Ruthenium Complexes: An Alternative to Platinum Drugs in Colorectal Cancer Treatment

Colorectal cancer (CRC) is one of the intimidating causes of death around the world. CRC originated from mutations of tumor suppressor genes, proto-oncogenes and DNA repair genes. Though platinum (Pt)-based anticancer drugs have been widely used in the treatment of cancer, their toxicity and CRC cells’ resistance to Pt drugs has piqued interest in the search for alternative metal-based drugs. Ruthenium (Ru)-based compounds displayed promising anticancer activity due to their unique chemical properties. Ru-complexes are reported to exert their anticancer activities in CRC cells by regulating different cell signaling pathways that are either directly or indirectly associated with cell growth, division, proliferation, and migration. Additionally, some Ru-based drug candidates showed higher potency compared to commercially available Pt-based anticancer drugs in CRC cell line models. Meanwhile Ru nanoparticles coupled with photosensitizers or anticancer agents have also shown theranostic potential towards CRC. Ru-nanoformulations improve drug efficacy, targeted drug delivery, immune activation, and biocompatibility, and therefore may be capable of overcoming some of the existing chemotherapeutic limitations. Among the potential Ru-based compounds, only Ru (III)-based drug NKP-1339 has undergone phase-Ib clinical trials in CRC treatment.


Introduction
Colorectal cancer (CRC) is a type of malignant neoplasm of the colon or rectum epithelial cell lining [1,2], which is recognized as the third most prevalent cancer worldwide and is the fourth leading cause of death [3,4]. It also accounts for about 10% of all yearly diagnosed cancers and cancer-related deaths globally [5]. Moreover, CRC has been documented as the second and third most common cancer in women and men, respectively [5,6]. CRC occurrence rate is high in most of the developed countries, whereas the rate is increasing rapidly in developing countries [6]. In 2020, more than 1.9 million individuals were estimated to be diagnosed, where 935,000 individuals would die among the CRC-diagnosed patients [7]. About 2.5 million people are predicted to be diagnosed with CRC by 2035 [5].
The conventional treatment strategies of CRC consist of surgical resection, radiation, and chemotherapy, which may extend the survival rate by only five years in 90% of stage I patients to 10% of stage IV patients [8,9]. Even though surgery has been an integral part of CRC treatment, it comes out with post-operative complications such as occurrence or acceleration in recurrence of tumor cells and/or development of liver metastasis [8]. Long-term use of chemotherapeutics and radiation induces peripheral neuropathy [10] and bowel dysfunction accompanied by increased frequency and urgency problems [11]. The limitations of the existing treatment strategies encourage researchers to develop effective therapeutic alternatives.
Over the past few decades, transition metal-based compounds have been extensively used in the anticancer medicinal chemistry area [12][13][14][15]. Platinum (Pt)-based medications such as cisplatin (CIS) and its analogs carboplatin (CAR) and oxaliplatin (OXA) (Figure 1) have been used worldwide in cancer treatment [16]. Additionally, some other Pt-based drugs, for example, miriplatin (Japan), nedaplatin (Japan), lobaplatin (China), and heptaplatin (Korea) (Figure 1) are used regionally in cancer treatment ( Figure 1) [17]. However, only OXA has been approved by the Food and Drug Administration (FDA) in CRC treatment [18] and stands out as the first-line therapy against CRC [19]. Despite being highly efficient, OXA has severe side effects [20] and drug resistance [21]. Such limitations inspire the search for alternative metal-based anticancer drugs. Among other transition metals, ruthenium (Ru) is a better alternative to Pt [22]. Ru displays both early and late transition metal properties due to its central position in the second row of the transition metal series [22]. The 4d subshell of Ru is partially filled and it contains many valencies that enable Ru to form a wide range of complexes via π bond formation, which can perform as anticancer agents against various tumor cell lines [23]. Ru-complexes showed promising anti-proliferative activity in vitro, in vivo, and in chemical model systems [24][25][26]. Moreover, the Ru-complex showed synergistic activity when combined with established anticancer agents and drugs [25,27]. Furthermore, Ru-complexes are widely used as phototherapeutic agents, biomolecular probes, and bioimaging reagents [28]. Luminescent Ru-complexes can differentiate DNA structures and have the potential to be used as molecular light switches for DNA [29]. Additionally, Ru nanoparticles (RuNPs) can be used as a cancer theranostic agent for the early diagnosis and treatment of CRC [30,31]. Nanostructured Ru-complexes offer improved anticancer activity under their targeted drug delivery and reduced side effects [32].
