Anticancer Ruthenium Research Articles

Binding of Organometallic Ruthenium Anticancer Complexes to DNA: Thermodynamic Base and Sequence Selectivity.

Organometallic ruthenium(II) complexes [(η6-arene)Ru(en)Cl][PF6] (arene = benzene (1), p-cymene (2), indane (3), and biphenyl (4); en = ethylenediamine) are promising anticancer drug candidates both in vitro and in vivo. In this paper, the interactions between ruthenium(II) complexes and 15-mer single- and double-stranded oligodeoxynucleotides (ODNs) were thermodynamically investigated using high performance liquid chromatography (HPLC) and electrospray ionization mass spectroscopy (ESI-MS). All of the complexes bind preferentially to G8 on the single strand 5′-CTCTCTT7G8T9CTTCTC-3′ (I), with complex 4 containing the most hydrophobic ligand as the most reactive one. To the analogs of I (changing T7 and/or T9 to A and/or C), complex 4 shows a decreasing affinity to the G8 site in the following order: -AG8T- (K: 5.74 × 104 M−1) > -CG8C- > -TG8A- > -AG8A- > -AG8C- > -TG8T- (I) ≈ -CG8A- (K: 2.81 × 104 M−1). In the complementary strand of I, the G bases in the middle region are favored for ruthenation over guanine (G) bases in the end of oligodeoxynucleotides (ODNs). These results indicate that both the flanking bases (or base sequences) and the arene ligands play important roles in determining the binding preference, and the base- and sequence-selectivity, of ruthenium complex in binding to the ODNs.

Structure-activity relationships of anticancer ruthenium(II) complexes with substituted hydroxyquinolines

8-Hydroxyquinolines (HQ), including clioquinol, possess cytotoxic properties and are widely used as ligands for metal-based anticancer drug research. The number and identity of substituents on the HQ can have a profound effect on activity for a variety of inorganic compounds. Ruthenium complexes of HQ exhibit radically improved potencies, and operate by a new, currently unknown, mechanism of action. To define structure-activity relationships (SAR), a family of 22 Ru(II) coordination complexes containing mono-, di- and tri-substituted hydroxyquinoline ligands were synthesized and their biological activity evaluated. The complexes exhibited promising cytotoxic activity against a cancer cell line, and the SAR data revealed the 2- and 7-positions as key sites for the incorporation of halogens to improve potency. The Ru(II) complexes potently inhibited translation, as demonstrated by an in-cell translation assay. The effects were seen at 2–15-fold higher concentrations than those required to observe cytotoxicity, suggesting that prevention of protein synthesis may be a primary, but not the exclusive mechanism for the observed cytotoxic activity.

Bioorthogonal Catalytic Activation of Platinum and Ruthenium Anticancer Complexes by FAD and Flavoproteins.

Recent advances in bioorthogonal catalysis promise to deliver new chemical tools for performing chemoselective transformations in complex biological environments. Herein, we report how FAD (flavin adenine dinucleotide), FMN (flavin mononucleotide), and four flavoproteins act as unconventional photocatalysts capable of converting PtIV and RuII complexes into potentially toxic PtII or RuII−OH2 species. In the presence of electron donors and low doses of visible light, the flavoproteins mini singlet oxygen generator (miniSOG) and NADH oxidase (NOX) catalytically activate PtIV prodrugs with bioorthogonal selectivity. In the presence of NADH, NOX catalyzes PtIV activation in the dark as well, indicating for the first time that flavoenzymes may contribute to initiating the activity of PtIV chemotherapeutic agents.

Bioorthogonal Catalytic Activation of Platinum and Ruthenium Anticancer Complexes by FAD and Flavoproteins

AbstractRecent advances in bioorthogonal catalysis promise to deliver new chemical tools for performing chemoselective transformations in complex biological environments. Herein, we report how FAD (flavin adenine dinucleotide), FMN (flavin mononucleotide), and four flavoproteins act as unconventional photocatalysts capable of converting PtIV and RuII complexes into potentially toxic PtII or RuII−OH2 species. In the presence of electron donors and low doses of visible light, the flavoproteins mini singlet oxygen generator (miniSOG) and NADH oxidase (NOX) catalytically activate PtIV prodrugs with bioorthogonal selectivity. In the presence of NADH, NOX catalyzes PtIV activation in the dark as well, indicating for the first time that flavoenzymes may contribute to initiating the activity of PtIV chemotherapeutic agents.

