Copper and zinc dismetabolism in the mouse brain upon chronic cuprizone treatment.

Recent reports describe successful treatment using copper chelation therapy in neurodegenerative animal models. However, the success claimed for chelation therapy in neurodegenerative diseases is still rather controversial. To acquire new information on copper metabolism/homeostasis, we utilized cuprizone, a very sensitive and selective copper-chelating agent with well-known neurotoxic properties, as a relevant chemical model in mice. Upon cuprizone treatment, mice developed a pronounced astrocytosis, with brain oedema and spongiosis characterised by vacuolisations of the neuropil predominantly in the white matter. In addition, cuprizone treatment severely altered copper and zinc homeostasis in the central nervous system (CNS) as well as in all other tissues examined, with increasing metal ion concentrations particularly in the CNS. Concomitant with this increase in the Cu and Zn concentration in the brain, metallothionein-I and -II were also highly immunoreactive in astrocyte, consistent with the astrocytosis and demyelination observed in our and other laboratories.

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.

Biological role of adduct formation of the ruthenium(III) complex NAMI-A with serum albumin and serum transferrin.

NAMI-A is an innovative ruthenium(III) complex with a very encouraging preclinical profile of metastasis inhibition, which is undergoing initial phases of clinical trials. To assess the pharmacological relevance of the drug fraction associated to plasma proteins, adducts of NAMI-A with either serum albumin or serum transferrin were prepared and their biological effects tested in vitro and in vivo. Specifically, adducts of NAMI-A with either serum albumin or serum transferrin, prepared and characterized at a ruthenium-to-protein molar ratio of 4:1, were evaluated in vitro on the KB human tumor cell line and in vivo on the MCa mammary carcinoma tumor. The effects of NAMI-A/protein adducts on cell viability and on cell cycle progression were found to be far smaller than those produced by free NAMI-A. GFAAS measurements point out that the amount of ruthenium that gets into cells is drastically reduced when NAMI-A is presented in its protein-bound form. In vivo use of NAMI-A adducts with albumin and transferrin resulted markedly less effective on lung metastasis reduction, than free NAMI-A. Overall, the present results suggest that binding to plasma proteins causes a drastic decrease of NAMI-A bioavailability and a subsequent reduction of its biological activity, implying that association to plasma proteins essentially represents a mechanism of drug inactivation.

Antiangiogenic properties of selected ruthenium(III) complexes that are nitric oxide scavengers.

The nitric oxide synthase (NOS) pathway has been clearly demonstrated to regulate angiogenesis. Increased levels of NO correlate with tumour growth and spreading in different experimental and human cancers. Drugs interfering with the NOS pathway may be useful in angiogenesis-dependent tumours. The aim of this study was to pharmacologically characterise certain ruthenium-based compounds, namely NAMI-A, KP1339, and RuEDTA, as potential NO scavengers to be used as antiangiogenic/antitumour agents. NAMI-A, KP1339 and RuEDTA were able to bind tightly and inactivate free NO in solution. Formation of ruthenium-NO adducts was documented by electronic absorption, FT-IR spectroscopy and (1)H-NMR. Pretreatment of rabbit aorta rings with NAMI-A, KP1339 or RuEDTA reduced endothelium-dependent vasorelaxation elicited by acetylcholine. This effect was reversed by 8-Br-cGMP. The key steps of angiogenesis, endothelial cell proliferation and migration stimulated by vascular endothelial growth factor (VEGF) or NO donor drugs, were blocked by NAMI-A, KP1339 and RuEDTA, these compounds being devoid of any cytotoxic activity. When tested in vivo, NAMI-A inhibited angiogenesis induced by VEGF. It is likely that the antitumour properties previously observed for ruthenium-based NO scavengers, such as NAMI-A, are related to their NO-related antiangiogenic properties.

Solution chemistry and DNA binding properties of MEN 10755, a novel disaccharide analogue of doxorubicin.

The behavior under physiological conditions of MEN 10755, a novel disaccharide analogue of doxorubicin, was investigated in detail by a variety of spectroscopic techniques including spectrophotometry, fluorescence, and (1)H NMR. The pH dependent properties of MEN 10755 were also analysed by spectrophotometry and potentiometry within the pH range 5--11. It is found that MEN 10755 behaves very similarly to doxorubicin and reproduces closely its pH dependent pattern. Like doxorubicin, MEN 10755 undergoes dimerization with a significantly smaller association constant. The interaction of MEN 10755 with calf thymus DNA was studied in detail. Spectrophotometric and fluorescence titrations of MEN 10755 with calf thymus DNA show spectral patterns almost identical to those obtained with doxorubicin implying that the binding mechanism and the stability of the resulting adducts are very similar. An apparent affinity constant of 1.2 x 10(6) was determined for the interaction of MEN 10755 with calf thymus DNA to be compared with the value of 3.3 x 10(6) measured for doxorubicin, under the same conditions. The effects of both anthracyclines on the thermal denaturation profiles of calf thymus DNA were also analyzed; both compounds turned out to stabilize to a similar extent the DNA double helix and to give rise to a characteristic two-step melting profile. The implications of the present results for the pharmacological activity and the mechanism of action of this novel and promising antitumor compound are discussed.

Binding of Antitumor Ruthenium(III) Complexes to Plasma Proteins.

Presently, there is large interest in analysing the interactions in vitro with plasma proteins of some novel antitumor ruthenium(III) complexes that are in preclinical or clinical phase. The joint application of separation and spectroscopic techniques provides valuable information on the nature and the properties of the resulting ruthenium/protein adducts. Recent work carried out in our laboratory points out that, under physiological conditions, some selected ruthenium(III) complexes bind plasma proteins tightly with a marked preference for surface imidazole groups. Representative examples of interactions of antitumor ruthenium(III) complexes with plasma proteins such as albumin and transferrin are given. Notably the antitumor ruthenium(III) complexes considered here bind proteins much tighter than DNA; it is proposed that protein binding of ruthenium(III) complexes will have a large impact on the biodistribution, the pharmacokinetics and the mechanism of action of these experimental drugs.