To overcome the limitation of Pt-drugs, Ru-complexes could be used as an alternative to Pt-based chemotherapeutic drugs in CRC. In this review, we scrutinized the potential of ruthenium-based drugs, drug candidates, and ruthenium nanoparticles in the treatment of CRC. Additionally, the molecular mechanism of action(s) such as effects on nucleic acids, cell proliferative pathways, and cell cycle are summarized and compared their efficiency with Pt-based drugs and other chemotherapeutic drugs i.e., 5-Fluorouracil (5-FLU), Doxorubicin (DOX), and Etoposide (ETP).

DNA Damage Mediated Apoptosis
Many Ru-complexes control the proliferation of cancer cells through DNA damage [94,95]. Some of the Ru-complexes bind with DNA via electrostatic attraction, major or minor grooves binding, intercalative binding mode, or by a combination of these two or more [96,97]. DNA binding modes of Ru-complexes can be confirmed by UV-Vis spectroscopy, viscosity investigation, and fluorescence spectrometry [97]. Intercalative binding opens a gap between the DNA base pair and inserts planar aromatic molecule of anticancer drug above and below the bases [98]. This results in unwinding and lengthening of the helix structure of DNA ( Figure 3→I) [97,99]. Ru(II)-complexes bind preferentially to N7 of guanosine and N3 of thymidine, but insubstantially to N3 of cytidine, and little to adenosine [100]. On the other hand, Ru(III)-complexes preferentially bind to phosphate groups of the DNA backbone. This is attributed to the strong electrostatic interaction between tricationic Ru(III) fragment and anionic phosphate groups [101]. Ru(III)-complexes also have been reported to bind to the N7 site of guanine [102] but in a less pronounced way than phosphate [101]. However, both Ru(II) and (III)-complexes have a common tendency to bind to the N7 site of guanine which is similar to CIS [103].
Oxidative stress plays a central role in Ru(III)-based drug trans-[tetrachloro-bis(1Hindazole)ruthenate(III)] or KP-1019 (8b) (Figure 6) mediated apoptosis in CRC cells. Kapitza et al. [119] reported that 8b induced H 2 O 2 formation in CRC cells which further reacts with mitochondrial membrane-embedded unsaturated fatty acids to induce depolar-ization of the mitochondrial membrane and mediated caspase-3 dependent PARP cleavage. 8b induced cytotoxicity in both SW480 and LT97 cells with an IC 50 value of 30 and 50 µM, respectively. In contrast, antioxidant N-acetylcysteine (NAC) (5 µM) decreased their potency, as evident from the increase of IC 50 values to 55 and 88 µM towards SW480 and LT97, respectively. This finding confirmed that ROS is involved in 8b-mediated apoptosis [119]. In vivo activity of 8b was evaluated by the chemoresistant MAC15A colon carcinoma in a rat model, closely similar to human colon cancer. Treatment of 8b (13 mg/kg) two times a week for 10 weeks reduced 8% of tumor size, where 5-FLU (40 mg/kg) reduces tumor size down to only 40% and at the same time, another Pt-based established drug CIS did not show any activity [137,138].
