KP1019, a new redox-active anticancer agent--preclinical development and results of a clinical phase I study in tumor patients

The promising drug candidate indazolium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] (KP1019) is the second Ru-based anticancer agent to enter clinical trials. In this review, which is an update of a paper from 2006 (Hartinger et al., J. Inorg. Biochem. 2006, 100, 891-904), the experimental evidence for the proposed mode of action of this coordination compound is discussed, including transport into the cell via the transferrin cycle and activation by reduction. The results of the early clinical development of KP1019 are summarized in which five out of six evaluated patients experienced disease stabilization with no severe side effects.     

Serum-protein interactions with anticancer Ru(III) complexes KP1019 and KP418 characterized by EPR

The compounds imidazolium [trans-[RuCl₄(1H-imidazole)₂] (KP418) and indazolium [trans-RuCl₄(1H-indazole)₂] (KP1019) both show significant anticancer activity, with the latter recently having completed phase I clinical trials. An important component of this success has been associated with targeted delivery of the complexes to cancer cells by serum proteins. In this study, electron paramagnetic resonance (EPR) measurements, combined with incubation under physiological conditions, and separation of protein-bound fractions, have been used to characterize the interactions of these complexes with human serum albumin (hsA), human serum transferrin (hsTf) apoprotein, and whole human serum. The strong EPR signals observed in these experiments demonstrate that both complexes are primarily retained in the 3+ oxidation state in the presence of serum components. Rapid, noncovalent binding of KP1019 was observed in the presence of both hsA and serum, indicating that the predominant interactions occur within the hydrophobic binding sites of hsA. This sequestering process correlates with the low levels of side effects observed in clinical trials of the complex. At longer incubation times, the noncovalently bound complexes are converted slowly to a protein-coordinated form. Noncovalent interactions are not observed in the presence apo-hsTf, where only slow binding of KP1019 via ligand exchange with the protein occurs. By contrast, hydrophobic interactions of KP418 with hsA only occur with the aquated products of the complex, a process that also dominates in serum. In the presence of apo-hsTf, KP418 interacts directly with the protein through exchange of ligands, as observed with KP1019.     

Influence of ascorbic acid on the activity of the investigational anticancer drug KP1019

Ascorbic acid has been previously discussed to have antitumor potential through its interaction with transition metal ions such as iron and copper. Furthermore, ascorbic acid may act as a reducing agent for Ru(III) compounds such as indazolium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] (KP1019), an investigational anticancer drug which is supposed to be activated by reduction, prior to binding to cellular target proteins. Therefore, we investigated the influence of ascorbic acid on the activity of this antitumor metal complex in cell culture studies. We show that co-incubation of equicytotoxic, constant amounts of KP1019 with high concentrations of ascorbic acid (50–700 μM) increases cytotoxicity of the ruthenium anticancer drug in the human colon carcinoma cell line SW480, human cervical carcinoma KB-3-1 cells, and the multidrug-resistant subline KBC-1, whereas addition of low concentrations (2.7–50 μM) has a strong chemoprotective effect in the human colon carcinoma cell line SW480, but not in multidrug-resistant KBC-1 cells. Although cellular uptake of KP1019 is not altered, ascorbic acid induce stronger interaction of the ruthenium compound with DNA both in SW480 cells and under cell-free conditions with plasmid DNA. Even if DNA interactions probably play a subordinate role in vivo given the extensive protein binding of the compound, our data exemplify that ascorbic acid enhances the reactivity of KP1019 with biomolecules. Moreover, we demonstrate that the levels of KP1019-generated reactive oxygen species are markedly decreased by co-incubation with ascorbic acid. Conclusively, our results indicate that application of high doses of ascorbic acid might increase the anticancer effects of KP1019.     

Selective anticancer activities of ruthenium(II)-tetrazole complexes and their mechanistic insights

BioMetals - Ruthenium-based metallotherapeutics is an interesting alternative for platinum complexes acting as anticancer agents after the entry of KP1019, NAMI-A, and TLD1339 in clinical trials....     

