Intercalator conjugates of pyrimidine locked nucleic acid-modified triplex-forming oligonucleotides: improving DNA binding properties and reaching cellular activities

Triplex-forming oligonucleotides (TFOs) are powerful tools to interfere sequence-specifically with DNA-associated biological functions. (A/T,G)-containing TFOs are more commonly used in cells than (T,C)-containing TFOs, especially C-rich sequences; indeed the low intracellular stability of the non-covalent pyrimidine triplexes make the latter less active. In this work we studied the possibility to enhance DNA binding of (T,C)-containing TFOs, aiming to reach cellular activities; to this end, we used locked nucleic acid-modified TFOs (TFO/LNAs) in association with 5′-conjugation of an intercalating agent, an acridine derivative. In vitro a stable triplex was formed with the TFO-acridine conjugate: by SPR measurements at 37°C and neutral pH, the dissociation equilibrium constant was found in the nanomolar range and the triplex half-life ∼10 h (50-fold longer compared with the unconjugated TFO/LNA). Moreover to further understand DNA binding of (T,C)-containing TFO/LNAs, hybridization studies were performed at different pH values: triplex stabilization associated with pH decrease was mainly due to a slower dissociation process. Finally, biological activity of pyrimidine TFO/LNAs was evaluated in a cellular context: it occurred at concentrations ∼0.1 μM for acridine-conjugated TFO/LNA (or ∼2 μM for the unconjugated TFO/LNA) whereas the corresponding phosphodiester TFO was inactive, and it was demonstrated to be triplex-mediated

Targeting chromosomal sites with locked nucleic acid-modified triplex-forming oligonucleotides: study of efficiency dependence on DNA nuclear environment

Triplex-forming oligonucleotides (TFOs) are synthetic DNA code-reading molecules that have been demonstrated to function to some extent in chromatin within cell nuclei. Here we have investigated the impact of DNA nuclear environment on the efficiency of TFO binding. For this study we have used locked nucleic acid-containing TFOs (TFO/LNAs) and we report the development of a rapid PCR-based method to quantify triplex formation. We have first compared triplex formation on genes located at different genomic sites and containing the same oligopyrimidine•oligopurine sequence. We have shown that efficient TFO binding is possible on both types of genes, expressed and silent. Then we have further investigated when gene transcription may influence triplex formation in chromatin. We have identified situations where for a given gene, increase of transcriptional activity leads to enhanced TFO binding: this was observed for silent or weakly expressed genes that are not or are only slightly accessible to TFO. Such a transcriptional dependence was observed for integrated and endogenous loci, and chemical and biological activations of transcription. Finally, we provide evidence that TFO binding is sequence-specific as measured on mutated target sequences and that up to 50% of chromosomal targets can be covered by the TFO/LNA in living cells

Gliclazide may have an antiapoptotic effect related to its antioxidant properties in human normal and cancer cells

Experimental and clinical studies suggest that gliclazide may protect pancreatic β-cells from apoptosis induced by an oxidative stress. However, the precise mechanism(s) of this action are not fully understood and requires further clarification. Therefore, using human normal and cancer cells we examined whether the anti-apoptotic effects of this sulfonylurea is due to its free radical scavenger properties. Hydrogen peroxide (H2O2) as a model trigger of oxidative stress was used to induce cell death. Our experiments were performed on human normal cell line (human umbilical vein endothelial cell line, HUVEC-c) and human cancer cell lines (human mammary gland cell line, Hs578T; human pancreatic duct epithelioid carcinoma cell line, PANC-1). To assess the effect of gliclazide the cells were pre-treated with the drug. The 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay was employed to measure the impact of gliclazide on cell viability. Generation of reactive oxygen species, mitochondrial membrane potential (∆Ψm), and intracellular Ca2+ concentration [Ca2+] were monitored. Furthermore, the morphological changes associated with apoptosis were determined using double staining with Hoechst 33258-propidium iodide (PI). Gliclazide protects the tested cells from H2O2-induced cell death most likely throughout the inhibition of ROS production. Moreover, the drug restored loss of ΔΨm and diminished intracellular [Ca2+] evoked by H2O2. Double staining with Hoechst 33258-PI revealed that pre-treatment with gliclazide diminished the number of apoptotic cells. Our findings indicate that gliclazide may protect both normal and cancer human cells against apoptosis induced by H2O2. It appears that the anti-apoptotic effect of the drug is most likely associated with reduction of oxidative stress

Disgregation time of spheroids (modified drop pills) using different vehicles and solid supports