Sodium salt of 8b, i.e., sodium trans-[tetrachloride-bis(1H-indazole)ruthenate(III)] or NKP-1339 (8c) (Figure 6) was prepared by Keppler et al. [139]. 8c was reported to increase the ROS level and, therefore, induced ER stress-mediated apoptosis in CRC cells ( Figure 3→VIII) [89]. Exposure of 8c (200 µM) to HCT116 and SW480 CRC cells elevated ROS concentration by 2-fold and 2.5-fold, respectively, after 1 h compared to control and led to apoptosis through ER stress [89]. ROS causes potential damage to proteins that piled up in the ER. Since cancer cells tend to demonstrate an increased level of oxidative stress and ER stress due to having enhanced and fast metabolic activity, hence excessively accumulated misfolded proteins led the ER to start unfolded protein response (UPR) which induced apoptosis after exceeding a certain threshold level ( Figure 3→VIII) [89,140]. The underlying mechanism is associated with three transmembrane receptors namely, PrKrlike ER kinase (PERK), activating transcription factor 6 (ATF6), and inositol-requiring protein 1α (IRE1α) which are bound by an ER-resident chaperone, glucose-regulated protein (GRP78), which has high affinity towards misfolded protein [89,140,141]. Upon release from GRP78, PERK phosphorylated eukaryotic translation initiation factor 2α (eIF2α), which increased the cap-independent translation of activating transcription factor 4 (ATF4) [140]. ATF4 consequently translocated to the nucleus and induced transcription factor C/eBP homologous protein (CHOP), which is involved in apoptosis ( Figure 3→VIII) ( Table 2) [141]. Besides, treatment of both HCT116 and SW480 with 8c (200 µM) for 6 h mediated translocation of transcription factor Nrf2 from the cytoplasm into the nucleus, which induced different genes containing an antioxidant response element (ARE) in their promoter site to exert antioxidant response [89]. The GRP78 chaperone was found to be regulated on the protein level but only had a slight influence on the mRNA level recommending involvement of ER-associated protein degradation (ERAD) in the mode of action of 8c [89,141]. Considering all observations, it can be said that ROS plays a vital role in inducing apoptosis in the CRC cells. Among three complexes (8a-c), 8a can be considered as the most potent in terms of IC 50 values. However, experimentation using standard chemotherapeutics drug(s) (e.g., OXA, 5-FLU) could make the study more significant.

Immunogenic Cell Death
8c is responsible to mediate ER stress that induces a cascade of events leading to CRC cells death along with providing critical signals to visualize dying cancer cells to the immune system. This consequently introduces sustained immune response against the CRC; a phenomenon termed immunogenic cell death (ICD) [120]. ICD is characterized by secretion of immune-modulatory damage-associated molecular patterns (DAMPs), such as pre-apoptotic calreticulin (CRT) surface-exposure, extracellular adenosine triphosphate (ATP), and high mobility group box 1 (HMGB-1) [142,143]. The ER stress triggers a cascade of reactions that activate PERK. This activated PERK phosphorylates eIF2α which in turn translocate CRT to the cell membrane that is generally located at the lumen of the endoplasmic reticulum of colon cancer cells [144]. Exposure of CRT on cell membrane elicits an "eat me" signal which induces maturation of phagocyte dendritic cell (DC) as well as uptake tumor antigens ( Figure 3→IX) [145][146][147]. The other DAMP, HMGB-1 protein, residing in the nucleus moves to the extracellular space in the course of ICD and attach to pattern recognition receptors (PRRs) such as toll-like receptor 4 (TLR-4), receptor for advanced glycation end products (RAGE), and nuclear factor-κB (NF-κB) of DCs and presented antigens from dying tumor cells. This also accelerates DC maturation and migration. HMGB-1 functions as a crucial DAMP showing immune-stimulatory and proinflammatory effects [147,148] Lastly, extracellular adenosine triphosphate (ATP), released from dying tumor cell expresses a "find me" signal [147]. Released ATP binds to the purinergic P2RX7 receptors of dendritic cells and activated the (NOD)-like receptor protein 3 (NLRP3) inflammasome. This, in turn, stimulates tumor-specific cytotoxic T cells to secrete IFN-γ [120,147]. IFN-γ have pro-apoptotic and anti-proliferative functions such as inhibiting tumor angiogenesis, inducing regulatory T-cell apoptosis, and influencing M1 pro-inflammatory macrophages activity to suppress tumor progression [149].
Wernitznig et al. [120] described that treatment with 8c (100 µM) for 24 h upsurged CRT expression by approximately 7%, 10%, and 7% as well as extracellular ATP level by around 3%, 2.5%, and 2.6% in HCT116, HT15, and HT29 cell membrane, respectively, compared to the control. However, 8c increased the CRT level by 3.75 and 1.25-fold compared to CIS and OXA, respectively, as well as ATP level than both CIS and OXA by 1.36-fold in HT29 cells. Furthermore, the same concentration in HCT116 enhanced the release of HMGB-1 into the cytoplasm (Table 2) [120]. These findings consolidate that 8c could inhibit CRC cells proliferation via immunogenic death as well as ROS-mediated apoptosis and could be used as an alternative to OXA.