Intracellular protein binding patterns of the anticancer ruthenium drugs KP1019 and KP1339

The ruthenium compound KP1019 has demonstrated promising anticancer activity in a pilot clinical trial. This study aims to evaluate the intracellular uptake/binding patterns of KP1019 and its sodium salt KP1339, which is currently in a phase I–IIa study. Although KP1339 tended to be moderately less cytotoxic than KP1019, IC₅₀ values in several cancer cell models revealed significant correlation of the cytotoxicity profiles, suggesting similar targets for the two drugs. Accordingly, both drugs activated apoptosis, indicated by caspase activation via comparable pathways. Drug uptake determined by inductively coupled plasma mass spectrometry (ICP-MS) was completed after 1 h, corresponding to full cytotoxicity as early as after 3 h of drug exposure. Surprisingly, the total cellular drug uptake did not correlate with cytotoxicity. However, distinct differences in intracellular distribution patterns suggested that the major targets for the two ruthenium drugs are cytosolic rather than nuclear. Consequently, drug–protein binding in cytosolic fractions of drug-treated cells was analyzed by native size-exclusion chromatography (SEC) coupled online with ICP-MS. Ruthenium–protein binding of KP1019- and KP1339-treated cells distinctly differed from the platinum binding pattern observed after cisplatin treatment. An adapted SEC-SEC-ICP-MS system identified large protein complexes/aggregates above 700 kDa as initial major binding partners in the cytosol, followed by ruthenium redistribution to the soluble protein weight fraction below 40 kDa. Taken together, our data indicate that KP1019 and KP1339 rapidly enter tumor cells, followed by binding to larger protein complexes/organelles. The different protein binding patterns as compared with those for cisplatin suggest specific protein targets and consequently a unique mode of action for the ruthenium drugs investigated.     

A comparative study of adduct formation between the anticancer ruthenium(III) compound HInd trans-[RuCl4(Ind)2] and serum proteins

Formation of adducts between the antitumor ruthenium(III) complex [HInd]trans-[RuCl(4)(Ind)(2)] (KP1019) and the plasma proteins serum albumin and serum transferrin was investigated by UV-vis spectroscopy, for metal-to-protein ratios ranging from 1:1 to 5:1. In both cases, formation of tight metal-protein conjugates was observed. Similar spectroscopic features were observed for both albumin and transferrin derivatives implying a similar binding mode of the ruthenium species to these proteins. Surface histidines are the probable anchoring sites for the bound ruthenium(III) ions in line with previous crystallographic results. In order to assess the stability of the KP1019-protein adducts the influence of pH, reducing agents and chelators was analysed by UV-vis spectroscopy. Notably, there was no effect of addition of EDTA on the UV-vis spectra of the conjugates. The pH-stability was high in the pH range 5-8. Experiments with sodium ascorbate showed that there was just some alteration of selected bands. The implications of the present results are discussed in relation to the pharmacological behavior of this novel class of antitumor compounds.     

Preclinical characterization of anticancer gallium(III) complexes: solubility, stability, lipophilicity and binding to serum proteins

The discovery and development of gallium(III) complexes capable of inhibiting tumor growth is an emerging area of anticancer drug research. A range of novel gallium coordination compounds with established cytotoxic efficacy have been characterized in terms of desirable chemical and biochemical properties and compared with tris(8-quinolinolato)gallium(III) (KP46), a lead anticancer gallium-based candidate that successfully finished phase I clinical trials (under the name FFC11), showing activity against renal cell cancer. In view of probable oral administration, drug-like parameters, such as solubility in water, saline and 0.5% dimethyl sulfoxide, stability against hydrolysis, measured as the rate constant of hydrolytic degradation in water or physiological buffer using a capillary zone electrophoresis (CZE) assay, and the octanol-water partition coefficient (logP) providing a rational estimate of a drug's lipophilicity, have been evaluated and compared. The differences in bioavailability characteristics between different complexes were discussed within the formalism of structure-activity relationships. The reactivity toward major serum transport proteins, albumin and transferrin, was also assayed in order to elucidate the drug's distribution pathway after intestinal absorption. According to the values of apparent binding rate constants determined by CZE, both KP46 and bis(2-acetylpyridine-4,4-dimethyl-3-thiosemicarbazonato-N,N,S)gallium(III) tetrachlorogallate(III) (KP1089) bind to transferrin faster than to albumin. This implies that transferrin would rather mediate the accumulation of gallium antineoplastic agents in solid tumors. A tendency of being faster converted into the protein-bound form found for KP1089 (due possibly to non-covalent binding) seems complementary to its greater in vitro antiproliferative activity.     