En el presente trabajo se evalúan los tiempos de disgregación de los esferoides seleccionados entre los obtenidos a partir de seis excipientes y doce polvos receptores diferentes, de acuerdo a las características morfológicas. También se analiza el comportamiento reológico de dos excipientes hidrosolubles utilizados (polietilenglicol 4.000 y 6.000). De la comparación de los resultados surge que los tiempos de disgregación se mantienen menores a los 5 minutos en el caso de los excipientes hidrosolubles ensayados, constituyendo una excepción los obtenidos con cubierta del derivado acrílico (Eudragit L-100). Estos resultados parecen apoyar la utilidad de esta nueva forma farmacéutica (esferoides) cuando se deseen vehiculizar principios muy activos con los excipientes y cubiertas adecuados para modular la velocidad de disolución.In this work disgregation rates of "spheroids" made with six vehicles and twelve receiver powders are evaluated, according to the morphological characteristics. The rheological behavior of two water-soluble vehicles (polyethilene glycol 4000 and 6000) is also analyzed. Results show that watersoluble vehicles provides disgregation time lower than five minutes, except those spheroids made with acrylic (Eudragit L-100) cover. Spheroids could be a really useful pharmaceutical form when very active principles need to be vehiculized, as dissolution rate can be adequately modulated by selecting appropriate vehicles and covers.Colegio de Farmacéuticos de la Provincia de Buenos Aire

Spheroids: effect of the fixation of the solid bed on the erosion time

Se verificó la cantidad de lecho sólido pulverizado que se fija a la gota de polímero que cae sobre el mismo y de qué manera ese polvo adherido influye en el tiempo de erosión de los esferoides formados. Los resultados permiten observar notorias diferencias respecto a la cantidad de polvo fijado, estudiando el comportamiento de once sustancias. Asimismo, los tiempos de erosión de los esferoides son distintos, aunque no guardan relación con la cantidad de polvo usado.The amount of powdered solid bed futed to the drop of a polymer that falls on it was determined, so as the influence than the mode of adherence exerts on the erosion time of the spheroids. The amount of powder adhered to the spheroid is not constant (eleven substances were tested) and the erosion time of different spheroids also varies, but seemingly there is no relation between both facts.Se verificó la cantidad & lecho sólido pulverizado que se fija a la gota de polímero que cae sobre el mismo y de qué manera ese polvo adherido influye en el tiempo de erosión de los esferoides formados. Los resultados permiten observar notorias diferencias respecto a la cantidad de polvo fijado, estudiando el comportamiento de once sustancias. Asimismo, los tiempos de erosión de los esferoides son distintos, aunque no guardan relación con la cantidad de polvo usado.Colegio de Farmacéuticos de la Provincia de Buenos Aire

Disgregation time of spheroids (modified drop pills) using different vehicles and solid supports

En el presente trabajo se evalúan los tiempos de disgregación de los esferoides seleccionados entre los obtenidos a partir de seis excipientes y doce polvos receptores diferentes, de acuerdo a las características morfológicas. También se analiza el comportamiento reológico de dos excipientes hidrosolubles utilizados (polietilenglicol 4.000 y 6.000). De la comparación de los resultados surge que los tiempos de disgregación se mantienen menores a los 5 minutos en el caso de los excipientes hidrosolubles ensayados, constituyendo una excepción los obtenidos con cubierta del derivado acrílico (Eudragit L-100). Estos resultados parecen apoyar la utilidad de esta nueva forma farmacéutica (esferoides) cuando se deseen vehiculizar principios muy activos con los excipientes y cubiertas adecuados para modular la velocidad de disolución.In this work disgregation rates of "spheroids" made with six vehicles and twelve receiver powders are evaluated, according to the morphological characteristics. The rheological behavior of two water-soluble vehicles (polyethilene glycol 4000 and 6000) is also analyzed. Results show that watersoluble vehicles provides disgregation time lower than five minutes, except those spheroids made with acrylic (Eudragit L-100) cover. Spheroids could be a really useful pharmaceutical form when very active principles need to be vehiculized, as dissolution rate can be adequately modulated by selecting appropriate vehicles and covers.Colegio de Farmacéuticos de la Provincia de Buenos Aire

Sequences of the oligopyrimidine•oligopurine DNA targets () and of the oligonucleotides () used in this study

<p><b>Copyright information:</b></p><p>Taken from "Intercalator conjugates of pyrimidine locked nucleic acid-modified triplex-forming oligonucleotides: improving DNA binding properties and reaching cellular activities"</p><p>Nucleic Acids Research 2005;33(13):4223-4234.</p><p>Published online 27 Jul 2005</p><p>PMCID:PMC1181241.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> (A) The wild-type PPT target sequence (boxed), Dra I recognition site (underlined) are shown, as well as the mutated PPT duplex presenting two mutations (in bold). (B) (T,C)-containing TFOs directed against the wild-type PPT duplex and control sequences. Small letters indicate LNA nucleotides and capitals DNA nucleotides. Cytosines in italic ( and ) are methylated at position 5; all cytosines (LNA and DNA) are 5-methylated. The abbreviated names are indicated near the sequences. (po) stands for phosphodiester. Last four TFOs are either acridine (Acr) or psoralen (Pso) 5′-conjugated TFO/LNAs