Cancer is a frequently lethal ilness caused by an abnormal cell growth with the ability to invade and spread throughout the organism; thus, it has the greatest incidence and mortality rate worldwide [1]. In the year 2020, the US was anticipated to witness 606,520 deaths and 1.8 million new cases [2]. About 4.5 million premature cancer-caused deaths were reported worldwide in 2016 [3]. Based on current data, the International Agency for Research on Cancer (IARC) forecasts that around 13 million cancer-related deaths will occur by 2030 [4].

There are hundreds different types of cancer affecting various organs, tissues, and cells (i.e., breast, bone, blood, colon, liver, lung, etc.) in the form of carcinomas, lymphomas, sarcomas, or leukemia, each one requiring specific treatment [5]. The most common clinical approaches for cancer treatment are chemotherapy, radiation therapy, surgery, hormone therapy, and targeted therapy with anticancer drugs [6].

The need for new and better pharmacological therapies for the treatment of cancer still exists. While there are succesful specific treatment frameworks against some types of cancer, such as anti-hormonal therapy in breast cancer [7], or monoclonal antibodies targeting aberrant receptors, the intrinsic heterogeneity found in cancer forces the use of highly toxic chemotherapeutic regimes [8].

The development of effective antitumor drugs with high selectivity and low toxicity is currently a major challenge for the scientific community. Indeed, the wide range of adverse effects resulting from cancer therapy have an impact on therapeutic adherence and general quality of life for patients and their families [9].

Cisplatin efficacy in the treatment of various cancers places coordinative chemistry among viable antitumor design alternatives. Although highly efficacious, treatment with cisplatin is still limited by side effects, inherited resistance, or acquired resistance, which has only partially been eliminated by the introduction of new Pt(II) drugs [10].

Other metal complexes containing ions such as copper, gold and zinc chelating agents have received great interest as anticancer agents ^[11][12][13][11,12,13]. Recently, the chemistry of ruthenium compounds has been intensively analyzed due to the interest in providing an alternative to cisplatin, because of their promising cytotoxic and potential anticancer properties ^[14][15][16][14,15,16].

2. New Platinum Compounds

While cisplatin is used widely in the clinic to treat many tumour types, clinical utility is limited by two major factors: (i) drug resistance, either intrinsic or acquired, and (ii) the principal dose-limiting side effects of nephrotoxicity and neurotoxicity. Unfortunately, although the initial tumour response to cisplatin in many tumours may be high, most patients will nevertheless relapse and die of their disease.

2.1. BBR 3464

BBR 3464 has notable preclinical characteristics. It is approximately fourty to eighty fold more potent than cisplatin on a molar-dose basis and is active in vivo in cisplatin-sensitive and -resistant tumors, as well as in insensitive xenografts. Additionally, BBR 3464 was shown to be more active than cisplatin in p53 mutant tumors. The characteristic DNA-interaction of BBR 3464 results in the inhibition of DNA replication and RNA transcription, with triggering of the apoptotic cascade leading to cell death. Unlike cisplatin, BBR 3464 leads to prolonged tumor-growth inhibition after the discontinuation of treatment, suggesting that the two drugs could differ significantly in their ability to perturb the cell cycle. As a distinctive feature, BBR 3464 achieves a high proportion of interstrand and intrastrand DNA adducts in contrast to the effects of cisplatin, which predominantly produces the latter type of DNA damage. While cisplatin-damaged DNA is recognized by HMG proteins, the conformational changes resulting from BBR 3464 interaction with DNA are not. This observation might explain the resistance to cisplatin, and the lack of resistance to BBR 3464 in tumors expressing mutations of the p53 oncosuppressor gene ^[17][59]. In vitro and in vivo studies have shown that BBR 3464 manifests its cytotoxic properties at concentrations 10 times lower than cisplatin and is effective on cell lines resistant to it ^[18][60]. Preclinical toxicology in mice, rats and dogs treated on a single or five-timed-daily refracted schedule showed that target organs for BBR 3464 toxicity was bone marrow, resulting in leukopenia, while renal tubulopathy was only minimal or slight. A slight pulmonary interstitial reaction, with fibroblast proliferation and inflammatory infiltration, was observed in mice and dogs. This unusual effect was more extensive after intravenous bolus, given as a single or weekly dose, than after slow infusion or a bolus given every two weeks. The schedule dependency of the lung toxicity prompted the clinical evaluation of a five-times-daily refracted schedule in addition to a single dose schedule. Phase I clinical studies established the value of the maximum tolerated dose as 0.12 mg· m⁻²·day⁻¹ at a daily administration rate, for 5 days. The toxicity of this compound is its main disadvantage. In the case of increasing the dose to 0.17 mg·m⁻²·day⁻¹, severe myelosuppression and gastrointestinal toxicity were recorded. On the other hand, no nephrotoxic effects were reported ^[17][59]. Phase II studies performed on patients with non-small cell lung carcinomas and ovarian tumors in advanced stages highlight the increased efficiency of BBR 3464 compared to therapeutic combinations of platinum compounds with taxanes, but also a lack of activity of the compound against gastric tumors and cancer small cell lung ^[19][20][21][61,62,63]. In the case of a phase II study regarding the effectiveness of this compound in the treatment of pancreatic cancer, although the completion deadline has been exceeded, the results have not yet been made public ^[22][64].