Anti-cancer drug KP1019 modulates epigenetics and induces DNA damage response in Saccharomyces cerevisiae

KP1019 comprises a class of ruthenium compounds having promising anticancer activity. Here, we investigated the molecular targets of KP1019 using Saccharomyces cerevisiae as a model organism. Our results revealed that in the absence of the N-terminal tail of histone H3, the growth inhibitory effect of KP1019 was markedly enhanced. Furthermore, H3K56A or rtt109Δ mutants exhibit hypersensitivity for KP1019. Moreover, KP1019 evicts histones from the mononucleosome and interacts specifically with histone H3. We have also shown that KP1019 treatment causes induction of Ribonucleotide Reductase (RNR) genes and degradation of Sml1p. Our results also suggest that DNA damage induced by KP1019 is primarily repaired through double-strand break repair (DSBR). In summary, KP1019 targets histone proteins, with important consequences for DNA damage responses and epigenetics.     

Recent developments in ruthenium anticancer drugs

Interest in Ru anticancer drugs has been growing rapidly since NAMI-A ((ImH(+))[Ru(III)Cl(4)(Im)(S-dmso)], where Im = imidazole and S-dmso = S-bound dimethylsulfoxide) or KP1019 ((IndH(+))[Ru(III)Cl(4)(Ind)(2)], where Ind = indazole) have successfully completed phase I clinical trials and an array of other Ru complexes have shown promise for future development. Herein, the recent literature is reviewed critically to ascertain likely mechanisms of action of Ru-based anticancer drugs, with the emphasis on their reactions with biological media. The most likely interactions of Ru complexes are with: (i) albumin and transferrin in blood plasma, the former serving as a Ru depot, and the latter possibly providing active transport of Ru into cells; (ii) collagens of the extracellular matrix and actins on the cell surface, which are likely to be involved in the specific anti-metastatic action of Ru complexes; (iii) regulatory enzymes within the cell membrane and/or in the cytoplasm; and (iv) DNA in the cell nucleus. Some types of Ru complexes can also promote the intracellular formation of free radical species, either through irradiation (photodynamic therapy), or through reactions with cellular reductants. The metabolic pathways involve competition among reduction, aquation, and hydrolysis in the extracellular medium; binding to transport proteins, the extracellular matrix, and cell-surface biomolecules; and diffusion into cells; with the extent to which individual drugs participate in various steps along these pathways being crucial factors in determining whether they are mainly anti-metastatic or cytotoxic. This diversity of modes of action of Ru anticancer drugs is also likely to enhance their anticancer activities and to reduce the potential for them to develop tumour resistance. New approaches to metabolic studies, such as X-ray absorption spectroscopy and X-ray fluorescence microscopy, are required to provide further mechanistic insights, which could lead to the rational design of improved Ru anticancer drugs.     

CZE-ICP-MS as a tool for studying the hydrolysis of ruthenium anticancer drug candidates and their reactivity towards the DNA model compound dGMP

Elucidating the mode of action and thereby opening the way to the design of chemotherapeutic agents is one of the major goals of metal-based anticancer research. Hydrolysis and DNA binding play an important role for pharmaceutical formulation and for exerting anticancer activity. Herein, for the first time the application of capillary zone electrophoresis–inductively-coupled plasma mass spectrometry (CZE–ICP-MS) for studying the hydrolytic stability and the binding of the ruthenium anticancer drug candidates KP418, KP1019, and RAPTA-C to dGMP is described. RAPTA-C was found to hydrolyze fastest and showed the highest reactivity toward the DNA model compound, whereas KP418 was the most stable compound in both these respects.     

