Antineoplásico potencial: síntese de derivado de salicilato de octila e5-fluoruracila para tratamento do câncer de pele

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Efeito citotóxico do sistema HRP/Indóis em células McCoy in vitro

Pós-graduação em Biociências e Biotecnologia Aplicadas à Farmácia - FCFAR

Sensibilização comportamental ao etanol e neuroadaptações envolvendo a proteína (DELTA)FOSB: efeitos do tratamento com N-Acetilcisteína em camundongos

Pharmacological treatments currently used in ethanol addiction are inefficient, requiring new drugs for this purpose. The pro-drug N-Acetylcysteine (N-Ac) has shown efficacy in the treatment of addiction to cocaine and nicotine in preclinical research and clinical pilot studies. When administered, N-Ac is subsequently converted to cysteine, and cystine, which has an action on the central nervous system. However, there are few data about the possible application of N-Ac in the ethanol addiction. Behavioral sensitization is the gradual increase of the psychostimulant effects induced by repeated administration of drugs of abuse, including ethanol, and its development has been linked to important neuroadaptations in addiction. These neuroadaptations occur in neural circuits that mediate the reinforcing properties of these drugs and may involve several proteins. The ΔFosB protein accumulates in neurons after repeated activation and mediates long lasting changes in response to drugs of abuse. These changes are manifested mainly in the nucleus accumbens (Acb) and prefrontal cortex (PFC) neurons. The aim of the study was to evaluate the effects of N-Ac treatment in the development of ethanol behavioral sensitization and in alterations in the ΔFosB protein in mice. Swiss male mice were exposed to a standardized behavioral sensitization protocol to the experimental conditions of the laboratory and treated with N-Ac. At the end of behavioral sensitization procedure, animals were euthanized and their brains removed for ΔFosB quantification by Western blotting. Two experiments of behavioral sensitization were performed to the standardization of the protocol. The first, although effective in demonstrating the development of behavioral sensitization, was not effective in allowing the evaluation of the expression of the behavioral sensitization. The age of the animals and the conditions of luminosity and color of locomotion apparatus were changed and a new...

Sensibilização comportamental ao etanol e neuroadaptações envolvendo a proteína (DELTA)FOSB: efeitos do tratamento com N-Acetilcisteína em camundongos

Pharmacological treatments currently used in ethanol addiction are inefficient, requiring new drugs for this purpose. The pro-drug N-Acetylcysteine (N-Ac) has shown efficacy in the treatment of addiction to cocaine and nicotine in preclinical research and clinical pilot studies. When administered, N-Ac is subsequently converted to cysteine, and cystine, which has an action on the central nervous system. However, there are few data about the possible application of N-Ac in the ethanol addiction. Behavioral sensitization is the gradual increase of the psychostimulant effects induced by repeated administration of drugs of abuse, including ethanol, and its development has been linked to important neuroadaptations in addiction. These neuroadaptations occur in neural circuits that mediate the reinforcing properties of these drugs and may involve several proteins. The ΔFosB protein accumulates in neurons after repeated activation and mediates long lasting changes in response to drugs of abuse. These changes are manifested mainly in the nucleus accumbens (Acb) and prefrontal cortex (PFC) neurons. The aim of the study was to evaluate the effects of N-Ac treatment in the development of ethanol behavioral sensitization and in alterations in the ΔFosB protein in mice. Swiss male mice were exposed to a standardized behavioral sensitization protocol to the experimental conditions of the laboratory and treated with N-Ac. At the end of behavioral sensitization procedure, animals were euthanized and their brains removed for ΔFosB quantification by Western blotting. Two experiments of behavioral sensitization were performed to the standardization of the protocol. The first, although effective in demonstrating the development of behavioral sensitization, was not effective in allowing the evaluation of the expression of the behavioral sensitization. The age of the animals and the conditions of luminosity and color of locomotion apparatus were changed and a new...