2.2. Satraplatin

Satraplatin was rationally designed such that the lipophilicity and stability were suitable for oral administration. The half-life of reduction of satraplatin by 5 mM ascorbate takes 50 min, which is an adequate time for absorption by the gastrointestinal mucosa in the platinum(IV) form once ingested ^[23][21]. Upon entry into the bloodstream, satraplatin undergoes reduction to give six distinct platinum(II) species. Ammine dichloro-(cyclohexyl-amine)platinum(II), derived from the loss of two acetate ligands, is the major metabolite and also exhibits the most potent anticancer activity ^[24][65]. Like cisplatin, satraplatin acts through the formation DNA cross-links, DNA distortion, and subsequent inhibition of DNA transcription and replication. The ability of satraplatin to overcome cisplatin resistance is thought to arise from the asymmetric nature of the DNA lesions, which unlike cisplatin adducts, can evade recognition by DNA repair proteins ^[23][21]. In preclinical studies, satraplatin exhibited a better toxicity profile than cisplatin, and showed activity in cisplatin-resistant human tumor cell lines ^[25][66]. In vivo studies in mice bearing murine ADJ/PC6 plasmacytoma, which we note was the same model used to identify carboplatin as a viable alternative to cisplatin, showed satraplatin to exhibit markedly superior antitumor efficacy relative to cisplatin and carboplatin. Also, in four ovarian carcinoma xenograft models of varying cisplatin and carboplatin resistance, satraplatin displayed activity similar to that of cisplatin and carboplatin, which were administered intravenously. In rodents, the dose-limiting toxicity of satraplatin was myelosuppression. It has beWen emphasized that less hepatotoxicity and fewer gastrointestinal effects were observed as compared to treatment with cisplatin or carboplatin ^[23][21]. In the first Phase I study, satraplatin was administered at doses ranging from 60–170 mg·m⁻² as a single oral dose. The pharmacokinetics data suggested that gastrointestinal absorption was being saturated, preventing the maximum tolerated dose from being reached. To improve absorption into the bloodstream, patients were treated on a five-times daily schedule with lower doses (30–140 mg·m⁻²) ^[26][67]. The dose-limiting toxicities were thrombocytopenia and neutropenia and in about 10% of the patients treated, nausea, vomiting, and diarrhea were also observed. Based on the Phase I studies, doses of 100–120 and 45–50 mg·m⁻² were recommended for repeated daily dosing for 5 and 14 days, respectively, in Phase II/III trials ^[23][21]. Several Phase II/III trials have been carried out to determine the efficacy of satraplatin. A Phase II study on metastatic NSCLC patients, in which satraplatin was administered as single daily 120 mg·m⁻² doses for 5 days on 3 week cycles, failed to provide any objective responses. Nevertheless, 46% of the patients were noted to express some palliation. A more advanced Phase II study on patients with small-cell lung cancer and squamous cell head and neck cancer, with escalated doses of satraplatin, produced a response rate of 38%, similar to that observed with cisplatin. Furthermore, this study found no signs of severe neurotoxicity or nephrotoxicity. Other Phase II studies in patients with relapsed ovarian cancer and advanced/recurrent squamous cancer of the cervix produced clinically beneficial or partial rates of response in several patients. The former study noted that the most common form of toxicity were neutropenia and thrombocytopenia ^[26][67]. Satraplatin has also been heavily studied as a potential second-line chemotherapeutic for patients with metastatic castration-resistant prostate cancer. Treatment with 120 mg·m⁻² satraplatin daily for 5 days, used in patients with castration-resistent prostate cancer who had undergone front-line hormone therapy, resulted in 62% of patients expressing stable disease or partial response ^[27][28][68,69]. Currently, a phase I clinical trial is underway regarding the efficacy of satraplatin in the treatment of prostate cancer without metastases ^[22][64].