Advances in developing tris(8-quinolinolato)gallium(iii) as an anticancer drug: critical appraisal and prospects

Gallium-based anticancer chemotherapeutics are appreciably progressing in clinical studies. A steady interest of drug developers and clinicians in gallium compounds is due to a proven ability of gallium cations to inhibit tumour growth, on the one hand, and enhanced bioavailability and moderate toxicity provided by the conversion of gallium into chelate complexes, on the other. One of the complexes suitable for a more convenient oral administration is tris(8-quinolinolato)gallium(iii) (KP46). Nominated from a range of gallium complexes for the clinical stage of development, KP46 has finished phase I trials with the outcome of promising tolerability and evidence of clinical activity in renal cell carcinoma. Therefore, there is obviously a need to codify and critically evaluate the continuing advances in the emergence of KP46 as a lead-drug candidate. Additionally, many questions remain unanswered regarding the relevant biological reactivity, modes of delivery and action and potential cell target(s) of KP46. The timely publication of the present review is also an attempt to shed light on these pertinent drug assets and to accelerate research activities towards further clinical development of KP46.     

Cellular uptake and subcellular distribution of ruthenium-based metallodrugs under clinical investigation versus cisplatin

The cellular uptake and subcellular distribution including adduct formation with genomic DNA and uptake into mitochondria of two ruthenium(iii)-based drugs in clinical trials, KP1019 and NAMI-A, and cisplatin, was investigated in cisplatin sensitive and resistant A2780 human ovarian carcinoma cells. These data indicate that reduced metal uptake into mitochondria in combination with increased binding towards low molecular weight components involved in detoxification mechanisms is essential for cisplatin resistance. The ruthenium drugs show distinct differences with respect to cisplatin, especially in the cisplatin resistant cells; in comparison to the sensitive cells, KP1019 exhibits higher cytotoxicity and an only slightly changed metabolism of the drug, whereas NAMI-A treatment results in increased intracellular ruthenium levels and a higher number of ruthenium-DNA adducts. In addition, size exclusion-inductively coupled mass spectrometry indicates that adduct formation with high molecular weight components in the particulate and nuclear fractions is crucial for the therapeutic effect of KP1019 in both cisplatin resistant and sensitive cell lines.     

Ti(IV) uptake and release by human serum transferrin and recognition of Ti(IV)-transferrin by cancer cells: understanding the mechanism of action of the anticancer drug titanocene dichloride

The organometallic anticancer agent titanocene dichloride, Cp(2)TiCl(2), is now in phase II clinical trials as an anticancer drug, but its mechanism of action is poorly understood. We show here that the interactions of Cp(2)TiCl(2) with human serum transferrin (hTF) and that of Ti(2)-hTF with adenosine triphosphate (ATP) have characteristics that could allow transferrin to act as a mediator for titanium delivery to tumor cells. Such reactions may therefore be important to the anticancer activity of this new class of drugs. Cp(2)TiCl(2) reacts rapidly with human apo-transferrin under physiological conditions (100 mM NaCl, 25 mM bicarbonate, and 4 mM phosphate, pH 7.4) with carbonate as a synergistic anion. The Cp ligands are released from the drug. Two-dimensional [(1)H, (13)C] NMR studies of epsilon-[(13)C]Met-hTF show that Ti(IV) loads the C-lobe first followed by the N-lobe and binds in the specific Fe(III) sites. The protein conformational changes induced by Ti(IV) appear to be similar to those induced by Fe(III). Carbonate can act as a synergistic anion in Ti(2)-hTF but does not appear to be essential. A specific Ti(IV)-hTF adduct is formed even in the absence of bicarbonate. When the pH of Ti(2)-hTF solutions is lowered, no Ti(IV) is released at the endosomal pH of ca. 5.0-5.5, but one Ti(IV) dissociates between pH 4.5-2.0. In contrast, in the presence of 1 mM ATP, all Ti(IV) is readily released from both lobes when the pH is lowered from 7.0 to 4.5. Moreover, Fe(III) displaces Ti(IV) rapidly from the C-lobe of Ti(2)-hTF (<5 min) but only slowly (days) from the N-lobe. Thus, the species Fe(C)Ti(N)-hTF might also provide a route for Ti(IV) entry into tumor cells via the transferrin receptor. Ti(2)-hTF effectively blocked cell uptake of radiolabeled (59)Fe-hTF into BeWo cells, a human placental choriocarcinoma cell line in culture. These results imply that titanium transferrin might be recognized by the transferrin receptor and be taken up into cancer cells.     