Controlled synthesis and size effects of multifunctional mesoporous silica nanosystem for precise cancer therapy

Nanomaterials-based drug delivery systems display potent applications in cancer therapy, owing to the enhanced permeability and retention effect and diversified chemical modification. In this study, we have tailored and synthesized different sized mesoporous silica nanoparticles (MSNs) through reactant control to investigate the relevancy of nanoparticle size toward anticancer efficacy and suppressing cancer multidrug resistance. The different sized MSNs loaded with anticancer ruthenium complex (RuPOP) and conjugated with folate acid (FA) to enhance the selectivity between cancer and normal cells. The nanosystem (Ru@MSNs) can specifically recognize HepG2 hepatocellular carcinoma cells, thus enhance accumulation and selective cellular uptake. The smaller sized (20 nm) Ru@MSNs exhibit higher anticancer activity against HepG2 cells, while the larger sized (80 nm) Ru@MSNs exhibit higher inhibitory effect against DOX-resistant hepatocellular carcinoma cells (R-HepG2). Moreover, Ru@MSNs induced ROS overproduction in cancer cells, leading to DNA damage and p53 phosphorylation, consequently promoting cancer cells apoptosis. Ru@MSNs (80 nm) also inhibited ABCB1 and ABCG2 expression in R-HepG2 cells to prevent drug efflux, thus overcome multidrug resistance. Ru@MSNs also inhibited tumor growth in vivo without obvious toxicity in major organs of tumor-bearing nude mice. Taken together, these results verify the size effects of MSNs nanosystem for precise cancer therapy.

Studies on hydrolysis mechanism of anticancer ruthenium drug ImH[trans-Ru(Im)2Cl4] via ABEEMσπ polarizable force field combined with QM and MD-FEP

We used ABEEMσπ(atom-bond electronegativity equalization method) polarizable force field(ABEEMσπ PFF) method combined with QM and molecular dynamics-free energy perturbation(MD-FEP) methods to investigate the function of water molecules in hydrolysis process of ImH[trans-Ru(Im)2Cl4](ICR). The activation free energies obtained via MD-FEP calculation are in fair agreement with the experimental data. In addition, QM/MM(ABEEM) rationally describes the charge distributions and the electrostatic interaction between molecules. ABEEMσπ fluctuating charge model has the following good characteristics: (1) not only atomic charge regions but also σ, π bond and lone pair charge regions are explicitly represented for a molecule; (2) the region charges are geometry dependent and calculated from time to time in the dynamic simulation without any iterative procedure so that its performance is time-saving compared with the Drude model and induced dipole model.

Differential Cytotoxicity, Cellular Uptake, Apoptosis and Inhibition of BRCA1 Expression of BRCA1-Defective and Sporadic Breast Cancer Cells Induced by an Anticancer Ruthenium(II)-Arene Compound, RAPTA-EA1.

The RAPTA-EA1 complex [ruthenium(II)-arene 1,3,5-triaza-7-phosphaadamantane (pta) complex with an arene-tethered ethacrynic acid ligand] has been reported to overcome drug resistance that developed due to the current use of platinum-based treatments. However, the exact mechanism of action of RAPTA-EA1 remains largely unexplored and unknown. Here we have further studied the effect of RAPTA-EA1 on BRCA1-defective HCC1937 breast cancer cells and compared its effects on BRCA1-competent MCF-7 breast cancer cells. HCC1937 and MCF-7 breast cancer cells were treated with the RAPTA-EA1 complex. The cytotoxicity of ruthenium-induced cells was evaluated by a MTT assay. Cellular uptake of ruthenium was determined by ICP-MS. Cell cycle and apoptosis were assessed using a flow cytometer. Expression of BRCA1 mRNA and its encoded protein was quantitated by a real-time RT-PCR and Western blotting. Differences in cytotoxicity were correlated with the differential accumulations of ruthenium and the induction of apoptosis. The ruthenium complex caused dramatically more damage to the BRCA1 gene in the BRCA1-defective HCC1937 cells than to the BRCA1-competent MCF-7 cells. It decreased the expression of BRCA1 mRNA in the BRCA1-competent cells, while in contrast, its expression increased in the BRCA1-defective cells. However, the expression of the BRCA1 protein was significantly reduced in both types of breast cancer cells. The results presented here have demonstrated a differential cellular response for the BRCA1-defective and BRCA1-competent breast cancer cells to RAPTA-EA1. These findings have provided more insight into the actions and development of the ruthenium-based compounds for use for the treatment of breast cancer.