2.3. Picoplatin

Picoplatin (codenamed AMD473 or ZD0473), structurally, is diammine dichloro-(2-methylpiridine) platinum(II). It was primarily designed to overcome one of the known mechanisms of platinum resistance-detoxification by intracellular thiols—through the introduction of a bulky methylpyridine ring to provide steric hindrance to direct interaction with platinum ^[29][30][58,70]. Preclinical evaluation of picoplatin confirmed that this drug was also able to overcome platinum complex resistance in cell lines with high levels of glutathione (GSH) ^[31][71]. In studies with human ovarian cell lines, it has been shown that increasing levels of reduced GSH are associated with increasing platinum resistance. In addition, lower levels of glutathione S-transferase (GST) activity have been shown to be associated with enhanced clinical response to platinum-based chemotherapy in head and neck cancer ^[32][72]. Additionally, picoplatin was able to overcome resistance because of decreased cellular uptake of the drug in some cell lines or enhanced DNA repair/increased tolerance of platinum adducts in others. Picoplatin forms interstrand cross-links, but does so at a rate intermediate to that of cisplatin and carboplatin, because of its reduced reactivity relative to cisplatin. Using a Taq polymerase stop assay to identify the site of DNA adducts, a novel pattern of DNA binding was identified in pBR322 DNA after incubation with 10 and 100 mM picoplatin for 2 h ^[31][71]. This finding may also, in part, account for the observed capability of picoplatin to circumvent adduct tolerance and DNA-repair mechanisms associated with resistance to cisplatin. Preclinical pharmacology studies in mice have demonstrated that the maximum tolerated dose is 45 mg·kg⁻¹, given as a single intraperitoneal administration, with the dose-limiting toxicity being myelosuppression. Owing to the limited aqueous solubility of picoplatin, preclinical toxicology studies were conducted by intraperitoneal injection following a pharmacokinetic demonstration of the equivalent bioavailability of intraperitoneal and intravenous administration. No renal toxicity was observed. Antitumour activity was noted in several tumour models including human ovarian carcinoma xenografts with acquired resistance to cisplatin and carboplatin. Picoplatin showed an improved antitumour effect compared with cisplatin and satraplatin against the CH1cisR xenograft (34 days growth delay vs 10.4 and 3.5 days, respectively). The antitumour activity was similar when picoplatin was given daily at 60 mg·kg⁻¹ for 5 days every week for 4 weeks or by weekly administration (300 mg·kg⁻¹ every 7 days for 4 weeks) ^[33][73]. Results from preclinical studies have highlighted the effectiveness of picoplatin against some types of ovarian cancer, mesothelioma, small cell lung cancer and non-small cell lung carcinomas resistant to cisplatin and oxaliplatin. It is interesting to mention that this compound manages to avoid all three major methods of resistance to cisplatin (deficient absorption, inactivation by thiol compounds and DNA repair mechanisms) ^[30][70]. On the basis of the preclinical antitumour activity seen with picoplatin, especially in models with acquired platinum resistance, and its lack of nonhematological toxicity, the drug was taken into clinical development. A Phase I trial was carry out at the Royal Marsden Hospital under the auspices of the Cancer Research UK Phase I/II Committee. The initial schedule chosen was a short intravenous infusion given once every 3 weeks. A pharmacokinetically guided dose-escalation scheme was used, for several reasons. Firstly, there is no evidence of metabolism of picoplatin in vivo, and thus, the free platinum AUC (area under the concentration vs. time curve) should reflect the biologically important species in both man and mouse. Secondly, previous experience with cisplatin and carboplatin has demonstrated the close relation between the AUC at maximum tolerated dose in mice and humans. It was hoped that this approach would reduce the number of dose escalations required to reach the maximum tolerated dose ^[34][74]. Following phase I studies, the maximum tolerated dose value was established as 150 mg·m⁻² and the picoplatin administration regimen as doses of 120 mg·m⁻², intravenously, once every 3 weeks, with the possibility of increasing the dose for patients who had not previously undergone antitumor treatment. The limiting toxic effects were neutropenia and thrombocytopenia, whereas alopecia, neurotoxicity and nephrotoxicity were not reported. Other adverse effects included nausea and vomiting, anorexia and metallic taste ^[29][58]. Phase II clinical trials in patients suffering from various types of lung tumors resulted in a response rate of 15.4% in small cell lung cancer resistant to other platinum compounds ^[35][75]. Other phase II studies have highlighted the fact that picoplatin is also active on ovarian tumors sensitive and resistant to other platinum compounds, bringing a benefit in terms of survival rate and the limitation of disease progression ^[36][76]. Similar results have been reported in the case of patients with mesothelioma and metastatic breast cancer; for approximately 50% of patients, a stop was found in the progression of the disease ^[37][38][77,78]. In the case of some phase II studies regarding the effectiveness of picoplatin in the treatment of colorectal and prostate cancer refractory to hormone therapy, although the completion deadline has been exceeded, the results have not yet been made public ^[22][64]. Only one phase III study was undertaken targeting small cell lung cancer. The study showed that patients who did not respond to treatment up to that point or who had experienced rapid disease progression benefited from an extension of life after treatment with picoplatin ^[39][32].