Anti-Metastasis Activity
Novel Ru(III)-based drug, Imidazolium-trans-tetrachloro(dimethylsulfoxide) imidazoleruthenium(III) or NAMI-A (12a) (Figure 6) was synthesized by Alessio et al. [163]. 12a displayed anti-proliferative property against CRC cells by virtue of its anti-metastasis activity. Unlike other Ru-based drugs, 12a focuses on the tumor microenvironment instead of showing direct cytotoxicity to the cell [164][165][166]. 12a interact with actin-like proteins of the cell surface and collagens of the extracellular matrix and reduced invasive cancer cells mobility [167]. 12a selectively targets surface adhesion receptor α5β1 integrin of colon cancer cells [88]. Highly invasive colon cell lines tend to express an increased level of α5β1 integrin [168], which is responsible for adhesion and migration of colon cancer cells through interacting with extracellular matrix proteins (ECM) [88,169,170]. According to Pelillo et al. [88], about 78% of cell adhesion is reduced by blocking α5β1 integrin site of HCT116 cells. 12a blocked both the steps of adhesion and migration of the tumor cells by impairing the contact between α5β1 integrin and fibronectin of HCT116 cells in an inverse concentration-dependent manner ( Figure 3→XII). 12a (1-10 µM) inversely decreased the attachment of fibronectin with α5β1 integrin by 38-25%. 12a (10-100 µM) also reduced the adhesion rate by 58-82%. Molecular insight of CRC revealed that 12a altered the expression of the genes encoding the α5 and β1 subunit and, therefore, decreased the number of α5β1 integrin receptor molecules (Table 2). 12a at a concentration of 1 µM downregulated α5 subunit encoding gene ITGA5 while 100 µM concentration upregulated ITGA5 expression up to 3.5-fold. Nonetheless, β1 subunit encoding gene ITGA1 did not respond to the alteration of concentrations [88]. Besides, the binding event activated autophosphorylation at the Tyr 397 site of the intracellular focal adhesion kinase (FAK) [170], which not only mediated tumor cell proliferation, survival, and migration [171] but also regulated the binding strength between integrins and ECM proteins [88]. 12a (1-0 µM) decreased nearly 70-15% level of p-Tyr 397 FAK [88].
The metastasis of CRC cells is influenced by the hepatic microenvironment [170]. Bergamo et al. [169] reported that normal epithelial colon cells and hepatocytes release different soluble factors involved in the transcription of genes of the tumor cells associated with tumor growth, invasion, and migration. 12a prevented transcription of those genes, thus inhibit the growth and dissemination of CRC cells. VEGF or MCP-1 either alone or in combination increased the migration ability of HCT116 cells. Exposure to 12a decreased VEGF or MCP-1 induced migration of HCT116 cells [169]. As Pelillo et al. did not use any standard chemotherapeutics drug(s) (e.g., OXA, 5-FLU), further investigations are required to determine the potency of 12a compared to standard drugs.

Lysosomal Dysfunction
Lysosomes contain various hydrolytic enzymes which degrade damaged proteins and organelles to regulate cellular functions [172]. However, releasing these hydrolase enzymes from lysosomes degrade other cytoplasmic organelles and lead to cell death [173]. Some Ru-complexes can be localized inside the lysosome, specifically where they cause lysosomal dysfunction [91,174,175]. Lysosomal dysfunction can be identified by unusual instigation of lysosomal enzymes, reduced lysosome-associated membrane proteins (LAMPs) expression as well as the permeability of lysosomes [176]. Disintegration of the lysosome induces the release of lysosomal hydrolases like cathepsin B from the lysosomal lumen to the cytosol, which makes the cells prone to lysosome-induced cell death ( Figure 3→XIII) [173].

Photodynamic Therapy
Photodynamic therapy (PDT) has emerged as a potential cancer therapy that is either used as a sole treatment or in combination with chemotherapy, surgery, and/or radiation [179]. PDT uses lights with appropriate wavelength to stimulate photosensitizer (PS) which mediates photochemical reaction to produce ROS and consequently kill tumors ( Figure 6) [180].
