cisplatin (A2780/S and A2780/R) following the Sulphorhodamine-B (SRB) procedure [8], and data analyzed through standard methods. Cells were exposed to the cytotoxic agent for 72 hours.
The activity of IRIM on the B 16 melanoma, MCF7 breast cancer and TS adenocarcinoma tumor cell lines was also analyzed, upon treatment 24 or 72 hours long. Cell growth of samples treated with increasing concentrations of IRIM -10 M, 10" M and 104Mwas monitored in comparison to controls through the microculture tetrazolium test (MTT) [9] or SRB method [8].

Interactions of lRIM with calf thymus DNA
The interactions of IRIM with calf thymus DNA in vitro was first analyzed by visible spectroscopy.
As previously reported [6], IRIM is soluble in a physiological buffer and gives rise to a characteristic absorption spectrum in the visible. Notably, the visible spectrum of IRIM does not change over a period of several hours, at room temperature, implying that the metal chromophore is relatively stable under physiological conditions [6]. In order to monitor the reaction of IRIM with DNA, the visible spectra of IRIM were recorded following addition of saturating amounts of calf thymus DNA. Notably the presence of DNA does not affect significantly the visible spectrum of IRIM implying that the metal chromophore is substantially conserved.
On the other hand ultradialysis experiments are suggestive of weak binding of IRIM to calf thymus DNA: either the fraction containing calf thymus DNA or the DNA-free fraction contain comparable amounts of IRIM following ultrafiltration.
The effects that IRIM produces on the structure and conformation of calf thymus DNA were further investigated through circular dichroism and DNA melting studies, The CD technique is indeed extremely sensitive to the conformation of DNA double helix. CD spectra of calf thymus DNA upon addition of increasing amounts of IRIM are shown in Figure 3 It clearly emerges that addition of increasing amounts of IRIM does not affect significantly the melting temperature of calf thymus DNA. In other word there is no evidence of stabilization or destabilization of the double helix following IRIM addition. It can just be observed that at the highest ratios the AT value is slightly increased. The DNA renaturation profiles show that the presence of IRIM does not favor at all the strand pairing process after melting, ruling out that IRIM is able to induce formation of interstrand crosslinks.

Cytotoxic effects of lRIm
The cytotoxic properties of IRIM in vitro were evaluated through two distinct experiments. They were first analyzed on the A2780 ovarian carcinoma cell line either sensitive or resistant to cisplatin in comparison to ICR and CDDP. Results are shown in Table 2.
It clearly emerges that IRIM produces a very modest growth inhibition effect; ICs0 values larger than 150 ktM were estimated on the cisplatin sensitive line to be compared with values of 80 laM for ICR and 2 [aM for cisplatin. Far larger ICs0 values for all complexes, within the same order of cytotoxic potency, were found in the case of the cisplatin resistant line A2780/R.