2.4. Ormaplatin

Ormaplatin is rapidly reduced to the corresponding dichloro (1,2-diaminocyclohexane) platinum(II) form in tissue culture medium (t_1/2 = 5–15 min) and undiluted rat plasma (t_1/2 = 3 s) ^[23][21]. The active platinum(II) species is similar to oxaliplatin. Ormaplatin displayed in vitro and in vivo activity against some cisplatin-resistant cancers and was taken forward to clinical trials ^[40][79]. Various doses, dose patterns, and modes of administration (intravenous and intraperitoneal) were investigated in six Phase I clinical trials; however, no Phase II clinical trials have been planned ^[41][42][80,81]. Ormaplatin was found to induce severe neurotoxicity at the maximum tolerated dose, and in some cases, a safe maximum tolerated dose could not be determined. Toxicity is thought to arise from fast reduction to the active platinum(II) form as a consequence of the axial chloride ligands ^[23][21].

2.5. Iproplatin

Iproplatin (also known as JM9 or CHIP), structurally, is dichloro-dihydroxy-bis(isopropylamine) platinum(IV). Iproplatin is structurally similar to ormaplatin in the sense that it contains two equatorial chloride groups which are cis to each other ^[23][21]. Carbon-14 labelling studies showed that the mechanism of action of iproplatin involves the reduction of the platinum(IV) center to platinum(II) followed by covalent bond formation with DNA. Iproplatin is less prone to reduction and deactivation by biological reducing agents than ormaplatin, presumably because of the presence of hydroxide axial ligands, allowing less hindered distribution throughout the body. Another advantage of iproplatin is its very high water solubility (44.1 mM), which allows simpler formulation and administration ^[43][82]. Iproplatin is one of the most clinically studied platinum agents to have not been approved for marketing, with 38 clinical trials ranging from Phase I to III having been concluded. Phase I studies revealed that the dose-limiting toxic effect was myelosuppression, which, in one study involving children, was partly correlated with the amount of prior therapy chemo- and radiotherapy received. The same study recommended intravenous doses of 324 mg·m⁻² over 2 h every 3–4 weeks for Phase II trials in children. Other studies proposed doses of 45–65 mg·m⁻² and 95 mg·m⁻² for patients treated on a five-times-daily schedule every three weeks and a four-times-weekly schedule with two-week break periods, respectively ^[44][83]. Phase II trials were carried out in patients with a variety of different cancer types, and Phase III trials were conducted in ovarian cancer patients and those with metastatic epidermoid carcinoma of the head and neck ^[45][46][84,85]. The ultimate conclusion of these studies was that iproplatin did not exhibit overall effectiveness that surpassed that of cisplatin or carboplatin and no further trials were undertaken ^[23][21].

3. New Copper Compounds

The differential response of normal and tumor cells exposed to Cu(II) ions is the starting point for obtaining new compounds with antineoplastic properties. Many of the copper complexes are active against tumor cell lines resistant to cisplatin and analogous compounds, and exhibit lower toxicity than established platinum derivatives as antitumor agents. Preclinical and clinical studies provide encouraging evidence to support the therapeutic potential of copper complexes despite their high toxicity. Due to the promising results obtained from in vitro and in vivo testing, some of these complexes have reached the clinical testing phase. In this context, Cu(I) and Cu(II) complexes present encouraging perspectives ^[47][86].