Ru ( Figure 7A) contain both photo-biological and photo-physical properties [92]. 14b was originally synthesized by Sherri MacFarland and this Ru(II)-based photosensitizer entered clinical trial to treat bladder cancer through PDT. A phase I clinical study was conducted with 14b (at 0.70 mgcm −2 dose) on six non-muscle-invasive bladder cancer patients (NCT03053635) and tumor relapse was not observed up to 180 days [181].
Ru(II) in 14a, and 14b makes the complexes specific towards cancer cells rather than normal cells and upon light irradiation increased singlet oxygen ( 1 O 2 ) quantum yield [182]. 14a and 14b induced fragmentation of DNA via photocleavage activities ( Figure 7B) [92,183]. According to Fong et al. [92], 14a (4 µM) and 14b (1 µM) exhibited photodynamic effects which cause photon-mediated complete death of CT-26 cells. However, both 14a (10 µM) and 14b (10 µM) showed minimal toxicity (less than 10%) in the dark. The maximum tolerated dose was recorded to 36 and 103 mg/kg for 14a and 14b, respectively. Besides, in vivo treatment of 14a and 14b modulated tumor cell regression. Four hours post-intrathecal administration of 14a (36 and 2 mg/kg), and 14b (53 and 5 mg/kg) in BALB/c mice, followed by irradiation with a continuous wave or pulsed light sources (λ = 525-530 nm, H = 192 Jcm −2 ) for 30 min (with 30 s on/off cycle) exhibited a higher tumor growth reducing efficacy after 24 h. 14a (2 mg/kg) and 14b (5 mg/kg) delayed the tumor growth for 8 and 9 days, respectively. Both the compounds increased survival time in a dose-dependent manner. However, 14b extended survival time by 5-fold compared to 14a [92]. Treatment with 14b also induced antitumor immunity in the colon cancer-containing mouse model [181]. Considering all observations, 14b could be considered as a promising PDT agent in CRC treatment. The Pt-based drug, Pt(II) 2,6-dipyrido-4-methyl-benzenechloride (PMB) also induced PDT-mediated DNA damage of CRC cells in a similar way [184]. Since 14b and PMB were not investigated under similar experimental conditions, thus the potency of 14b and PMB could not be compared.

CRC Diagnosis
RuNPs-based nanoformulations could facilitate the early diagnosis of CRC [30,185,186]. Xu et al. [30] constructed hollow mesoporous RuNPs (HMRuNPs) which are efficient in in vivo tumor imaging, drug loading, and combined treatment for CRC. Hollow mesoporous Ru containing fluorescent complex with anticancer activity (RBT) and bispecific antibodies (SS-Fc, anti-CD16, and anti-CEA) (15a) selectively accumulated into CRC cells by both active and passive targeting. Active targeting is mediated through antibody SS-Fc which can detect carcinoembryonic (CEA) antigen on CRC cell lines (i.e., HCT116, SW480, HT29, and Lovo) and attach with natural killer (NK) cells to induce an immune response. Passive targeting facilitates drug accumulation by the EPR effect. Moreover, the same compounds also have therapeutic effects. Treating BALB/c mice containing systemically administrated CT26-CEA tumor with 15a (5 mg/kg) for every three days for a total of three treatments released RBT that generated ROS and appointed NK cells to initiate immune response, which in turn led to apoptosis and necrosis in CRC [30].

CRC Treatment
Conventional chemotherapy, as well as radiation therapy, conveys side effects and other limitations such as drug resistance [187]. The application of nanoformulations could overcome such shortcomings [188,189]. The advent of RuNPs offers excellent anticancer activity due to the high photothermal conversion rate, multiple oxidation states, and valence states [30]. Heffeter et al. [190] reported that micelle-like carriers (MC-8b) and nanoform of the established drug 8b sidestepped the limitations of 8b aqueous solutions undergo rapid hydrolysis to yield water-insoluble 8b aqua complex, [mer,trans-[Ru(III)Cl 3 (Hind) 2 (H 2 O)]. Nevertheless, MC-8b (0.3 mg/mL 8b) solutions were stable at 4 • C for three months regarding precipitation. After 72 h of incubation, MC-8b was found to be more active than 8b against HCT116 cells (3.91-fold) and Lovo cells (4.88-fold) in terms of their IC 50 values. Moreover, MC-8b (IC 50 = 41 µM) provided rapid onset of anticancer activity than 8b (IC 50 value of 135 µM) within only 1 h in HCT116 cells. Additionally, treatment of HCT116 cells with MC-8b (25 µM) exhibited higher apoptosis potential than 8b by 6-fold. Western blotting indicated that MC-8b increased the expression of p53, phosphorylated P38, and JNK while decreased caspase-7 and PARP expression [190].