DISCUSSION
Metal complexes other than platinum are today actively investigated as potential cytotostatic and antitumor drugs. In this frame, much work has been devoted to the analysis of ruthenium(III) complexes that showed in some cases promising biological and pharmacological profiles. A ruthenium(III) complex-NAMI Ais presently undergoing phase clinical trials. Structural analogues of ruthenium(III) complexes may be prepared by using rhodium(III) or iridium(III) as the central metal/on. We are exploiting iridium(III) analogues of known antitumor ruthenium(III) complexes as novel antitumor agents; the aim is to define the chemical and biological consequences resulting from the replacement of the ruthenium(III) center with the more inert iridium(III) center.
In this paper we report on the biological behaviour of IRIM, trans bisimidazole tetrachloro iridate(III), compared to ICR. Specifically two aspects of the pharmacological behaviour of IRIM have been stressed: i) the interaction with DNA; ii) the cytotoxic effects on a number of human tumor cell lines. i) The interaction with DNA DNA was selected since it represents a major target for platinum(II) antitumor compounds and is the probable target for several other antitumor metal complexes. The interaction of IRIM with calf thymus DNA was monitored through a number of classical screening techniques including circular dichroism, analysis of DNA thermal denaturation and dialysis experiments. This approach permits to establish whether i) the metal chromophore undergoes transformations upon binding; ii) the conformation of DNA is modified; iii) the double helix is stabilized, destabilized or unaffected; iv) interstrand crosslinks are formed.
G. Marcon, A. Casini, P. Mura, L.Messori and P. Orioli Biological Properties of lrim, the Iridium(Ill) Analogue of (Imidazolium(Bisimidazole) Tetrachlororuthenate) (ICR) Overall, IRIM produces modest effects on DNA in the sense that the metal chromophore is virtually unaffected by addition of DNA; the stability of the double helix is only weakly decreased even at the highest ratios; formation of interstrand crosslinks is not observed. Some evidence of interaction is obtained from CD spectra indicating the occurrence of slight conformational changes of the DNA solution structure. A possible interpretation of these results stems on the fact that IRIM is far less reactive than ICR since ligand exchange reactions are much slower. In the case of ruthenium(III) complexes like ICR and NAMI, it is assumed that reactions with biologically relevant targets take place following chloride hydrolysis. It is straightforward to hypothesize that IRIM does not form coordinative bonds with DNA since it is not able to hydrolyze to a significant extent under physiologically occuring conditions. The only type of reactivity with DNA that can take place in the absence of hydrolysis is some groove interaction. In the light of the present results, it can be stated that IRIM exhibits very modest DNA modifying properties.
ii) The cytotoxic properties.
The lack of DNA modifying properties does not necessarily imply that IRIM must be discarded a priori from any further pharmacological evaluation. Indeed, apart from DNA binding, other mechanisms can be responsible for the antitumor activity, such as binding to proteins involved in DNA methabolism or implicated in malignant transformation and maintenance of the transformed phenotype. With this in mind, we further tested the biological properties of IRIM by analyzing its cytotoxic properties toward selected human tumor cell lines. Four major lines were considered, namely A2780 ovarian carcinoma, B16 melanoma, MCF7 breast cancer and TS adenocarcinoma tumor cell lines. We found that IRIM shows very modest cell growth inhibition effects. The comparison with cisplatin is striking: on the A2780 line the ICs0 value of cisplatin is 2 laM, that of IRIM is 140 laM; nevertheless, it is worthwhile noting that the ICs0 value of ICR on the same cell line is 80 tM, close to that of IRIM. Indeed the cytotoxicity test has been longly considered and it must be stressed that many compounds with good tumor cell-killing activity have been discovered, but few have found clinical utility. On the other side, in recent years, several non cytotoxic compounds-among which some ruthenium(III) complexes turned out to exhibit relevant anticancer properties. Thus, we must approach the cytotoxicity test considering the following limits: 1) a lack of discrimination between effects on tumor and normal tissue, 2) the cell-cycle dependency of many cytotoxic drugs and 3) the frequent susceptibility of these substances to induced drug resistance. To conclude, in our opinion, before stating that IRIM and similar iridium(III) complexes are not useful as antitumor agents, it is necessary to perform more advanced in vitro and in vivo pharmacological studies.

CONCLUSIONS
In vitro interactions of IRIM with calf thymus DNA were analyzed through various physicochemical techniques. It is found that IRIM induces small conformational changes on DNA. Overall the observed effects are much weaker than those induced by cisplatin. Notably DNA modifications brought about by ICR are larger than those produced by IRIM. It is hypothesized that the interaction of IRIM with DNA is essentially non-covalent in nature whereas ICR is able to form coordinative bonds. The weak DNA modifying properties correlate with low cytotoxicity on a number of human tumor cell lines. ACKNOWLEDGMENTS Dr. S. Carotti (Laboratory of Chemoterapy, Pharmacology, University of Florence) is acknowledged for helping us with the cytotoxicity tests.