3.1. Elesclomol

Elesclomol is an injectable, small molecule. It has been developed as a sodium salt formulation for single-agent use or for combination use with other anti-cancer drugs. The free acid form of elesclomol is the active ingredient in both elesclomol and sodium elesclomol. While in the bloodstream, elesclomol binds to copper (II) ions present in the serum. Cancer cells efficiently take up this complex, unlike free elesclomol. Once inside the cell, the copper in the complex undergoes a redox reaction whereby Cu(II) is reduced to Cu(I). This reaction, which is mediated by elesclomol, creates ROS and oxidative stress in the cell. The anti-cancer activity of elesclomol is attributed to its ability to directly increase this oxidative stress ^[48][87]. Cancer cells already have an elevated level of oxidative stress relative to most normal cells. It was hypothesized that the further increase in ROS induced by elesclomol would exceed a critical threshold in cancer cells, enhancing the sensitivity to traditional cytotoxic chemotherapeutic agents and triggering tumor cell death while sparing most normal cells ^[49][88]. Elesclomol exerts its activity by disrupting the metabolism of mitochondria in cancer cells. This activity requires the presence of oxygen that results in energy metabolism being driven primarily through oxidative phosphorylation in mitochondria. Under hypoxic conditions, energy metabolism occurs through glycolysis in cytoplasm, rather than in mitochondria. Under hypoxic conditions, often associated with elevated lactate dehydrogenase (LDH) levels, elesclomol’s activity is diminished. These observations are consistent with findings in a phase 3 metastatic melanoma study, where elesclomol activity was found only in subjects with normal baseline LDH levels ^[48][49][50][87,88,89]. Elesclomol and Cu(II)-elesclomol are both highly active in vitro and typically inhibit tumor cell growth at low (nanomolar) concentrations. In preclinical models, elesclomol has demonstrated synergistic anti-tumor activity with both paclitaxel and docetaxel, as well as single-agent activity ^[49][88]. Elesclomol showed antitumor activity against a broad range of cancer cell types and substantially enhanced the efficacy of chemotherapeutic agents such as paclitaxel in human tumor xenograft models ^[51][90]. In a phase I clinical trial, in combination with paclitaxel in patients with refractory solid tumors, elesclomol was well tolerated, with a toxicity profile similar to that observed with single agent paclitaxel ^[52][91]. In a double-blinded, randomized, controlled phase II clinical trial in 81 patients with stage IV metastatic melanoma, elesclomol, in combination with paclitaxel doubled median progression-free survival compared with paclitaxel alone. Cutaneous melanoma is a highly malignant tumor derived from melanocytes, the pigment-producing cells in the epidermis of the skin. If diagnosed and surgically removed while localized in the outermost skin layer, melanoma is potentially curable. However, for patients with deeper lesions or metastatic disease, the prognosis is poor, with an expected median survival of only 6 to 9 months for patients with stage IV metastatic melanoma ^[53][92]. Interestingly, under hypoxic conditions, often associated with elevated LDH levels, the activity of elesclomol is diminished. Thus, the combination of elesclomol with paclitaxel proved effective only in patients with normal LDH levels, and no changes were observed in those with elevated LDH levels. Despite intensive efforts to improve disease prognosis, little progress has been made ^[48][51][87,90]. In another phase I study, elesclomol, given in combination with paclitaxel in women with refractory ovarian cancer, obtained a favorable opinion at this stage, with the combination being well tolerated. Currently, elesclomol combined with paclitaxel is in a phase II study in patients with ovarian, fallopian tube or peritoneal cancer that is recurrent or resistant to cisplatin treatment. Only patients with normal LDH levels were selected for the study ^[49][88]. Researchers have also planned to start a phase I study for the combined administration of elesclomol+docetaxel+prednisone in patients with metastatic prostate cancer ^[54][93].