Besides, Zhu et al. [31] developed other Ru-based nanozymes, hollow Ru@CeO 2 yolk-shell nanozymes in conjugation with antitumor drug Ru-complex (RBT) along with resveratrol (Res) and coated with DEPG (16a) which exhibited anti-metastasis and antitumor activity in orthotopic CRC through dual-chemotherapy/Photothermal therapy (PTT) with in situ oxygen supply [31]. Moreover, recurrence of more than 60% of post-surgical colorectal tumors is associated with the liver while more than 35% of all metastases occur solely in the liver [191,192]. 16a with near infrared (NIR) efficiently inhibited intestinal, lung, and liver metastasis. 16a contains antitumor Ru drugs, RBT, and Res, which exerted dual chemotherapeutic efficiency, while Ru@CeO 2 holds efficient light-to-heat conversion potency. At the same time, 16a catalyzed H 2 O 2 to O 2 in the tumor microenvironment (TME) and thereby overcame hypoxia by achieving in situ O 2 supply and reduce HIF-1α hypoxic staining signal. Treatment with 16a (5 mg/kg) for every three days for a total of three treatments overcame tumor hypoxia and obtained dual-chemotherapy/PTT in BALB/c mice bearing CT-26 cells. The excellent biocompatibility of the nanozyme is achieved due to the DPEG coating that prevented the occurrence of hemolysis even at a high dose concentration [31].

Phase I Dose-Escalation Studies (Phase Ib Clinical Trials)
Dose escalation is an integral part of the phase I study which carefully looks for the optimal dose of a new drug to avoid therapeutic overdoses. In a dose-escalation study, the dosage of a drug is gradually increased until the side effects appear. Such a study is conducted on humans to assess the pharmacokinetics, pharmacodynamics, and safety of a new drug [199]. Rademaker-Lakhai et al. [200] conducted a phase I doseescalation study with 12a on seven CRC patients (n = 24, having solid tumors). 12a at a dose concentration of 2.4-38.4 mg/m 2 /day caused no drug-induced toxicity. However, a dose concentration of 76.8-115 mg/m 2 /day resulted in causing diarrhea, phlebitis, and fatigue, while 400-500 mg/m 2 /day dose caused skin blisters lasting up to several months, which caused extreme pain. Considering all these data, the prescribed dose was set as 300 mg/m 2 /day. However, this study did not provide effects of 12a specifically on CRC patients rather gave a generalized overview of the effects of 12a on 24 patients. Furthermore, in the phase I dose-escalation study 12a stabilized disease for 21 weeks in a patient with lung cancer which prompted to organize a phase I/II trial on 32 non-small cell lung cancer patients in combination with gemcitabine [200,201]. Lentz et al. [202] performed a phase I dose-escalation study with 8b on two CRC patients (n = 7, having various types of solid tumors). Intravenous administration of 8b escalating from 25 to 600 mg (equivalent to 5-120.8 mg of Ru) twice weekly over 3 weeks caused no dose-limiting toxicity. Nonetheless, 8b comes up with limitations regarding low solubility that makes it challenging to obtain proper dosage. Hence, its analogous sodium salt 8c is largely used in clinical trials, which provides 35-fold better solubility [138]. In the study of Thompson et al. [203], 34 patients having solid tumors were treated with 8c among whom 10 CRC patients were reported. 8c (20-780 mg/m 2 ) was infused in intravenous route on day 1, 8, and 15 of 28 days cycles. The maximum tolerated dose (MTD) was reported 625 mg/m 2 with minor side effects [203,204]. However, none of the aforementioned phase I dose-escalation studies of 12a, 8a, and 8b are listed on the clinicaltrial.gov website (www.clinicaltrial.gov) [205].
