Alma Revilla-Vázquez

A Proof-Of-Principle Study of Epigenetic Therapy Added to Neoadjuvant Doxorubicin Cyclophosphamide for Locally Advanced Breast Cancer

Background Aberrant DNA methylation and histone deacetylation participate in cancer development and progression; hence, their reversal by inhibitors of DNA methylation and histone deacetylases (HDACs) is at present undergoing clinical testing in cancer therapy. As epigenetic alterations are common to breast cancer, in this proof-of-concept study demethylating hydralazine, plus the HDAC inhibitor magnesium valproate, were added to neoadjuvant doxorubicin and cyclophosphamide in locally advanced breast cancer to assess their safety and biological efficacy. Methodology This was a single-arm interventional trial on breast cancer patients (ClinicalTrials.gov Identifier: NCT00395655). After signing informed consent, patients were typed for acetylator phenotype and then treated with hydralazine at 182 mg for rapid-, or 83 mg for slow-acetylators, and magnesium valproate at 30 mg/kg, starting from day –7 until chemotherapy ended, the latter consisting of four cycles of doxorubicin 60 mg/m2 and cyclophosphamide 600 mg/m2 every 21 days. Core-needle biopsies were taken from primary breast tumors at diagnosis and at day 8 of treatment with hydralazine and valproate. Main Findings 16 patients were included and received treatment as planned. All were evaluated for clinical response and toxicity and 15 for pathological response. Treatment was well-tolerated. The most common toxicity was drowsiness grades 1–2. Five (31%) patients had clinical CR and eight (50%) PR for an ORR of 81%. No patient progressed. One of 15 operated patients (6.6%) had pathological CR and 70% had residual disease <3 cm. There was a statistically significant decrease in global 5mC content and HDAC activity. Hydralazine and magnesium valproate up- and down-regulated at least 3-fold, 1,091 and 89 genes, respectively. Conclusions Hydralazine and magnesium valproate produce DNA demethylation, HDAC inhibition, and gene reactivation in primary tumors. Doxorubicin and cyclophosphamide treatment is safe, well-tolerated, and appears to increase the efficacy of chemotherapy. A randomized phase III study is ongoing to support the efficacy of so-called epigenetic or transcriptional cancer therapy.

Global DNA hypermethylation-associated cancer chemotherapy resistance and its reversion with the demethylating agent hydralazine

BackgroundThe development of resistance to cytotoxic chemotherapy continues to be a major obstacle for successful anticancer therapy. It has been shown that cells exposed to toxic concentrations of commonly used cancer chemotherapy agents develop DNA hypermetylation. Hence, demethylating agents could play a role in overcoming drug resistance.MethodsMCF-7 cells were rendered adriamycin-resistant by weekly treatment with adriamycin. Wild-type and the resulting MCF-7/Adr cells were analyzed for global DNA methylation. DNA methyltransferase activity and DNA methyltransferase (dnmt) gene expression were also determined. MCF-7/Adr cells were then subjected to antisense targeting of dnmt1, -3a, and -b genes and to treatment with the DNA methylation inhibitor hydralazine to investigate whether DNA demethylation restores sensitivity to adriamycin.ResultsMCF-7/Adr cells exhibited the multi-drug resistant phenotype as demonstrated by adriamycin resistance, mdr1 gene over-expression, decreased intracellular accumulation of adriamycin, and cross-resistance to paclitaxel. The mdr phenotype was accompanied by global DNA hypermetylation, over-expression of dnmt genes, and increased DNA methyltransferase activity as compared with wild-type MCF-7 cells. DNA demethylation through antisense targeting of dnmts or hydralazine restored adriamycin sensitivity of MCF-7/Adr cells to a greater extent than verapamil, a known inhibitor of mdr protein, suggesting that DNA demethylation interferes with the epigenetic reprogramming that participates in the drug-resistant phenotype.ConclusionWe provide evidence that DNA hypermethylation is at least partly responsible for development of the multidrug-resistant phenotype in the MCF-7/Adr model and that hydralazine, a known DNA demethylating agent, can revert the resistant phenotype.

Microneedles: A Valuable Physical Enhancer to Increase Transdermal Drug Delivery

Transdermal drug delivery offers an attractive alternative to the conventional drug delivery methods of oral administration and injection. However, the stratum corneum acts as a barrier that limits the penetration of substances through the skin. Recently, the use of micron‐scale needles in increasing skin permeability has been proposed and shown to dramatically increase transdermal delivery. Microneedles have been fabricated with a range of sizes, shapes, and materials. Most in vitro drug delivery studies have shown these needles to increase skin permeability to a broad range of drugs that differ in molecular size and weight. In vivo studies have demonstrated satisfactory release of oligonucleotides and insulin and the induction of immune responses from protein and DNA vaccines. Microneedles inserted into the skin of human subjects were reported to be painless. For all these reasons, microneedles are a promising technology to deliver drugs into the skin. This review presents the main findings concerning the use of microneedles in transdermal drug delivery. It also covers types of microneedles, their advantages and disadvantages, enhancement mechanisms, and trends in transdermal drug delivery.