3.2. Casiopeinas: Casiopeina III and Casiopeina II-gly

These are chelated complexes of Cu(II) with 4,7-dimethyl-1,10-phenanthroline and glycocol (CasII-gly) or with 4,7-dimethyl-1,10-phenanthroline and acetylacetone (CasIII) [55]. Casiopeinas are a group of copper-based chemical compounds with cytotoxic, genotoxic, antiproliferative and antineoplastic activity, as demonstrated in vitro and in vivo ^[56][42]. Casiopeinas were developed based on the rationale that copper compounds, unlike other metallic-based therapies, are more readily metabolized; this property decreases the incidence of side effects found in several other chemotherapies ^[57][94]. The main cytotoxic effect that representatives of this class have demonstrated is the activation of pro-apoptotic processes in malignant cells. There are at least two ways in which these compounds act on tumor cells: intercalation into the DNA structure followed by preventing its proper replication, and biochemical mechanisms leading to programmed cell death (apoptosis). Casiopeinas increase the level of ROS produced near DNA, most likely through a redox mechanism involving the Cu(II) ion. These species, once produced, will react with the DNA molecule causing damage to its structure most often by launching a radical attack at the level of the deoxyribose residue. Additionally, by increasing the level of ROS in the mitochondria, dysfunctions occur through the oxidation of thiol residues at the level of mitochondrial proteins and, finally, cell apoptosis occurs. Other mechanisms of action attributed to this class of Cu(II) chelate complexes, discovered later, involve DNA fragmentation and cell death through caspase-dependent pathways, or inhibition of energy metabolism and mitochondrial toxicity. In addition, CasII-gly exhibits an anti-tumor effect by inhibiting the cell cycle, regulating the transformation processes in fibroblasts or reducing the phenomenon of uncontrolled migration of tumor cells ^[57][58][94,95]. CasIII was entered into a phase I clinical trial, to test for acute myeloid leukemia and colon cancer. CasII-gly, because it blocks the migration and proliferation of HeLa cells, was entered into clinical trials to treat cervical cancer ^[59][96]. CasII-gly is currently in phase I clinical trials looking at its toxicity in humans. Tests have shown that it inhibits energy metabolism and induces a high cardiotoxic effect, a fact that will probably cause the clinical trials to be stopped ^[55][56][42,55]. The advantage observed in the case of clinical trials performed on Casiopeinas consists in an increase in the immunity of the patients; this is accompanied by a mechanism of high protection of the liver through the repair of damaged cells, compared to other cytotoxic drugs. A CasII-gly-based therapy may prevent the problematic side effects of chemotherapy, which often compromise patients’ health ^[60][54].

4. New Ruthenium Compounds

The success of cisplatin stimulated the scientific world in the development of other metal complexes with antitumor activity that are superior to platinum complexes, have lower toxicity or are active on other types of tumors. In this context, ruthenium complexes seem to be promising. Two such compounds have been entered into clinical trials. Despite their structural and chemical similarities, the two Ru(III) complexes show distinct antitumor behaviors. In preclinical studies, NAMI-A has demonstrated an inhibitory effect against the formation of cancer metastases in a variety of animal tumor models, but does not show direct cytotoxic effects on primary tumors, while KP1019 exhibits antitumor activity against a wide range of primary tumors humans through a cytotoxic mechanism of apoptosis induction ^{[61][62][63][64]}[57,97,98,99].