The development of anticancer ruthenium(ii) complexes: from single molecule compounds to nanomaterials.

Cancer is rapidly becoming the top killer in the world. Most of the FDA approved anticancer drugs are organic molecules, while metallodrugs are very scarce. The advent of the first metal based therapeutic agent, cisplatin, launched a new era in the application of transition metal complexes for therapeutic design. Due to their unique and versatile biochemical properties, ruthenium-based compounds have emerged as promising anti-cancer agents that serve as alternatives to cisplatin and its derivertives. Ruthenium(iii) complexes have successfully been used in clinical research and their mechanisms of anticancer action have been reported in large volumes over the past few decades. Ruthenium(ii) complexes have also attracted significant attention as anticancer candidates; however, only a few of them have been reported comprehensively. In this review, we discuss the development of ruthenium(ii) complexes as anticancer candidates and biocatalysts, including arene ruthenium complexes, polypyridyl ruthenium complexes, and ruthenium nanomaterial complexes. This review focuses on the likely mechanisms of action of ruthenium(ii)-based anticancer drugs and the relationship between their chemical structures and biological properties. This review also highlights the catalytic activity and the photoinduced activation of ruthenium(ii) complexes, their targeted delivery, and their activity in nanomaterial systems.

Cytogenotoxic effects of two potential anticancer Ruthenium(III) Schiff Bases complexes

Introduction: Treatment of cancer has been subject of great interest. Researchers are continuously searching for new medicines. In this sense, ruthenium complexes have big potential. Some evidences suggest that ruthenium compounds possess anticancer activities. We synthesized two recently published ruthenium(III) complexes with bidentate O,N and tridentate O,O,N Schiff bases derived from 5-substituted salicylaldehyde and aminophenol or anilineare. These compounds showed affinity for binding to the DNA molecule, however, insufficient data are available regarding their possible toxic effects on biological systems.Methods: In the present study we evaluated genotoxic, cytotoxic, and cytostatic effects of Na[RuCl2(L1)2] and Na[Ru(L2)2], using the Allium cepa assay.Results: Different toxic effects were observed depending on the substance, tested concentration, and endpoint measured. In general, the tested compounds significantly lowered the root growth and mitotic index values as compared to the control group. Additionally, a wide range of abnormal mitotic stages, both clastogenic and non-clastogenic were observed in the treated cells. Na[RuCl2(L1)2] significantly increased the frequency of sticky metaphases, chromosome bridges, micronuclei, impaired chromosome segregation, as well as number of apoptotic and necrotic cells over the controls. In contrast, Na[Ru(L2)2] did not show significant evidence of genotoxicity with regard to chromosome aberrations and micronuclei, however, significant differences were detected in the number of apoptotic and necrotic cells when the highest concentration was applied.Conclusions: In this study we demonstrated antiproliferative effects of Na[RuCl2(L1)2] and Na[Ru(L2)2]. At clinical level, these results could be interesting for further studies on anticancer potential of the ruthenium(III) complexes using animal models.

Thirty Years of the Drug Candidate NAMI‐A and the Myths in the Field of Ruthenium Anticancer Compounds: A Personal Perspective

As anticipated in the title, this contribution is basically divided into two, strictly connected, parts. The first is a personal overview of the ruthenium drug candidate NAMI‐A, almost 30 years after its synthesis and the discovery of its unprecedented antimetastatic properties in animal models at nontoxic dosages. The sections relating to the chemical and biological behavior of the complex, and the hypotheses on its mechanism(s) of action, are kept to a minimum, whereas more space is devoted to discussion of the results of the clinical investigations. The second part deals in detail with a number of undemonstrated misconceptions (or myths) that, over the years, have thrived around NAMI‐A and other ruthenium drug candidates, thus negatively affecting the whole field of Ru anticancer drugs.