Electroporation as an Efficient Physical Enhancer for Skin Drug Delivery

Transdermal drug delivery offers an attractive alternative to the conventional drug delivery methods of oral administration and injection. However, the stratum corneum acts as a barrier that limits the penetration of substances through the skin. Application of high‐voltage pulses to the skin increases its permeability (electroporation) and enables the delivery of various substances into and through the skin. The application of electroporation to the skin has been shown to increase transdermal drug delivery. Moreover, electroporation, used alone or in combination with other enhancement methods, expands the range of drugs (small to macromolecules, lipophilic or hydrophilic, charged or neutral molecules) that can be delivered transdermally. The efficacy of transport depends on the electrical parameters and the physicochemical properties of drugs. The in vivo application of high‐voltage pulses is well tolerated, but muscle contractions are usually induced. The electrode and patch design is an important issue to reduce the discomfort of the electrical treatment in humans. This review presents the main findings in the field of electroporation—namely, transdermal drug delivery. Particular attention is paid to proposed enhancement mechanisms and trends in the field of topical and transdermal delivery.

Nanocarrier Systems for Transdermal Drug Delivery

The nanomedicine which is the application of technologies on the scale of 1 to 500 nm to diagnose and treat diseases, it has become a very relevant topic nowadays. During the last century, there has been a lot of new research and patents regarding nanomedicine in health sciences [1]. The objective of nanomedicine is to diagnose and preserve the health without side effects with noninvasive treatments. To reach these goals, nanomedicine offers a lot of new tools and capabilities. The manipulation that nanomedicine provides to the drugs and other materials in the nanometer scale can change the basic properties and bioactivity of materials. The solubility, increment in surface area, control release and site-targeted delivery are some characteristics that nanotechnology can manipulate on drug delivery systems.

High Performance Liquid Chromatography Fluorescence Method for the Determination of Seleno-Amino Acids in Ovine Blood Plasma

This study was performed to validate an effective high performance liquid chromatography-fluorescence detection method for the separation of seleno-amino acids and sulfur amino acids in biological samples using pre-column derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. Standard calibration and standard addition procedures were developed, but the quantification of seleno-amino acids was carried out by a second method. The coefficient of variation for precision was < 15% and accuracies were 96 to 98%. Recoveries in the extraction method were between 63 and 99%. This method was applied to the contribution of seleno-amino acids containing methylselenocysteine and selenomethionine in plasma tissue of lambs during infusion study. The method demonstrated good selectivity, sensitivity, precision, and accuracy.

Pathophysiological response to experimental oral overdose of different forms of selenium in lambs

Abstract Twelve male lambs (30.5±2.1 kg) with cannulas in the rumen were divided into three groups to evaluate the accidental toxicity of selenium (Se) via digestive tract. The first group received intraruminal bolus without a Se source; the second group received intraruminal bolus with sodium selenite (SS: Na2SeO3); the third group received intraruminal bolus with barium selenate (BS: BaSeO4). The ruminal boluses were immediately degraded by a manufacturing defect, releasing an amount of 366 mg Se via rumen. All lambs had tachypnea (80 cycles per minute), a metallic smell in the oral cavity and laminitis foot. Analyses of necropsy and histopathology showed frequencies of lesions in the myocardium and skeletal muscle, lesions in the myocardium were more common in lambs intoxicated with SS or BS than the control group (P<0.05); the tissues were swollen and there were hyaline fibres (Zenker degeneration), as well as fragments and proliferation of the nuclei with necrosis. Se concentration in lambs intoxicated with SS and BS were: myocardium: 0.389 and 0.332; skeletal muscle: 0.583 and 0.492; and kidney: 2.871 and 2.841 μg/g fresh tissue, respectively. The Se concentration in blood increased over 2.5 times in the intoxicated lambs with both sources of Se (0.14 vs. 0.36 μg/mL) from baseline to the 22 days of sampling. There was a lower correlation (r=0.46) in SS intoxication group than the control and BS group (r≥0.93). As a conclusion, high dosages of 366 mg Se or 12 mg Se/kg BW in an accidental dosage did not cause a severe mortality but all lambs immediately showed reduced feed intake, metallic smell exhalation and depression.