4.1. NAMI-A

The proposed mechanisms of action of NAMI-A in metastasis control include the following: limiting actin dependent adhesion in vitro ^[65][100]; limiting in vitro tumor cell motility via cytoskeleton remodeling (the activation of collagen receptor integrin β1 on the cell surface results in RhoA activation and, subsequently, in rearrangement of the cytoskeleton in vitro ^[66][67][101,102]; and exerting anti-invasive effects in vitro and in vivo by promoting capsule formation (NAMI-A increases the extracellular matrix around tumor cells and tumor vasculature by triggering fibrotic reactions, regulates TGFβ1 expression, binds to collagen and stimulates collagen production ^[68][69][70][103,104,105] and anti-angiogenic effect (e.g., NAMI-A inhibits the angiogenic effect induced by vascular endothelial growth factor in vitro) ^[71][106]. NAMI-A transiently blocks cell cycle progression in vitro at G2M phase ^[72][73][107,108]. The mechanism might be activation of Chk1, resulting in the inhibition of CDC25 and, subsequently, in inactive phosphorylated CDC2, thereby preventing mitotic entry ^[66][101]. In vitro, NAMI-A inhibits the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathway and c-myc transcription ^[74][75][109,110]; DNA binding—although the intrastrand adduct formation of NAMI-A is significantly less than that of cisplatin, Ru-G and Ru-AG intrastrand adducts—was observed in vitro ^[76][111]. The AG:CG adduct ratio was four times higher for NAMI-A compared to cisplatin. NAMI-A sporadically forms interstrand crosslinks, whereas the formation of DNA protein crosslinks is comparable to cisplatin ^[66][101]. Due to its fast ligand exchange kinetics, it was found that NAMI-A is not significantly internalized by cells, but rather, binds to extracellular collagen matrix and to cell surface integrins, leading to increased adhesion and reduced cancer cell spread. If these results are consistent with the ability of NAMI-A to inhibit the growth of new metastases, its activity against already-developed metastases is probably due to its antiangiogenic properties ^[77][112]. Contrary to cisplatin, the cytotoxic effect of NAMI-A is not remarkable (on average, 1053 times less than cisplatin) ^[78][113]; its cytotoxicity has been found to be correlated with DNA binding (which is also the case for cisplatin) ^[66][76][101,111]. In preclinical studies, administration of NAMI-A in more frequent smaller dosages showed more prominent antimetastatic effects. Notably, the action of NAMI-A seems to be independent of the type of primary tumor or the stage of growth of metastases. NAMI-A is capable not only of preventing the formation of metastases, but also of inhibiting their growth once established ^[79][56]. Preclinical animal studies using NAMI-A have shown selective activity against lung metastases from a variety of primary tumors in murine models. NAMI-A reduced the weight of lung metastases more than their number. Since larger concentrations of NAMI-A in the lungs than in other tissues was ruled out, this finding was assumed to be related to the selective interference of NAMI-A with the growth of metastases already established in the lungs ^[80][114]. Toxicologic studies in dogs and mice have revealed an acceptable toxicity profile. The calculated half-life was approximately 18 h. Toxicity was observed at concentrations greater than 50 mg/kg/day, and in mice that survived treatment, was reversed within 3 weeks of the end of the treatment ^[81][115]. NAMI-A was the first ruthenium compound which entered into clinical trials. A phase I study was performed with NAMI-A as a single agent, given as an infusion over 3 h daily for 5 consecutive days every 3 weeks in patients with different types of solid tumors (including colorectal cancer, non-small cell lung cancer, melanoma, ovarian cancer, pancreatic cancer and mesothelioma). In total, 24 patients were treated at 12 different dose levels (2.4–500 mg/m²/day). All 24 patients underwent refractory to conventional treatment. The advised dose for further testing was 300 mg/m²/day. Adverse events included only mild hematologic toxicity, quite disabling nausea, vomiting, and diarrhea; furthermore, patientsexperienced stomatitis, fatigue, common toxicity criteria grade 1 and 2 creatinine increase, fever and sensitivity reactions to NAMI-A. Finally, phlebitis at the infusion site was observed when NAMI-A was administered intravenously without a port-a-cath. There was also painful blister formation on hands, fingers and toes, although no part of the formal common toxicity criteria was considered dose-limiting. Twenty out of twenty-four patients were evaluable for response evaluation. One patient (4%) with non-small cell lung cancer experienced stable disease for 21 weeks, and nineteen patients (79%) showed disease progression. The transport of NAMI-A was achieved via binding to plasma proteins. It was found that NAMI-A accumulates in white blood cells, but accumulation was not directly proportional to daily dose or total drug exposure. Studies of this phase have shown stabilization of the disease in patients with advanced lung cancer. This result, together with the excellent activity shown by NAMI-A against lung metastases in mice, led to the recommendation of NAMI-A for a phase II study in the treatment of non-small cell lung cancer. As the combination of cisplatin and gemcitabine is widely used for the first-line treatment of non-small cell lung cancer, researhers decided to test a similar combination with NAMI-A ^[72][77][79][56,107,112]. A phase I/II study in which NAMI-A was given in combination with gemcitabine to 32 patients with advanced non-small cell lung cancer was performed. Phase I of the study was directed towards establishing the optimal dose of the combination of NAMI-A and gemcitabine (given at the typical dose of 1000 mg m^–2). The maximum tolerated dose of NAMI-A was found to be 300 mg· m^–2 in the 28-day cycle (3 h infusion on days 1, 8, and 15, and gemcitabine given on days 2, 9, and 16) and 450 mg·m^–2 in the 21-day cycle (NAMI-A administered on days 1 and 8, and gemcitabine on days 2 and 9). A further dose escalation of NAMI-A to 600 mg m^–2 was found to induce dose-limiting toxicity. Besides neutropenia, the main non-hematological adverse events involved elevated liver enzymes, transient creatinine elevation, nausea, vomiting, constipation, diarrhea, fatigue and renal toxicity. Blister formation on fingers was observed only at 600 mg· m^–2. The 21-day regimen was used for the phase II part of the study, in which 15 patients were treated with the maximum tolerated dose of NAMI-A established in phase I with the aim of assessing the antitumor activity according to the response of evaluation criteria for solid tumors ^[82][116]. Out of the 27 patients evaluable for response, partial remission was observed in one case (at 300 mg·m^–2 in the 21-day schedule) and stable disease for at least 6–8 weeks in 10 patients. These results were not sufficient to warrant further expansion of the phase II cohort with additional 12 patients. Overall, the efficacy of the treatment was lower than expected for gemcitabine alone and it was declared to be “insufficiently effective for further use” ^[72][77][107,112].

