Novel bis-platinum complexes endowed with an improved pharmacological profile.

Multinuclear platinum complexes are characterized by a peculiar DNA binding mode and higher cytotoxic potency than the mononuclear complexes, and efficacy against a wide range of preclinical tumor models. To reduce the high irreversible plasma protein binding and improve the chemical and metabolic drug stability, novel bis-platinum complexes were designed starting from the parent compound CT-3610. The novel second-generation bis-platinum complexes utilize alkylcarboxylate as leaving groups to improve their pharmacokinetic and pharmacodynamic profiles, thus overcoming the limitations of the previously developed multinuclear compounds. The selected compounds [CT-47518 and CT-47463, respectively (bis-capronate) platinum and (bis-butyrate) platinum], have similar in vitro degradation kinetics in human and murine plasma and, above all, an increased stability when compared to CT-3610, particularly in human plasma. In addition, both compounds exhibited a marked cytotoxic potency as compared with cisplatin and oxaliplatin. Interestingly, they were capable of overcoming resistance mediated by DNA mismatch repair defects in different cellular models. The complexes showed marked antitumor efficacy in Pt-refractory tumor xenografts, with remarkable activity in terms of tumor growth inhibition and tumor growth delay. The improved stability profile in human plasma compared to early bis- and triplatinum complexes together with the marked activity in cellular systems as well as in in vivo models, make CT-47518 and CT-47463 attractive candidates for further development.

Modulation of survival pathways in ovarian carcinoma cell lines resistant to platinum compounds.

Because cytotoxic stress elicits various signaling pathways that may be implicated in cell survival or cell death, their alterations may have relevance in the development of platinum-resistant phenotype. Thus, in the present study, we investigated cell response to the epidermal growth factor receptor (EGFR) inhibitor gefitinib of ovarian carcinoma cell lines, including cells selected for resistance to cisplatin (IGROV-1/Pt1) and oxaliplatin (IGROV-1/OHP). Resistant sublines exhibited a marked decrease in sensitivity to gefitinib and resistance to apoptosis. Gefitinib was capable of inhibiting the phosphorylation of EGFR in all the studied cell lines. The Akt and extracellular signal-regulated kinase 1/2 (ERK1/2) kinases, which act downstream of EGFR, were constitutively active in the three cell lines, but phospho-ERK1/2 levels were increased in the two resistant sublines. This feature was associated with reduced sensitivity to the MEK1/2 inhibitor U0126. Pretreatment of resistant cells with U0126 resulted in restoration of sensitivity to gefitinib. Gefitinib was more effective in inhibiting ERK1/2 and Akt phosphorylation in IGROV-1 cells than in IGROV-1/OHP and IGROV-1/Pt1 cells. Phospho-p38 was up-regulated in the resistant sublines, indicating the concomitant activation of distinct mitogen-activated protein kinases. The up-regulation of phospho-p38 was associated with a peculiar localization of EGFR, which, in resistant sublines, was mainly internalized. In conclusion, our results indicate that the development of resistance to platinum drugs is associated with multiple alterations including deregulation of survival pathways activated by EGFR resulting in a reduced cellular response to gefitinib.

Different accumulation of cisplatin, oxaliplatin and JM216 in sensitive and cisplatin-resistant human cervical tumour cells.

The significance of reduced drug accumulation in resistance to cisplatin was investigated by using cisplatin, oxaliplatin and JM216 (hydrophobicity rank: JM216>oxaliplatin>cisplatin) in human squamous cell carcinoma cell line A431 and its cisplatin-resistant counterpart A431/Pt. While cisplatin showed a resistance factor of 2.6, oxaliplatin and JM216 circumvented the resistance. Platinum accumulation after cisplatin exposure was lower (2.4-fold) in A431/Pt than in A431 cells, whereas a similar accumulation was found in the two cell lines when oxaliplatin or JM216 were used, thereby suggesting the capability of the latter drugs to bypass the accumulation defect. In the A431 cell line platinum accumulated to a similar extent after exposure to cisplatin, oxaliplatin or JM216, while in A431/Pt cells, Platinum accumulation depended on the hydrophobicity of the drug, and an increased hydrophobicity favours the uptake. No difference in efflux of cisplatin was found between the two cell lines. The values of platinum-DNA binding in A431 cells were similar for cisplatin and JM216 and higher than those of oxaliplatin. In A431/Pt cells: (i) Pt-DNA binding levels of JM216 remained as in sensitive ones; (ii) Pt-DNA levels of cisplatin and oxaliplatin were very similar and nearly two-fold lower than those of JM216. Such results, in this cell system characterized by a low level of cisplatin resistance, support a model whereby platinum uptake occurs by a mechanism of facilitated diffusion, perhaps involving a gated channel, which can be lost during the selection of the drug-resistant variant(s). The hydrophobicity of the drug can be the key to bypass resistance.

Cobalt Oxide Nanoparticles: Behavior towards Intact and Impaired Human Skin and Keratinocytes Toxicity.