The polyamine transport system as a target for anticancer drug development

The vast majority of anticancer drugs in clinical use are limited by systemic host toxicity due to their non-specific side effects. These shortcomings have led to the development of tumour specific drugs which target a single-deregulated pathway or over expressed receptor in cancer cells. Whilst this approach has achieved clinical success, we have also learnt that targeting a single entity in cancer is rarely curative due to the large number of deregulated pathways, receptors and kinases which are also present, in addition to the target. An attractive alternative to improve targeting would be to harness the already established activity of known anticancer drugs by attaching them to a molecule that is transported into cancer cells via a selective transport system. One possibility for this approach is the polyamine pathway. This review provides a brief overview of the polyamine pathway and how, over the years, it has proved an exciting target for the development of novel anticancer agents. However, the focus of this article will be on the properties of the polyamine transport system and how these features could potentially be exploited to develop a novel and selective anticancer drug delivery system.     

Ruthenium versus platinum: interactions of anticancer metallodrugs with duplex oligonucleotides characterised by electrospray ionisation mass spectrometry

The binding of the ruthenium-based anticancer drug candidates KP1019, NAMI-A and RAPTA-T towards different double-stranded oligonucleotides was probed by electrospray ionisation mass spectrometry and compared with that of the widely used platinum-based chemotherapeutics cisplatin, carboplatin and oxaliplatin. It was found that the extent of adduct formation decreased in the following order: cisplatin > oxaliplatin > NAMI-A > RAPTA-T > carboplatin > KP1019. In addition to the characterisation of the adducts formed with the DNA models, the binding sites of the metallodrugs on the oligonucleotides were elucidated employing top-down tandem mass spectrometry and were found to be similar for all the metallodrugs studied, irrespective of the sequence of the oligonucleotide. A strong preference for guanine residues was established.     

LC- and CZE-ICP-MS approaches for the in vivo analysis of the anticancer drug candidate sodium trans-[tetrachloridobis(1H-indazole)ruthenate(III)] (KP1339) in mouse plasma

Ruthenium-indazole complexes are promising anticancer agents undergoing clinical trials. KP1339 is administered intravenously (i.v.), where serum proteins are the first available biological binding partners. In order to gain a better insight into the mode of action, mice were treated with different doses of KP1339 i.v. and sacrificed at different time points. The blood plasma was isolated from blood samples and analyzed by capillary zone electrophoresis (CZE) and size exclusion/anion exchange chromatography (SEC-IC) both combined on-line to inductively coupled plasma-mass spectrometry (ICP-MS). The performance of the analytical methodology was compared and the interaction of KP1339 with mouse plasma proteins characterized in vivo. Interestingly, the samples of the mice treated with 50 mg kg(-1) and terminated after 24 h showed a ca. 4-fold lowered albumin content and increased ruthenation of albumin aggregates as compared to the untreated control group and the 40 mg kg(-1) group. The majority of Ru was bound to albumin and the stoichiometry of the KP1339 protein binding was determined through the molar Ru/S ratio. In general, good agreement of the data obtained with both techniques was achieved and the SEC-IC method was found to be more sensitive as compared to the CZE-ICP-MS approach, whereas the latter benefits from the shorter analysis time and lower sample consumption.     