A Macrocyclic Ruthenium(III) Complex Inhibits Angiogenesis with Down‐Regulation of Vascular Endothelial Growth Factor Receptor‐2 and Suppresses Tumor Growth In Vivo

A macrocyclic ruthenium(III) complex [RuIII (N2 O2 )Cl2 ]Cl (Ru-1) is reported as an inhibitor of angiogenesis and an anti-tumor compound. The complex is relatively non-cytotoxic towards endothelial and cancer cell lines in vitro, but specifically inhibited the processes of angiogenic endothelial cell tube formation and cancer cell invasion. Moreover, compared with known anti-cancer ruthenium complexes, Ru-1 is distinct in that it suppressed the expression of vascular endothelial growth factor receptor-2 (VEGFR2), and the associated downstream signaling that is crucial to tumor angiogenesis. In addition, in vivo studies showed that Ru-1 inhibited angiogenesis in a zebrafish model and suppressed tumor growth in nude mice bearing cancer xenografts.

BRCA1-Associated Triple-Negative Breast Cancer and Potential Treatment for Ruthenium-Based Compounds.

Triple-negative breast cancer (TNBC) is defined by the absence of expression of estrogen receptor (ER), progesterone receptor (PR), and a lack of overexpression or amplification of human epidermal growth factor receptor 2 (HER2). The clinicopathological characteristics of TNBC include a high grading, a high rate of cell proliferation and a greater degree of chromosomal rearrangement. Patients with triple-negative breast cancer are more likely to be drug resistant and more difficult to treat, and are also frequently BRCA1 mutants. Methylation of the BRCA1 promoter region is associated with a reduction of the BRCA1 mRNA level. TNBC patients with a methylated BRCA1 had a better disease-free survival compared with those with non-methylated BRCA1. From a therapeutic perspective, the expression level of BRCA1 has been a major determinant of the responses to different classes of chemotherapy. BRCA1-dysfunctional tumors are hypersensitive to DNA damaging chemotherapeutic agents like platinum drugs. Although platinum based drugs are currently widely used as conventional chemotherapeutic drugs in breast cancer chemotherapy, their use has several disadvantages. It is therefore of interest to seek out alternative therapeutic metal-based compounds that could overcome the limitations of these platinum based drugs. Ruthenium-based compounds could be the most promising alternative to the platinum drugs. This review highlights the use of BRCA1 as a predictive marker as well as for a potential drug target for anticancer ruthenium compounds.

Lipooligosaccharides as Amphiphiles to Build Liposomes for Effective Drug Delivery: The Case of Anticancer Ruthenium Complex‐Based Aggregates

AbstractLiposomes are complex aggregates, often including polyethylene glycol (PEG) to expand their life span in vivo, although their full biocompatibility is still questioned. With the aim to suitably replace PEG within liposomal formulations, here we propose a new liposomes formulation, which includes an amphiphilic molecule of natural origin: the lipooligosaccharide (LOS) from the Gram‐negative bacterium Rhizobium rubi. LOS architecture is bifunctional: the lipid moiety at one terminus promotes its insertion into the liposome, the other terminus is hydrophilic and in this case presents an oligosaccharide motif similar to the human Lewis B antigen. Liposomes were prepared by co‐formulating de‐O‐acylated LOS (de‐LOS) with the lipid 1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phosphocholine (POPC) and the anticancer nucleolipid‐based Ru(III) complex, ToThyRu. In‐depth microstructural characterization shows that de‐LOS containing liposomes are stable aggregates. In vitro preliminary bioscreens have disclosed their negligible toxic profile and a good uptake in MCF‐7 and HaCaT cells. The results validate the use of lipooligosaccharides in formulating liposomes and pave the way to their use in drug delivery applications.

Ruthenium Complexes: An Emerging Ground to the Development of Metallopharmaceuticals for Cancer Therapy.