Untersuchungen zur Charakterisierung der Bystander-Effekte bei einer in vivo Therapie mit Suizidgenen

Untersuchungen zur Charakterisierung der Bystander-Effekte bei einer in vivo Therapie mit Suizidgenen Bei Tumoren eines syngenen Prostatakarzinoms der Ratte (Dunning R3327 AT-1), die zuvor ex vivo mit einem Fusionsgen aus Cytosin Deaminase und Tymidinkinase (AT-1/CDglyTK) transfiziert wurden, konnte durch Kombinationsbehandlung mit Ganciclovir (GCV) und 5-Fluorocytosin (5-FC) komplette Remission und Langzeitüberleben erzielt werden. Dagegen ergaben sich bei Applikation nur einer Pro-Drug lediglich lokale Tumorkontrollraten von 83% (GCV) und 57% (5-FC). Noch geringere therapeutische Effekte einer Kombinationstherapie mit GCV und 5-FC wurden beobachtet, wenn in Anlehnung an die klinische Situation der Anteil suizidgen-tragender Zellen in den Tumoren auf < 20% abgesenkt wurde. Molekularbiologische Analysen dieser Mischtumore zeigen eine Verminderung membranständiger Connexinproteine, welche für den interzellulären Transport phosphorylierter GCV-Metabolite über Gap-junctions erforderlich sind. Pharmakodynamische Untersuchungen mittels 19F-NMR belegen eine effiziente Metabolisierung von 5-FC zu 5-Fluorouracil (5-FU) und den anschließenden Einbau der F-Nukleotide in die DNA. Dennoch sind die intrazellulären und sezernierten 5-FU Konzentrationen für eine Inaktivierung benachbarter Zellen im Sinne eines „lokalen-Bystander-Effektes“ nicht ausreichend. Bei gleichzeitiger Therapie von AT-1 und AT-1/CDglyTK Tumoren, kommt es nicht zur Regression des AT-1 Tumors und damit nicht zu einem „Distalen-Bystander-Effekt“. Dagegen führt die Induktion eines immunologischen Gedächtnisses zu deutlich verminderten Angehraten bei später injizierten AT-1 Tumoren. Die Suizidgen-Therapie ist ein erfolgversprechender Ansatz zur Behandlung maligner Erkrankungen, bei dem die lokalen und distalen Bystander-Effekte individueller Tumoren den therapeutischen Erfolg maßgeblich mitbestimmen.

Conatumumab (AMG 655) coated nanoparticles for targeted pro-apoptotic drug delivery

Colloidal nanoparticle drug delivery systems have attracted much interest for their ability to enable effective formulation and delivery of therapeutic agents. The selective delivery of these nanoparticles to the disease site can be enhanced by coating the surface of the nanoparticles with targeting moieties, such as antibodies. In this current work, we demonstrate that antibodies on the surface of the particles can also elicit key biological effects. Specifically, we demonstrate the induction of apoptosis in colorectal HCT116 cancer cells using PLGA nanoparticles coated with Conatumumab (AMG 655) death receptor 5-specific antibodies (DR5-NP). We show that DR5-NP preferentially target DR5-expressing cells and present a sufficient density of antibody paratopes to induce apoptosis via DR5, unlike free AMG 655 or non-targeted control nanoparticles. We also demonstrate that DR5-targeted nanoparticles encapsulating the cytotoxic drug camptothecin are effectively targeted to the tumour cells, thereby producing enhanced cytotoxic effects through simultaneous drug delivery and apoptosis induction. These results demonstrate that antibodies on nanoparticulate surfaces can be exploited for dual modes of action to enhance the therapeutic utility of the modality. (C) 2011 Elsevier Ltd. All rights reserved.

A convergent solution-phase synthesis of the macrocycle Ac-Phe-[Orn-Pro-D-Cha-Trp-Arg], a potent new antiinflammatory drug

Relatively few cyclic peptides have reached the pharmaceutical marketplace during the past decade, most produced through fermentation rather than made synthetically. Generally, this class of compounds is synthesized for research purposes on milligram scales by solid-phase methods, but if the potential of macrocyclic peptidomimetics is to be realized, low-cost larger scale solution-phase syntheses need to be devised and optimized to provide sufficient quantities for preclinical, clinical, and commercial uses. Here, we describe a cheap, medium-scale, solution-phase synthesis of the first reported highly potent, selective, and orally active antagonist of the human C5a receptor. This compound, Ac-Phe[Orn-Pro-D-Cha-Trp-Arg], known as 3D53, is a macrocyclic peptidomimetic of the human plasma protein C5a and displays excellent antiinflammatory activity in numerous animal models of human disease. In a convergent approach, two tripeptide fragments Ac-Phe-Orn-(Boc)-Pro-OH and H-D-Cha-Trp(For)-Arg-OEt were first prepared by high-yielding solution-phase couplings using a mixed anhydride method before coupling them to give a linear hexapeptide which, after deprotection, was obtained in 38% overall yield from the commercially available amino acids. Cyclization in solution using BOP reagent gave the antagonist in 33% yield (13% overall) after HPLC purification. Significant features of the synthesis were that the Arg side chain was left unprotected throughout, the component Boe-D-Cha-OH was obtained very efficiently via hydrogenation Of D-Phe with PtO2 in TFA/water, the tripeptides were coupled at the Pro-Cha junction to minimize racemization via the oxazolone pathway, and the entire synthesis was carried out without purification of any intermediates. The target cyclic product was purified (>97%) by reversed-phase HPLC. This convergent synthesis with minimal use of protecting groups allowed batches of 50100 g to be prepared efficiently in high yield using standard laboratory equipment. This type of procedure should be useful for making even larger quantities of this and other macrocyclic peptidomimetic drugs.