The only registered study was conducted by Burris et al. (trial registration number: NCT01415297) [206]. The phase Ib clinical trial was conducted to investigate MTD of 8c (20-780 mg/m 2 ) on 11 CRC patients (n = 46 patients having advanced solid tumors) and found similar MTD (625 mg/m 2 ). However, ≥20% of the patients experienced adverse events that emerged from treatment which include fatigue, nausea, vomiting, dehydration, and anemia. In total, 59% of patients experienced ≤grade 2 and 37% of patients experienced grade 3 adverse effects though no patient was reported to have grade 4 adverse effects. It should be noted that both the studies did not present specific descriptions of the adverse effects on CRC patients [206].
Pt-based drugs, CIS, CAR, and picoplatin (PIC) have been used in combination with other chemotherapeutic drugs in clinical trials of CRC [205]. A Phase I clinical trial (NCT00465725) on various solid tumors, including CRC was studied using PIC only as an anti-proliferative agent [205]. A phase I clinical and pharmacological study with PIC revealed the MTD (150 mg/m 2 ). Moderate level of anorexia, nausea, vomiting and transient metallic taste was evident and there was no significant sign of alopecia [207]. Peripheral neuropathy is the main disadvantage of OXA-based chemotherapy [208][209][210], whereas some Ru-based complexes (mentioned in this section) or PIC did not show such neurotoxic effects [211]. Therefore, Ru-based complexes or PIC could be used as an alternative to OXA in CRC patients with compromised neuronal function.
Koch et al. [218] reported that some Ru-complexes (i.e., bipyridine, terpyridine, and phenanthroline Ru-complexes) acted as cholinesterase inhibitors in vitro and induced hind limb paralysis, respiratory distress, and death in respiratory failure as well as block curariform at the neuromuscular junction in in vivo mice model [218]. Furthermore, Ru is reported to inhibit Ca 2+ uptake by mitochondria which possibly contributed to β-adrenergic and neuromuscular blocking [219][220][221]. Kruszyna et al. [221] described that some Runitrosyl complexes at a concentration of (55-63 mg/kg) induced rapid death (after 10 min) while the rest of the complexes mediated death after 4-7 days a concentration ranging from 8.9 to 127 mg/kg. Ru is also retained in muscle and bone, rising concern about their long-term effect [221]. Thus, the toxicity of Ru-based drug candidates is a considerable issue before clinical applications. Ru-complexes including 1c, 1d, 4a, 4f, 6a, and 6b, displayed higher cytotoxic potential than OXA [27,77,87,[115][116][117] while 5-FLU were found to be less potent than 4a and 4f in vitro and 3d and 8b in vivo [27,81,115,138]. Although the anticancer activity of Ru-complexes has been explored largely, their extended toxicity studies were not investigated. Therefore, preclinical chronic toxicity studies should be performed before considering these as potential drugs of CRC.

Conclusions
Ru-complexes displayed promising anticancer activities in the treatment of CRC. Ru(III) and Ru(II) were the most investigated oxidation states against all cancer cells including CRC cells, where the former one acts as a prodrug and converts to Ru(II) in the tumor microenvironment. Ru(II)-complexes displayed more reactivity compared to Ru(III) complexes. Many Ru-complexes were found to be more efficient than the Ptbased drugs; therefore, Ru could be used as an alternative to Pt-based drugs. Moreover, some Ru-complexes were reported to be more effective compared to the conventional chemotherapeutic drug (5-FLU) which has been used as first-line treatment against CRC. Though Ru conjugation with organic molecules could enhance anticancer activity through a synergistic effect [82][83][84], sometimes Ru-complexes are found to be less potent compared to parent organic molecules against CRC cells [90,222]. While conjugation of Ru-compounds with RuNps enhanced cellular uptake, selectivity, and drug delivery in CRC cells. Therefore, higher attention should be given to this field. Finally, extensive preclinical studies should be formed to confirm the efficacy, elucidating the potential mechanism of action(s), and toxicity of Ru-complexes or Ru-nanoformulations before considering them as potent drug candidates against CRC.

Conflicts of Interest:
The authors declare no conflict of interest.