Anna Gasparini

Modified peptide nucleic acids are internalized in mouse macrophages RAW 264.7 and inhibit inducible nitric oxide synthase

Overexpression of inducible nitric oxide synthase causes the production of high levels of nitric oxide, which, under pathological conditions, leads to immunosuppression and tissue damage. The results recently obtained using peptide nucleic acids, rather than traditional oligonucleotides as antigen and antisense molecules, prompted us to test their efficacy in the regulation of nitric oxide production, thereby overcoming the obstacle of cellular internalization. The cellular permeability of four inducible nitric oxide synthase antisense peptide nucleic acids of different lengths was evaluated. These peptide nucleic acids were covalently linked to a hydrophobic peptide moiety to increase internalization and to a tyrosine to allow selective 125I radiolabelling. Internalization experiments showed a 3–25‐fold increase in the membrane permeability of the modified peptide nucleic acids with respect to controls. inducible nitric oxide synthase inhibition experiments on intact stimulated macrophages RAW 264.7 after passive permeation of the two antisense peptide nucleic acids 3 and 4 demonstrated a significant decrease (43–44%) in protein enzymatic activity with respect to the controls. These data offer a basis for developing a good alternative to conventional drugs directed against inducible nitric oxide synthase overexpression.

Synthesis and characterization of a specific peptide nucleic acid that inhibits expression of inducible NO synthase

Inducible nitric oxide synthase (iNOS) is modulated at the transcriptional level. Overexpression of this protein may result in high levels of nitric oxide leading to tissue damage and immunosuppression. In order to reduce the pathological effects of NO overproduction many efforts have been devoted to the identification of specific inhibitors of iNOS. The discovery of peptide nucleic acids (PNA), a novel class of molecules able to selectively interact with nucleic acids, prompted us to attempt a new way for the regulation of NO production. Here we describe the synthesis, characterization and in vitro effects of a PNA molecule bearing a homopyrimidine sequence complementary to the 5′ coding region of murine iNOS mRNA. This PNA shows specific interactions with iNOS mRNA in RNase protection assays and is able to block the synthesis of iNOS protein selectively in a rabbit reticulocyte lysate system. These results strengthen the view of a possible pharmacological application of PNA as a compound able to interfere with a specific enzymatic activity even at low concentrations.

Enhanced formation of reactive species from cis-diammine-(1,1-cyclobutanedicarboxylato)- platinum(ii) (carboplatin) in the presence of oxygen free radicals

Experiments were designed to investigate the influence of oxygen free radicals on the rate of conversion of the anticancer drug cis-diammine-(1,1-cyclobutanedicarboxylato)platinum(II) (CBDCA) to reactive species able to bind to DNA. A system containing the Fe-EDTA chelate and ascorbate was used to generate free radicals. The rate of drug conversion to by-products, during incubation in chloride-free phosphate buffer at 37 degrees, was determined by HPLC analysis and found to be approximately 10 times faster in the presence of the free radical generating system, compared to CBDCA alone. The hydroxyl radical scavenger, mannitol, was able to reduce the rate of CBDCA conversion significantly, while an enhancing effect was observed in the presence of superoxide dismutase. The platinum containing species, which are formed in the presence of free radicals, were demonstrated to react with isolated salmon sperm DNA. The rate of platinum binding to DNA during incubation of CBDCA in the presence of the Fe-EDTA/ascorbate system was markedly enhanced. No effect on platinum binding to DNA during incubation with cis-diamminedichloroplatinum(II) (CDDP) in the same experimental conditions was observed, thus excluding an increased susceptibility of DNA itself to binding of platinum, due to DNA damage induced by free radicals. These findings support the hypothesis that the increased conversion of CBDCA, previously observed in our laboratory, which occurs in the presence of hemoglobin could be mediated by a Fenton-like reaction resulting in oxygen free radical production, thus providing potential clues to improvements in the clinical use of this drug.

Enhanced intracellular activation of carboplatin in hemoglobin-synthesizing versus undifferentiated erythroleukemic cells.

The formation of DNA interstrand cross-links following exposure of target cells to the antineoplastic drugs Cisplatin and Carboplatin was measured in murine erythroleukemic cells either native or induced to differentiate and to synthesize hemoglobin by treatment with hexamethylene bisacetamide. The uptake of both drugs was identical in the uninduced and the induced cells, thus excluding differences in permeability due to differentiation. Both cell types formed comparable levels of DNA interstrand cross-links when submitted to treatment with Cisplatin. Conversely, significant differences were observed after exposure to Carboplatin. Specifically, the induced, hemoglobin containing cells showed significantly enhanced intracellular activation of the drug and subsequent binding to DNA, compared to the uninduced cells. These data suggest that, in agreement with previous results obtained with erythrocytes, the hemoglobin present in the committed cells enhances the aquation process that mediates the activation of Carboplatin.

Synthesis, Characterization and Erythrocyte Encapsulation of an Azidothymidine Homodinucleotide

A new nucleoside analogue, di(thymidine-3′-azido-2′,3′-dideoxy-D-riboside)-5′-5′-p1-p2-pyrophosphate (AZTp2AZT), was designed and synthesized to overcome some limitations of conventional antiretroviral chemotherapy based upon nucleoside analogues (AZT, ddC, ddI). Indeed, the triphosphorylated form of these nucleoside analogues is able to inhibit the infectivity and replication of Human Immunodeficiency Virus (HIV-1), but the main problems in the use of these drugs concern their limited phosphorylation in some cells (e.g., macrophages) and their cytotoxic side effects. AZTp2AZT was encapsulated in human erythrocytes according to a conservative procedure of hypotonic shock-isotonic resealing and reannealing and showed a remarkable stability and a slow conversion to 5′-monophosphate (AZT-MP) and to AZT. This azidothymidine homodinucleotide seems to have chemical and biochemical properties enabling its profitable utilization in the erythrocyte-encapsulated form.

Interactions of Carboplatin with Human Erythrocytes and Murine Erythroleukemic Cells

Platinum coordination complexes are extensively used in antineoplastic therapy against a broad spectrum of solid tumors.1,2 Besides Cisplatin (CDDP)*, the precursor of this platinum compounds, the second generation molecule Carboplatin (CBDCA) has been introduced in clinical trial recently.3