Combination of MEK-ERK inhibitor and hyaluronic acid has a synergistic effect on anti-hypertrophic and pro-chondrogenic activities in osteoarthritis treatment

We hypothesised that a potentially disease-modifying osteoarthritis (OA) drug such as hyaluronic acid (HA) given in combination with anti-inflammatory signalling agents such as mitogen-activated protein kinase kinase–extracellular signal-regulated kinase (MEK-ERK) signalling inhibitor (U0126) could result in additive or synergistic effects on preventing the degeneration of articular cartilage. Chondrocyte differentiation and hypertrophy were evaluated using human OA primary cells treated with either HA or U0126, or the combination of HA + U0126. Cartilage degeneration in menisectomy (MSX) induced rat OA model was investigated by intra-articular delivery of either HA or U0126, or the combination of HA + U0126. Histology, immunostaining, RT-qPCR, Western blotting and zymography were performed to assess the expression of cartilage matrix proteins and hypertrophic markers. Phosphorylated ERK (pERK)1/2-positive chondrocytes were significantly higher in OA samples compared with those in healthy control suggesting the pathological role of that pathway in OA. It was noted that HA + U0126 significantly reduced the levels of pERK, chondrocyte hypertrophic markers (COL10 and RUNX2) and degenerative markers (ADAMTs5 and MMP-13), however, increased the levels of chondrogenic markers (COL2) compared to untreated or the application of HA or U0126 alone. In agreement with the results in vitro, intra-articular delivery of HA + U0126 showed significant therapeutic improvement of cartilage in rat MSX OA model compared with untreated or the application of HA or U0126 alone. Our study suggests that the combination of HA and MEK-ERK inhibition has a synergistic effect on preventing cartilage degeneration.

IL-23 is pro-proliferative, epigenetically regulated and modulated by chemotherapy in non-small cell lung cancer

Background IL-23 is a member of the IL-6 super-family and plays key roles in cancer. Very little is currently known about the role of IL-23 in non-small cell lung cancer (NSCLC). Methods RT-PCR and chromatin immunopreciptiation (ChIP) were used to examine the levels, epigenetic regulation and effects of various drugs (DNA methyltransferase inhibitors, Histone Deacetylase inhibitors and Gemcitabine) on IL-23 expression in NSCLC cells and macrophages. The effects of recombinant IL-23 protein on cellular proliferation were examined by MTT assay. Statistical analysis consisted of Student's t-test or one way analysis of variance (ANOVA) where groups in the experiment were three or more. Results In a cohort of primary non-small cell lung cancer (NSCLC) tumours, IL-23A expression was significantly elevated in patient tumour samples (p<0.05). IL-23A expression is epigenetically regulated through histone post-translational modifications and DNA CpG methylation. Gemcitabine, a chemotherapy drug indicated for first-line treatment of NSCLC also induced IL-23A expression. Recombinant IL-23 significantly increased cellular proliferation in NSCLC cell lines. Conclusions These results may therefore have important implications for treating NSCLC patients with either epigenetic targeted therapies or Gemcitabine. © 2012 Elsevier Ireland Ltd.

Integrated genome-wide chromatin occupancy and expression analyses identify key myeloid pro-differentiation transcription factors repressed by Myb

To gain insight into the mechanisms by which the Myb transcription factor controls normal hematopoiesis and particularly, how it contributes to leukemogenesis, we mapped the genome-wide occupancy of Myb by chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-Seq) in ERMYB myeloid progenitor cells. By integrating the genome occupancy data with whole genome expression profiling data, we identified a Myb-regulated transcriptional program. Gene signatures for leukemia stem cells, normal hematopoietic stem/progenitor cells and myeloid development were overrepresented in 2368 Myb regulated genes. Of these, Myb bound directly near or within 793 genes. Myb directly activates some genes known critical in maintaining hematopoietic stem cells, such as Gfi1 and Cited2. Importantly, we also show that, despite being usually considered as a transactivator, Myb also functions to repress approximately half of its direct targets, including several key regulators of myeloid differentiation, such as Sfpi1 (also known as Pu.1), Runx1, Junb and Cebpb. Furthermore, our results demonstrate that interaction with p300, an established coactivator for Myb, is unexpectedly required for Myb-mediated transcriptional repression. We propose that the repression of the above mentioned key pro-differentiation factors may contribute essentially to Myb's ability to suppress differentiation and promote self-renewal, thus maintaining progenitor cells in an undifferentiated state and promoting leukemic transformation. © 2011 The Author(s).