Skin absorption and toxicity on keratinocytes of cobalt oxide nanoparticles (Co3O4NPs) have been investigated. Co3O4NPs are commonly used in industrial products and biomedicine. There is evidence that these nanoparticles can cause membrane damage and genotoxicity in vitro, but no data are available on their skin absorption and cytotoxicity on keratinocytes. Two independent 24 h in vitro experiments were performed using Franz diffusion cells, using intact (experiment 1) and needle-abraded human skin (experiment 2). Co3O4NPs at a concentration of 1000 mg/L in physiological solution were used as donor phase. Cobalt content was evaluated by Inductively Coupled-Mass Spectroscopy. Co permeation through the skin was demonstrated after 24 h only when damaged skin protocol was used (57 ± 38 ng·cm⁻²), while no significant differences were shown between blank cells (0.92 ± 0.03 ng cm⁻²) and those with intact skin (1.08 ± 0.20 ng·cm⁻²). To further investigate Co3O4NPs toxicity, human-derived HaCaT keratinocytes were exposed to Co3O4NPs and cytotoxicity evaluated by MTT, Alamarblue and propidium iodide (PI) uptake assays. The results indicate that a long exposure time (i.e., seven days) was necessary to induce a concentration-dependent cell viability reduction (EC50 values: 1.3 × 10-4 M, 95% CL = 0.8-1.9 × 10⁻4 M, MTT essay; 3.7 × 10⁻5 M, 95% CI = 2.2-6.1 × 10⁻5 M, AlamarBlue assay) that seems to be associated to necrotic events (EC50 value: 1.3 × 10⁻4 M, 95% CL = 0.9-1.9 × 10⁻4 M, PI assay). This study demonstrated that Co3O4NPs can penetrate only damaged skin and is cytotoxic for HaCat cells after long term exposure.

Development of resistance to a trinuclear platinum complex in ovarian carcinoma cells.

BBR3464 is a trinuclear platinum complex that exhibits a potent cytotoxicity and efficacy against cisplatin-resistant tumors. To better understand the determinants of cellular resistance to BBR3464, we selected a resistant ovarian carcinoma cell line after exposure to the complex. The resistant cells (A2780/BBR3464) exhibited a high level of resistance to the selecting agent, but a marginal cross-resistance to cisplatin. Although cellular accumulation of BBR3464 was similar in parental and in resistant cells, DNA platination was decreased in A2780/BBR3464 cells, suggesting a reduced drug accessibility to DNA. This behavior reflected a partial drug inactivation at cytoplasmic level, as a consequence of increased levels of nucleophilic molecules including metallothioneins and human neurofilament low, but not glutathione. A2780/BBR3464 cells also exhibited a reduced susceptibility to apoptosis, which was consistent with reduced expression of Bax, and an alteration of DNA mismatch repair system, as reflected by lack of expression of MLH1 and PMS2, which could impair the recognition/repair of DNA lesions. Whereas both platinum drugs induced G2/M arrest in the parental cells, BBR3464, but not cisplatin, caused a late G1 arrest of resistant cells. Cisplatin induced an appreciable increase of p21(WAF1) levels in both models, in contrast to BBR3464 that produced a substantial upregulation of p21(WAF1) only in parental cells. An inverse relationship with p21(WAF1) modulation was found for CHK1 in parental cells treated with both agents and in resistant cells treated with cisplatin. This pattern of response is consistent with a regulatory loop involving p53 and p21(WAF1) at G2 checkpoint. In contrast, no modulation of CHK1 was found in A2780/BBR3464 treated with the triplatinum compound. These findings, indicating a different activation of regulatory pathways at DNA damage checkpoints in response to cisplatin and BBR3464, support an altered ability of resistant cells to recognize or tolerate sublethal lesions induced by BBR3464.

Determinaçäo de manganês em fluidos biológicos por ETA-AAS ZEEMAN - I - Amostras de soro / Determination of Manganese in biological fluids by ETA-AAS ZEEMAN - I - Serum samples

Um método é proposto para determinaçäo de Mn em amostras de soro, utilizando-se ETA-AAS, com correçäo Zeeman, combinado à plataforma de L'Vov, e emprego de Mg (NO3)2 0,2 por cento como modificador de matriz. A técnica prevê diluiçäo das amostras biológicas (1 + 1 v/v) com soluçäo aquosa do modificador de matriz, e posterior introduçäo de 20 uL desta soluçäo em forno de grafite. O limite de detecçäo obtido com o método foi de 0,08 ug/l de Mn, enquanto as recuperaçöes médias do metal foram avaliadas em 96,9 por cento. O coeficiente de variaçäo foi de 5,2 por cento para um teor de Mn de soro de 5 ug/l. O procedimento descrito foi utilizado no estudo de valores de referência para Mn em amostras de soro de populaçäo da Província de Brescia (Itália)

Determinaçäo de manganês em fluidos biológicos por ETA-AAS ZEEMAN - II - Amostras de urina / Determination of manganese in biological fluids by ETA- AAS ZEEMAN - II - Urine samples

Um método é proposto para determinaçäo de Mn em amostras de urina, utilizando-se ETA-AAS, com correçäo Zeeman, combinado à plataforma de L'Vov, e emprego de Mg (NO3)2 0,2 por cento como modificador de matriz. A técnica prevê diluiçäo das amostras biológicas (1+1,v/v) com soluçäo aquosa do modificador de matriz, e posterior introduçäo de 20 ul desta soluçäo em forno de grafite. O limite de detecçäo obtido com o método foi de 0,08 ug/l de Mn, enquanto as recuperaçöes médias do metal foram avaliadas em 95,6 por cento. O CV determinado foi de 4,7 por cento para uma concentraçäo urinária de 2 ug/l. O procedimento descrito foi utilizado no estudo de valores de referência para Mn em amostras de urina de populaçäo da Província de Brescia (Itália)