GLOBOCAN 2012 estimates 14.1 million new cancer cases and 8.2 million cancer-related deaths worldwide. Cancer is rapidly becoming a major public health concern in India as well, with the number of new cancer cases anticipated to double within the next 20 years. The percentage of currently approved metallodrugs is very low, in contrast to the majority of drugs available as organic compounds. The search for alternative drugs to cisplatin, carboplatin and other derivatives is highly needed due to their severe side effects including nephrotoxicity and neurotoxicity. Ruthenium, among other transition metal complexes appears to be a possible candidate for cancer therapy in the near future. The most significant rationale is ruthenium's octahedral chemistry and greater propensity to undergo redox reactions. The hypoxic environment of tumors favors the reduction of inert ruthenium (III) to active ruthenium (II) which opens new prospects for the development of novel prodrugs. Although studies suggest that ruthenium complexes penetrate well within the tumor cells and bind effectively to DNA, its binding to proteins is not very well explained. Ruthenium complexes are presently receiving great attention in the fields of biological, pharmaceutical and medicinal chemistry as anticancer agents. This review poses a comprehensive overview of the studies on competent anticancer ruthenium complexes and the role of these metal complexes in relation to their anticancer properties as well as those under clinical trials.

Synthesis of novel anticancer ruthenium–arene pyridinylmethylene scaffolds via three-component reaction

A novel three components approach for the synthesis of bioactive Ru–arene pyridinylmethylene complexes has been developed using pyridine carboxaldehyde, amino pyridine and dichloro (p-cymene) ruthenium(II) dimer as starting materials. These scaffolds were screened for their anticancer activity against breast cancer (MCF7) and human Epitheloid Cervix Carcinoma (HeLa) cell line. It was established that compounds [(η6-pcymene)RuCl(κ2-N,N-(3,5-dinitro-pyridin-2-yl)-pyridin-2-ylmethylene-amine)]PF6 (4o), [(η6-pcymene)RuCl(κ2-N,N-N-(3,5-dibromo-pyridin-2-yl)-pyridin-2-ylmethylene-amine)]PF6 (4c), [(η6-pcymene)RuCl(κ2-N,N-(3,5-dibromo-6-methylpyridin-2-yl)-pyridin-2-ylmethylene-amine)]PF6 (4j) and [(η6-pcymene)RuCl(κ2-N,N-3(3-bromo-5-methyl-pyridin-2-yl)-pyridin-2-ylmethylene-amine)]PF6 (4b) were significantly active against both the cell lines.

Testing the photo-sensitive nanocomposite system for potential controlled metallo-drug delivery

Photo-responsive drug release systems are promising for drug delivery applications due to many benefits compared to conventional chemotherapy such as targeted, controlled release of a drug and reduced toxicity to healthy tissues. In this work, we report synthesis of the nanocomposite system based on carrier TiO2 nanoparticles and potential anticancer ruthenium complex, with light controllable release properties. Nanocomposite system showed biological activity and induced the generation of free radicals, which are implied in the efficient cell killing. The drug release tests demonstrated sustained release of the transition metal complex from the surface implying the potency for the controlled drug delivery system. Taking into account photoactivity of the Ru-complex, in the next step we have investigated the influence of green light on the rate of the complex release, and the results showed dependence of the Ru-complex release from the surface of TiO2 nanoparticles on the applied laser energy. Therefore, these characteristics make this nanocomposite system promising for the photo-responsive chemotherapy.

Amberlite IR-120(H)-mediated “on water” synthesis of novel anticancer ruthenium(ii)–p-cymene 2-pyridinylbenzothiazole (BTZ), 2-pyridinylbenzoxazole (BOZ) & 2-pyridinylbenzimidazole (BIZ) scaffolds

A class of novel Ru(ii)–p-cymene BTZ, BOZ & BIZ complexes have been synthesized “on water” using Amberlite IR-120(H) resin and their anticancer activities were evaluated.

DNA Damage Response Checkpoint Activation Drives KP1019 Dependent Pre-Anaphase Cell Cycle Delay in S. cerevisiae.

Careful regulation of the cell cycle is required for proper replication, cell division, and DNA repair. DNA damage–including that induced by many anticancer drugs–results in cell cycle delay or arrest, which can allow time for repair of DNA lesions. Although its molecular mechanism of action remains a matter of debate, the anticancer ruthenium complex KP1019 has been shown to bind DNA in biophysical assays and to damage DNA of colorectal and ovarian cancer cells in vitro. KP1019 has also been shown to induce mutations and induce cell cycle arrest in Saccharomyces cerevisiae, suggesting that budding yeast can serve as an appropriate model for characterizing the cellular response to the drug. Here we use a transcriptomic approach to verify that KP1019 induces the DNA damage response (DDR) and find that KP1019 dependent expression of HUG1 requires the Dun1 checkpoint; both consistent with KP1019 DDR in budding yeast. We observe a robust KP1019 dependent delay in cell cycle progression as measured by increase in large budded cells, 2C DNA content, and accumulation of Pds1 which functions to inhibit anaphase. Importantly, we also find that deletion of RAD9, a gene required for the DDR, blocks drug-dependent changes in cell cycle progression, thereby establishing a causal link between the DDR and phenotypes induced by KP1019. Interestingly, yeast treated with KP1019 not only delay in G2/M, but also exhibit abnormal nuclear position, wherein the nucleus spans the bud neck. This morphology correlates with short, misaligned spindles and is dependent on the dynein heavy chain gene DYN1. We find that KP1019 creates an environment where cells respond to DNA damage through nuclear (transcriptional changes) and cytoplasmic (motor protein activity) events.