Introduction of high-content screening method for studying drug-induced mitochondrial dysfunction in hepatocytes

Drug induced liver injury is one of the frequent reasons for the drug removal from the market. During the recent years there has been a pressure to develop more cost efficient, faster and easier ways to investigate drug-induced toxicity in order to recognize hepatotoxic drugs in the earlier phases of drug development. High Content Screening (HCS) instrument is an automated microscope equipped with image analysis software. It makes the image analysis faster and decreases the risk for an error caused by a person by analyzing the images always in the same way. Because the amount of drug and time needed in the analysis are smaller and multiple parameters can be analyzed from the same cells, the method should be more sensitive, effective and cheaper than the conventional assays in cytotoxicity testing. Liver cells are rich in mitochondria and many drugs target their toxicity to hepatocyte mitochondria. Mitochondria produce the majority of the ATP in the cell through oxidative phosphorylation. They maintain biochemical homeostasis in the cell and participate in cell death. Mitochondria is divided into two compartments by inner and outer mitochondrial membranes. The oxidative phosphorylation happens in the inner mitochondrial membrane. A part of the respiratory chain, a protein called cytochrome c, activates caspase cascades when released. This leads to apoptosis. The aim of this study was to implement, optimize and compare mitochondrial toxicity HCS assays in live cells and fixed cells in two cellular models: human HepG2 hepatoma cell line and rat primary hepatocytes. Three different hepato- and mitochondriatoxic drugs (staurosporine, rotenone and tolcapone) were used. Cells were treated with the drugs, incubated with the fluorescent probes and then the images were analyzed using Cellomics ArrayScan VTI reader. Finally the results obtained after optimizing methods were compared to each other and to the results of the conventional cytotoxicity assays, ATP and LDH measurements. After optimization the live cell method and rat primary hepatocytes were selected to be used in the experiments. Staurosporine was the most toxic of the three drugs and caused most damage to the cells most quickly. Rotenone was not that toxic, but the results were more reproducible and thus it would serve as a good positive control in the screening. Tolcapone was the least toxic. So far the conventional analysis of cytotoxicity worked better than the HCS methods. More optimization needs to be done to get the HCS method more sensitive. This was not possible in this study due to time limit.

Cyclin dependent protein kinases as drug targets – potential in cardiovascular diseases

Complications of atherosclerosis such as myocardial infarction and stroke are the primary cause of death in Western societies. The development of atherosclerotic lesions is a complex process, including endothelial cell dysfunction, inflammation, extracellular matrix alteration and vascular smooth muscle cell (VSMC) proliferation and migration. Various cell cycle regulatory proteins control VSMC proliferation. Protein kinases called cyclin dependent kinases (CDKs) play a major role in regulation of cell cycle progression. At specific phases of the cell cycle, CDKs pair with cyclins to become catalytically active and phosphorylate numerous substrates contributing to cell cycle progression. CDKs are also regulated by cyclin dependent kinase inhibitors, activating and inhibitory phosphorylation, proteolysis and transcription factors. This tight regulation of cell cycle is essential; thus its deregulation is connected to the development of cancer and other proliferative disorders such as atherosclerosis and restenosis as well as neurodegenerative diseases. Proteins of the cell cycle provide potential and attractive targets for drug development. Consequently, various low molecular weight CDK inhibitors have been identified and are in clinical development. Tylophorine is a phenanthroindolizidine alkaloid, which has been shown to inhibit the growth of several human cancer cell lines. It was used in Ayurvedic medicine to treat inflammatory disorders. The aim of this study was to investigate the effect of tylophorine on human umbilical vein smooth muscle cell (HUVSMC) proliferation, cell cycle progression and the expression of various cell cycle regulatory proteins in order to confirm the findings made with tylophorine in rat cells. We used several methods to determine our hypothesis, including cell proliferation assay, western blot and flow cytometric cell cycle distribution analysis. We demonstrated by cell proliferation assay that tylophorine inhibits HUVSMC proliferation dose-dependently with an IC50 value of 164 nM ± 50. Western blot analysis was used to determine the effect of tylophorine on expression of cell cycle regulatory proteins. Tylophorine downregulates cyclin D1 and p21 expression levels. The results of tylophorine’s effect on phosphorylation sites of p53 were not consistent. More sensitive methods are required in order to completely determine this effect. We used flow cytometric cell cycle analysis to investigate whether tylophorine interferes with cell cycle progression and arrests cells in a specific cell cycle phase. Tylophorine was shown to induce the accumulation of asynchronized HUVSMCs in S phase. Tylophorine has a significant effect on cell cycle, but its role as cell cycle regulator in treatment of vascular proliferative diseases and cancer requires more experiments in vitro and in vivo.