Arabidopsis kinesin KP1 specifically interacts with VDAC3, a mitochondrial protein, and regulates respiration during seed germination at low temperature

The involvement of cytoskeleton-related proteins in regulating mitochondrial respiration has been revealed in mammalian cells. However, it is unclear if there is a relationship between the microtubule-based motor protein kinesin and mitochondrial respiration. In this research, we demonstrate that a plant-specific kinesin, Kinesin-like protein 1 (KP1; At KIN14 h), is involved in respiratory regulation during seed germination at a low temperature. Using in vitro biochemical methods and in vivo transgenic cell observations, we demonstrate that KP1 is able to localize to mitochondria via its tail domain (C terminus) and specifically interacts with a mitochondrial outer membrane protein, voltage-dependent anion channel 3 (VDAC3). Targeting of the KP1-tail to mitochondria is dependent on the presence of VDAC3. When grown at 4° C, KP1 dominant-negative mutants (TAILOEs) and vdac3 mutants exhibited a higher seed germination frequency. All germinating seeds of the kp1 and vdac3 mutants had increased oxygen consumption; the respiration balance between the cytochrome pathway and the alternative oxidase pathway was disrupted, and the ATP level was reduced. We conclude that the plant-specific kinesin, KP1, specifically interacts with VDAC3 on the mitochondrial outer membrane and that both KP1 and VDAC3 regulate aerobic respiration during seed germination at low temperature.     

Selective Enhancing Effect of Early Mitotic Inhibitor 1 (Emi1) Depletion on the Sensitivity of Doxorubicin or X-ray Treatment in Human Cancer Cells

Chemotherapy and radiation in addition to surgery has proven useful in a number of different cancer types, but the effectiveness in normal tissue cannot be avoided in these therapies. To improve the effectiveness of these therapies selectively in cancer tissue is important for avoiding side effects. Early mitotic inhibitor 1 (Emi1) is known to have the function to inhibit anaphase-promoting complex/cyclosome ubiquitin ligase complex, which ubiquitylates the cell cycle-related proteins. It recently has been shown that Emi1 knockdown prevents transition from S to G₂ phase by down-regulating geminin via anaphase-promoting complex/cyclosome activation. At present, anticancer drugs for targeting DNA synthesis to interfere with rapidly dividing cells commonly are used. As Emi1 depletion interferes with completion of DNA synthesis in cancer cells, we thought that Emi1 knockdown might enhance the sensitivity for anticancer agents. Here, we confirmed that Emi1 siRNA induced polyploidy for preventing transition from S to G₂ phase in several cancer cell lines. Then, we treated Emi1 depleted cells with doxorubicin. Interestingly, increased apoptotic cells were observed after doxorubicin treatment in Emi1 siRNA-treated cancer cells. In addition, Emi1 depletion enhanced the sensitivity of x-ray irradiation in cancer cells. Importantly, synergistic effect of Emi1 knockdown in these combination therapies was not observed in normal cells. These results suggest that Emi1 siRNA can be a useful tool for enhancing of sensitivity of cancer cells to anticancer reagents and radiation.     

Characterization of glyceraldehyde-3-phosphate dehydrogenase as a novel transferrin receptor

A majority of cells obtain of transferrin (Tf) bound iron via transferrin receptor 1 (TfR1) or by transferrin receptor 2 (TfR2) in hepatocytes. Our study establishes that cells are capable of acquiring transferrin iron by an alternate pathway via GAPDH.These findings demonstrate that upon iron depletion, GAPDH functions as a preferred receptor for transferrin rather than TfR1 in some but not all cell types. We utilized CHO-TRVb cells that do not express TfR1 or TfR2 as a model system. A knockdown of GAPDH in these cells resulted in a decrease of not only transferrin binding but also associated iron uptake. The current study also demonstrates that, unlike TfR1 and TfR2 which are localized to a specific membrane fraction, GAPDH is located in both the detergent soluble and lipid raft fractions of the cell membrane. Further, transferrin uptake by GAPDH occurs by more than one mechanism namely clathrin mediated endocytosis, lipid raft endocytosis and macropinocytosis. By determining the kinetics of this pathway it appears that GAPDH-Tf uptake is a low affinity, high capacity, recycling pathway wherein transferrin is catabolised. Our findings provide an explanation for the detailed role of GAPDH mediated transferrin uptake as an alternate route by which cells acquire iron.