4.2. KP1019 and KP1339

The mode of action of KP1019 is very distinct from that of NAMI-A. These differences probably arise from the observed kinetic differences in the aquation processes and ruthenium activation. A key point is represented by the large difference in ruthenium uptake in the two cases, leading to significantly higher ruthenium concentrations in the cytosol for KP1019. Accordingly, the in vivo activity of KP1019 on primary tumor growth is believed to be predominantly due to cytotoxic effects on tumor cells arising from direct interference with cell signaling and metabolic pathways; in other words, KP1019 behaves as a classical cytotoxic drug. Furtheremore, one of the recent interpretation of the molecular mechanism of KP1339 tends to rule out direct DNA damage as the main determinant of its cytotoxic action. In contrast, the postulated mode of action involves strong interactions with cytosol proteins, leading to ROS overproduction, oxidative stress and endoplasmic reticulum stress through targeting the chaperone protein GRP78. Eventually, this cellular damage triggers apoptosis through a mitochondrial pathway ^[83][84][117,118]. Based on a quite complex investigative strategy relying on transcriptomics and a genetic screening approach in a budding-yeast model, various genetic targets and a plethora of cellular pathways targeted by KP1019 were identified. Then, the actions produced by KP1019 in yeast were compared with those produced by the same ruthenium drug in Hela cells, and reasonable extrapolations were made. On the grounds of the obtained results, a comprehensive model depicting the mode of action of KP1019 and the targeted cellular pathways was proposed. According to this model, KP1019 induces ROS generation, causes DNA damage (and thus cell cycle arrest), activates mitogen-activated protein kinase signaling, alters intracellular metal ion and lipid homeostasis and also affects the chromatin assembly. Cells activate transcriptional responses to alleviate cellular damage ^[85][119] Moreover, it was found that the toxicity potential of KP1019 is enhanced in the presence of various metal ions but suppressed by the supplementation of Fe²⁺ ions, and reduced glutathione, N-acetylcysteine, and ethanolamine. Thus, this model postulates for KP1019 a realistic multifactorial mechanism mediated by a variety of yet-unknown molecular targets ^[64][99]. KP1019 is moderately cytotoxic in vitro. Therefore, when tested against a panel of chemosensitive cell lines and their chemoresistant sublines, IC50 values in the range of 50 to 180 µM were measured. When compared with its sodium salt, KP1339, in a few cancer cell lines, KP1019 tended to be moderately more cytotoxic. Nevertheless, the significant correlation between the cytotoxicity profiles suggests that the two complexes have similar modes of action. For both compounds, no correlation between total ruthenium uptake and cytotoxicity was found ^[86][120]. Both KP1019 and KP1339 were found to be moderately cytotoxic (30–95 µM), but more cytotoxic than cisplatin, in SW480 and HT29 colorectal carcinoma cells, upon 24 h exposure; moreover, they induced apoptosis, predominantly via the intrinsic mitochondrial pathway. Upon 72 h exposure, cisplatin is much more efective than the two ruthenium compounds ^[87][121]. Studies concerning the antimetastatic ability of KP1019 in vitro gave controversial results. The complex revealed some anti-invasive activity in monolayer cultures of breast cancer cell lines, causing the significant reduction of cell migration and invasion ^[88][122]. KP1019 was tested in vitro against more than 50 primary tumors explanted from humans; in this highly predictive model, the complex proved a positive response rate higher than 70% ^[64][99]. KP1019 has antitumor activity in colon cancer in rats. Treatment with KP1019 resulted in a 95% reduction in tumor volume, with no mortality and no significant weight loss. In addition, its efficacy was superior to 5-fluorouracil, the standard agent used against colorectal cancer ^[89][123]. The time-dependent tissue distribution of KP1339 (given i.v.) in non-tumor-bearing BALB/c nude mice was recently determined ^[90][124]. The highest (and comparable) ruthenium concentrations were found in the liver, lungs, kidneys and thymus, followed by the spleen and colon (~50% less). Consistent with the trend of total ruthenium in blood plasma, the peak levels in the mentioned tissues were found one to six hours after administration and decreased slowly with time, with the exception of the spleen, where the highest amount was found 24 h post-injection. Based on this promising activity, the two ruthenim compounds were selected for further clinical evaluation ^[64][99].