María Elena Bravo-Gómez

Copper Compounds in Cancer Chemotherapy

Transitional metals have a large variety of coordination numbers and geometries, accessible redox states in physiological conditions and a wide range of thermodynamic and reactivity properties which can be successfully tuned by selection of suitable ligands. These characteristics can be used to develop new drugs with numerous advantages over the organic based drugs. Historically, research in this field has focus on platinum and DNA targeting; however, anticancer drug research may be expanded to include alternative metal compounds with different mode of action resulting in markedly different cytotoxic response profiles. Cooper complexes with selected ligands are being extensively studied as agents for the treatment of cancer. Current research on copper compounds as antitumoral compounds is being reviewed in this chapter particularly focused on the family of copper Casiopeinas.

Genotoxicity of the copper antineoplastic coordination complexes casiopeinas.

Casiopeinas is the generic name of a group of coordination complexes with a central copper atom bound to organic ligands, designed to be an alternative to cancer therapy. Indeed, some of these compounds can reduce implanted tumors in mice. Casiopeinas were expressly designed to interact with the genetic material, so the aim of the present work is to determine if these compounds have genotoxic activity. The results indicate that casiopeinas produce DNA fragmentation and base oxidation and suggest that their mode of action is related to reactive oxygen species (ROS) generation after copper reduction.

Comparison between the antiproliferative effect and intracellular glutathione depletion induced by Casiopeína IIgly and cisplatin in murine melanoma B16 cells.

BACKGROUND Casiopeína IIgly (Cas IIgly) [Cu(4,7-dimethyl-1,10-phenanthroline)(glycinate)]NO(3) induce oxidative damage in different human tumour cell strains, as the known anticancer agent cisplatin (CDDP) does. PURPOSE To compare glutathione (GSH) depletion induced by Cas IIgly and CDDP in murine melanoma B16 cells and its relationship with their antiproliferative effect. MATERIALS AND METHODS Cell growth was determined according to the sulforhodamine B assay. Intracellular GSH levels were measured by the reduction of Ellmans reagent (DTNB). RESULTS Cas IIgly IC50 in B16 cells was 54.5 μM (24.21 μg/mL), which depleted GSH from 1092 to 585 ng per million cells in a 30 min incubation period. In the other hand, CDDP was less toxic at the same conditions with an IC50 equal to 197.76 μM (59.33 μg/mL), and depleted GSH to 50% of the normal only after a longer exposure period (4h). The addition of 1.8mM ascorbic acid (Asc) or 1mM buthionine sulfoximine (BSO) enhanced Cas IIgly toxicity, whereas it was prevented by 100 U/mL catalase. BSO sensitised B16 cells to CDDP, but neither Asc or catalase modified CDDP effects. CONCLUSIONS The antiproliferative effect of both drugs correlated to intracellular GSH levels. Unlike CDDP, GSH depletion induced by Cas IIgly occurs earlier, it is enhanced by ascorbic acid and preventable by catalase. Redox cycles, feasible only with Cas IIgly, may be an important difference in their mode of action.

Pharmacokinetics and hematotoxicity of a novel copper-based anticancer agent: casiopeina III-Ea, after a single intravenous dose in rats.

Casiopeina III‐Ea is a mixed chelate copper (II) complex that has shown cytotoxic and antitumor activity in vitro and in vivo. The aim of this study was to investigate the pharmacokinetics of total copper derived from casiopeina III‐Ea administered by intravenous bolus injection to Wistar rats. Other objective was to evaluate the hematotoxicity produced by this compound in those animals. Wistar rats received a single intravenous dose of 4 mg/kg of casiopeina III‐Ea. Blood samples were taken and pharmacokinetics evaluated. Furthermore, erythrocyte copper levels were determined to identify a potential target and Zn levels were analyzed to determine a possible change. For the evaluation of hematotoxicity, both blood and urine samples were collected for hematological and biochemical analyses; moreover, Fe determination was performed. Blood copper and zinc levels, red blood cell copper levels as well as copper, zinc, and iron amounts excreted into urine were analyzed by ICP‐MS. The blood concentration–time profile of copper derived from casiopeina III‐Ea was fitted to a two‐compartment model with a zero‐order input. Cumulative amounts of Cu, Zn, and Fe excreted into rat urine after administration of casiopeina III‐Ea were different with respect to control. Hematological and biochemical data indicated a hemolytic toxicity. Pharmacokinetic analysis of total copper derived from casiopeina III‐Ea provided a general knowledge about distribution and elimination process of this compound. Additionally, the systemic exposure of the copper derived from casiopeina III‐Ea accounts for the hematotoxicity of this complex at test dose.