Literature Review: Investigating Drug Transport at the Blood-Brain Barrier and the Effects of P-glycoprotein on the Brain Distribution of Drugs.

The blood-brain barrier (BBB) is a unique barrier that strictly regulates the entry of endogenous substrates and xenobiotics into the brain. This is due to its tight junctions and the array of transporters and metabolic enzymes that are expressed. The determination of brain concentrations in vivo is difficult, laborious and expensive which means that there is interest in developing predictive tools of brain distribution. Predicting brain concentrations is important even in early drug development to ensure efficacy of central nervous system (CNS) targeted drugs and safety of non-CNS drugs. The literature review covers the most common current in vitro, in vivo and in silico methods of studying transport into the brain, concentrating on transporter effects. The consequences of efflux mediated by p-glycoprotein, the most widely characterized transporter expressed at the BBB, is also discussed. The aim of the experimental study was to build a pharmacokinetic (PK) model to describe p-glycoprotein substrate drug concentrations in the brain using commonly measured in vivo parameters of brain distribution. The possibility of replacing in vivo parameter values with their in vitro counterparts was also studied. All data for the study was taken from the literature. A simple 2-compartment PK model was built using the Stella™ software. Brain concentrations of morphine, loperamide and quinidine were simulated and compared with published studies. Correlation of in vitro measured efflux ratio (ER) from different studies was evaluated in addition to studying correlation between in vitro and in vivo measured ER. A Stella™ model was also constructed to simulate an in vitro transcellular monolayer experiment, to study the sensitivity of measured ER to changes in passive permeability and Michaelis-Menten kinetic parameter values. Interspecies differences in rats and mice were investigated with regards to brain permeability and drug binding in brain tissue. Although the PK brain model was able to capture the concentration-time profiles for all 3 compounds in both brain and plasma and performed fairly well for morphine, for quinidine it underestimated and for loperamide it overestimated brain concentrations. Because the ratio of concentrations in brain and blood is dependent on the ER, it is suggested that the variable values cited for this parameter and its inaccuracy could be one explanation for the failure of predictions. Validation of the model with more compounds is needed to draw further conclusions. In vitro ER showed variable correlation between studies, indicating variability due to experimental factors such as test concentration, but overall differences were small. Good correlation between in vitro and in vivo ER at low concentrations supports the possibility of using of in vitro ER in the PK model. The in vitro simulation illustrated that in the simulation setting, efflux is significant only with low passive permeability, which highlights the fact that the cell model used to measure ER must have low enough paracellular permeability to correctly mimic the in vivo situation.

Study of interaction between drug enantiomers and serum albumin by capillary electrophoresis

The interaction between drugs and human serum albumin (HSA) was investigated by capillary electrophoresis (CE). It involves stereoselectivity, drug displacement and synergism effects. Under protein-drug binding equilibrium, the unbound concentrations of drug enantiomers were measured by frontal analysis (FA). The stereoselectivity of verapamil (VER) binding to HSA was proved by the different free fractions of two enantiomers. In physiological pH (7.4, ionic strength 0.17 phosphate buffer) when 300 mu M (+/-) VER were equilibrated with 500 mu M HSA, the concentration of unbound S-VER was about 1.7 times its antipode. The binding constants of two enantiomers, KR-VER and KS-VER, were 2670 and 850 M-1, respectively. However, no obvious stereoselective binding of propranolol (PRO) to HSA was observed. Trimethyl-beta-cyclodextrin (45 mM) was used as a chiral selector in pH 2.5 phosphate buffer. Several drug systems were studied by the method. When ibuprofen (IBU) was added into VER-HSA solution. R-VER was partially displaced while S-VER was not displaced at all. A binding synergism effect between bupivacaine (BUP) and verapamil was observed and further study suggested that verapamil and bupivacaine occupy different binding site of HSA (site II and site III, respectively).