Functionalized Multiwalled Carbon Nanotubes as Carriers of Ruthenium Complexes to Antagonize Cancer Multidrug Resistance and Radioresistance.

Multidrug resistance and radioresistance are major obstacles for successful cancer therapy. Due to the unique characteristics of high surface area, improved cellular uptake, and the possibility to be easily bound with therapeutics, carbon nanotubes (CNTs) have attracted increasing attention as potential nanodrug delivery systems. In this study, a CNT-based radiosensitive nanodrug delivery system was rationally designed to antagonize the multidrug resistance in hepatocellular carcinoma. The nanosystem was loaded with a potent anticancer ruthenium polypyridyl complex (RuPOP) via π-π interaction and formation of a hydrogen bond. The functionalized nanosystem (RuPOP@MWCNTs) enhanced the cellular uptake of RuPOP in liver cancer cells, especially drug-resistant R-HepG2 cells, through endocytosis. Consistently, the selective cellular uptake endowed the nanosystem amplified anticancer efficacy against R-HepG2 cells but not in normal cells. Interestingly, RuPOP@MWCNTs significantly enhanced the anticancer efficacy of clinically used X-ray against R-HepG2 cells through induction of apoptosis and G0/G1 cell cycle arrest, with the involvement of ROS overproduction, which activated several downstream signaling pathways, including DNA damage-mediated p53 phosphorylation, activation of p38, and inactivation of AKT and ERK. Moreover, the nanosystem also effectively reduces the toxic side effects of loaded drugs and prolongs the blood circulation in vivo. Taken together, the results demonstrate the rational design of functionalized carbon nanotubes and their application as effective nanomedicine to overcome cancer multidrug resistance.

Activation of p53 mediated glycolytic inhibition-oxidative stress-apoptosis pathway in Dalton's lymphoma by a ruthenium (II)-complex containing 4-carboxy N-ethylbenzamide.

There is a general agreement that most of the cancer cells switch over to aerobic glycolysis (Warburg effect) and upregulate antioxidant enzymes to prevent oxidative stress induced apoptosis. Thus, there is an evolving view to target these metabolic alterations by novel anticancer agents to restrict tumor progression in vivo. Previously we have reported that when a non toxic dose (10 mg/kg bw i.p.) of a novel anticancer ruthenium(II)-complex containing 4-carboxy N-ethylbenzamide; Ru(II)-CNEB, was administered to the Dalton's lymphoma (DL) bearing mice, it regressed DL growth by inducing apoptosis in the DL cells. It also inactivated M4-LDH (M4-lactate dehydrogenase), an enzyme that drives anaerobic glycolysis in the tumor cells. In the present study we have investigated whether this compound is able to modulate regulation of glycolytic inhibition-apoptosis pathway in the DL cells in vivo. We observed that Ru(II)-CNEB could decline expression of the inducible form of 6-phosphofructo-2-kinase (iPFK2: PFKFB3), the master regulator of glycolysis in the DL cells. The complex also activated superoxide dismutase (the H2O2 producing enzyme) but declined the levels of catalase and glutathione peroxidase (the two H2O2 degrading enzymes) to impose oxidative stress in the DL cells. This was consistent with the enhanced p53 level, decline in Bcl2/Bax ratio and activation of caspase 9 in those DL cells. The findings suggest that Ru(II)-CNEB is able to activate oxidative stress-apoptosis pathway via p53 (a tumor supressor protein) mediated repression of iPFK2, a key glycolytic regulator, in the DL cells in vivo.