Genotoxic assessment of the copper chelated compounds Casiopeinas: Clues about their mechanisms of action

Casiopeinas is the generic name of a group of copper chelated complexes designed to be used as antineoplastic. Some of these compounds have shown promising results, and in fact, one of them named Casiopeina III-ia has completed preclinical trials and is ready to start clinical phase I in Mexico. As part of the tests that have to be done to every molecule intended to be used in humans, bacterial assays are required because of their sensitivity, speed and reproducibility and among them, Ames test and the SOS Chromotest are widely used to evaluate DNA damage. With the aim to contribute to complete safety information related to genotoxicity and support the hypothesis about their mode of action, four different Casiopeinas (Cas II-gly, Cas III-Ea, Cas III-ia and Cas III-Ha) were tested for genotoxicity with these assays, as well as differential cytotoxicity upon Escherichia coli mutants defectives in some DNA repair mechanisms. However, although it is well known that these molecules produce DNA breakage, the results of the Chromotest and Ames test were negative. Despite this is controversial, a possible explanation is that there is a direct interaction between DNA and the Casiopeinas tested.

Antineoplastic copper coordinated complexes (Casiopeinas) uncouple oxidative phosphorylation and induce mitochondrial permeability transition in cardiac mitochondria and cardiomyocytes

Copper-based drugs, Casiopeinas (Cas), exhibit antiproliferative and antineoplastic activities in vitro and in vivo, respectively. Unfortunately, the clinical use of these novel chemotherapeutics could be limited by the development of dose-dependent cardiotoxicity. In addition, the molecular mechanisms underlying Cas cardiotoxicity and anticancer activity are not completely understood. Here, we explore the potential impact of Cas on the cardiac mitochondria energetics as the molecular mechanisms underlying Cas-induced cardiotoxicity. To explore the properties on mitochondrial metabolism, we determined Cas effects on respiration, membrane potential, membrane permeability, and redox state in isolated cardiac mitochondria. The effect of Cas on the mitochondrial membrane potential (Δψm) was also evaluated in isolated cardiomyocytes by confocal microscopy and flow cytometry. Cas IIIEa, IIgly, and IIIia predominately inhibited maximal NADH- and succinate-linked mitochondrial respiration, increased the state-4 respiration rate and reduced membrane potential, suggesting that Cas also act as mitochondrial uncouplers. Interestingly, cyclosporine A inhibited Cas-induced mitochondrial depolarization, suggesting the involvement of mitochondrial permeability transition pore (mPTP). Similarly to isolated mitochondria, in isolated cardiomyocytes, Cas treatment decreased the Δψm and cyclosporine A treatment prevented mitochondrial depolarization. The production of H2O2 increased in Cas-treated mitochondria, which might also increase the oxidation of mitochondrial proteins such as adenine nucleotide translocase. In accordance, an antioxidant scavenger (Tiron) significantly diminished Cas IIIia mitochondrial depolarization. Cas induces a prominent loss of membrane potential, associated with alterations in redox state, which increases mPTP opening, potentially due to thiol-dependent modifications of the pore, suggesting that direct or indirect inhibition of mPTP opening might reduce Cas-induced cardiotoxicity.

Genomic action of permanently charged tamoxifen derivatives via estrogen receptor-α

Tamoxifen is a selective estrogen receptor modulator widely used in oncology and reproductive endocrinology. In order to decrease its non-desirable effects and elucidate mechanisms of action, permanently charged tamoxifen derivatives (PCTDs) have been reported. Whether PCTDs have genomic effects remains controversial. Since the clinical relevance of tamoxifen, the necessity to have new anticancer drugs, and in order to gain insights into the mechanisms of action of PCTDs, we obtained six quaternary ammonium salts derived from tamoxifen including three new compounds. We characterized them by nuclear magnetic resonance, X-ray diffraction, electron microscopy, and/or high performance liquid chromatography, and detected them in cell lysates by liquid chromatography coupled to mass spectrometry. We evaluated their binding to estrogen receptor-alpha (ERalpha, their effect on the transcriptional activity mediated by ERalpha (gene reporter assays), and the proliferation of cancer cells (MCF-7 and cells from a cervical cancer primary culture). Structural studies demonstrated the expected identity of the molecules. All PCTDs did bind to ERalpha, one of them induced ERalpha-mediated transcription while two others inhibited such genomic action. Accordingly, PCTDs were detected in cell lysates. PCTDs inhibited cell proliferation, noteworthy, two of them displayed higher inhibition than tamoxifen. Structure-activity analysis suggests that PCTDs permanent positive charge and the length of the aliphatic chain might be associated to the biological responses studied. We suggest genomic effects as a mechanism of action of PCTDs. The experimental approaches here used could lead to a better design of new therapeutic molecules and help to elucidate molecular mechanisms of new anticancer drugs.

Effect of [Cu(4,7-dimethyl-1,10-phenanthroline)(acetylacetonato)]NO3, Casiopeína III-Ea, on the activity of cytochrome P450

Casiopeína III-Ea (Cas III-Ea(1)) is a copper complex with antiproliferative and antitumor activities, designed to act via alternative mechanisms of action different from Cisplatin. This compound has also been well characterized in preclinical test and pharmacokinetic analysis, being a good candidate for clinical phases. Since very little is known about the processes of biotransformation of therapeutic metal based drugs, this paper report the first approach to the study of the interaction between metal complex Cas III-Ea and cytochromes P450 with the aim to find out possible biotransformation pathways for this complexes and feasible drug-drug interactions. Results showed that Cas III-Ea is a strong irreversible competitive inhibitor of CYP1A1 (IC50 = 7.5 ± 1.0 μM; Ki = 240 nM). The magnitude of values indicate that it is necessary to be taken into account such effect when analyzing possible drug interactions with these new drugs in order to prevent adverse reactions derived from this inhibition.

Casiopeina III-Ea, a copper-containing small molecule, inhibits the in vitro growth of primitive hematopoietic cells from chronic myeloid leukemia

Several novel compounds have been developed for the treatment of different types of leukemia. In the present study, we have assessed the in vitro effects of Casiopeina III-Ea, a copper-containing small molecule, on cells from patients with Chronic Myeloid Leukemia (CML). We included primary CD34+ Lineage-negative (Lin-) cells selected from CML bone marrow, as well as the K562 and MEG01 cell lines. Bone marrow cells obtained from normal individuals - both total mononuclear cells as well as CD34+ Lin- cells- were used as controls. IC50 corresponded to 0.5μM for K562 cells, 0.63μM for MEG01 cells, 0.38μM for CML CD34+ lin- cells, and 1.0μM for normal CD34+ lin- cells. Proliferation and expansion were also inhibited to significantly higher extents in cultures of CML cells as compared to their normal counterparts. All these effects seemed to occur via a bcr-abl transcription-independent mechanism that involved a delay in cell division, an increase in cell death, generation of Reactive Oxygen Species and changes in cell cycle. Our results demonstrate that Casiopeina III-Ea possesses strong antileukemic activity in vitro, and warrant further preclinical (animal) studies to assess such effects in vivo.

Abstract CT408: Phase I study of one mixed chelates copper(II) compound, Casiopeína CasIIIia with antitumor activity and its mechanism of action

Metal complexes have gained a growing interest as pharmaceuticals for their use as diagnostic agents or as chemotherapeutic drugs[1]. In our group some efforts have been done in the development of anticancer agents employing essential metals such as the family of copper (II) compounds known as Casiopeinas®, with a general formula [Cu (N-N) (X-Y) H2O] NO3 where N-N is a diimine (phen or bipy) and X-Y is a bidentate ligand (acac, salal, aminoacidate, peptide, benzimidazol). These compounds have shown cytostatic, cytotoxic and antineoplastic activity in vitro and in vivo[2]. Also albumin/Casiopeinas interaction is discussed. Among those one have selected that is CasIII-ia as the most adequate to go into clinical trial Phase I. Although the mechanism of action is still not completely elucidated, experimental evidence suggests the interaction of coordination compounds with DNA (nuclear or mitochondrial) and their components and the generation of reactive oxygen species (ROS) as the main action pathways. Induction of apoptosis has been proved to be the main death tumoral cell pathway. DNA cleavage capacity, compared with the 4, 4´-diimine analogs, then is suggested an intercalation process as the first step for the DNA damage. Also calculations have been done in order to modeling the interaction between Casiopeinas and DNA [3, 4]. The clinical protocol phase I of the dimethyl bpy derivative with acac Cas IIIia is presented and the selection criteria of the drug. [1] S. H. van Rijt, P. J. Sadler, Drug Discov. Today, 14 (2009) 1089-1097. [2] L. Ruiz-Azuara, M.E. Bravo-Gomez, Curr. Med. Chem., 17 (2010) 3606-3615. [3] R. Galindo-Murillo, J. Hernandez-Lima, M. Gonzalez-Rendon, F. Cortes-Guzman, L. Ruiz-Azuara, R. Moreno-Esparza Phys. Chem. Chem. Phys., 13 (2011) 14511-14516. [4] R. Galindo-Murillo, L. Ruiz-Azuara, R. Moreno-Esparza, F. Cortes-Guzman Phys. Chem. Chem. Phys., 14 (2012) 15539-15546. Citation Format: Lena Ruiz-Azuara, Gerard Bastian, Maria Elena Bravo-Gomez, Roberto Carlos Canas, Marcos Flores-Alamo, Ines Fuentes, Carmen Mejia, Juan Carlos Garcia-Ramos, Alberto Serrano. Phase I study of one mixed chelates copper(II) compound, Casiopeina CasIIIia with antitumor activity and its mechanism of action. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr CT408. doi:10.1158/1538-7445.AM2014-CT408