Preliminary biocompatibility investigation of magnetic albumin nanosphere designed as a potential versatile drug delivery system

Background: The magnetic albumin nanosphere (MAN), encapsulating maghemite nanoparticles, was designed as a magnetic drug delivery system (MDDS) able to perform a variety of biomedical applications. It is noteworthy that MAN was efficient in treating Ehrlich's tumors by the magnetohyperthermia procedure. Methods and materials: In this study, several nanotoxicity tests were systematically carried out in mice from 30 minutes until 30 days after MAN injection to investigate their biocompatibility status. Cytometry analysis, viability tests, micronucleus assay, and histological analysis were performed. Results: Cytometry analysis and viability tests revealed MAN promotes only slight and temporary alterations in the frequency of both leukocyte populations and viable peritoneal cells, respectively. Micronucleus assay showed absolutely no genotoxicity or cytotoxicity effects and histological analysis showed no alterations or even nanoparticle clusters in several investigated organs but, interestingly, revealed the presence of MAN clusters in the central nervous system (CNS). Conclusion: The results showed that MAN has desirable in vivo biocompatibility, presenting potential for use as a MDDS, especially in CNS disease therapy.

Proteasome Inhibition Represses Unfolded Protein Response and Nox4, Sensitizing Vascular Cells to Endoplasmic Reticulum Stress-Induced Death

Background: Endoplasmic reticulum (ER) stress has pathophysiological relevance in vascular diseases and merges with proteasome function. Proteasome inhibition induces cell stress and may have therapeutic implications. However, whether proteasome inhibition potentiates ER stress-induced apoptosis and the possible mechanisms involved in this process are unclear. Methodology/Principal Findings: Here we show that proteasome inhibition with MG132, per se at non-lethal levels, sensitized vascular smooth muscle cells to caspase-3 activation and cell death during ER stress induced by tunicamycin (Tn). This effect was accompanied by suppression of both proadaptive (KDEL chaperones) and proapoptotic (CHOP/GADD153) unfolded protein response markers, although, intriguingly, the splicing of XBP1 was markedly enhanced and sustained. In parallel, proteasome inhibition completely prevented ER stress-induced increase in NADPH oxidase activity, as well as increases in Nox4 isoform and protein disulfide isomerase mRNA expression. Increased Akt phosphorylation due to proteasome inhibition partially offset the proapoptotic effect of Tn or MG132. Although proteasome inhibition enhanced oxidative stress, reactive oxygen species scavenging had no net effect on sensitization to Tn or MG132-induced cell death. Conclusion/Relevance: These data indicate unfolded protein response-independent pathways whereby proteasome inhibition sensitizes vascular smooth muscle to ER stress-mediated cell death. This may be relevant to understand the therapeutic potential of such compounds in vascular disease associated with increased neointimal hyperplasia.

Activation of endogenous p53 by combined p19Arf gene transfer and nutlin-3 drug treatment modalities in the murine cell lines B16 and C6

Background: Reactivation of p53 by either gene transfer or pharmacologic approaches may compensate for loss of p19Arf or excess mdm2 expression, common events in melanoma and glioma. In our previous work, we constructed the pCLPG retroviral vector where transgene expression is controlled by p53 through a p53-responsive promoter. The use of this vector to introduce p19Arf into tumor cells that harbor p53wt should yield viral expression of p19Arf which, in turn, would activate the endogenous p53 and result in enhanced vector expression and tumor suppression. Since nutlin-3 can activate p53 by blocking its interaction with mdm2, we explored the possibility that the combination of p19Arf gene transfer and nutlin-3 drug treatment may provide an additive benefit in stimulating p53 function. Methods: B16 (mouse melanoma) and C6 (rat glioma) cell lines, which harbor p53wt, were transduced with pCLPGp19 and these were additionally treated with nutlin-3 or the DNA damaging agent, doxorubicin. Viral expression was confirmed by Western, Northern and immunofluorescence assays. p53 function was assessed by reporter gene activity provided by a p53-responsive construct. Alterations in proliferation and viability were measured by colony formation, growth curve, cell cycle and MTT assays. In an animal model, B16 cells were treated with the pCLPGp19 virus and/or drugs before subcutaneous injection in C57BL/6 mice, observation of tumor progression and histopathologic analyses. Results: Here we show that the functional activation of endogenous p53wt in B16 was particularly challenging, but accomplished when combined gene transfer and drug treatments were applied, resulting in increased transactivation by p53, marked cell cycle alteration and reduced viability in culture. In an animal model, B16 cells treated with both p19Arf and nutlin-3 yielded increased necrosis and decreased BrdU marking. In comparison, C6 cells were quite susceptible to either treatment, yet p53 was further activated by the combination of p19Arf and nutlin-3. Conclusions: To the best of our knowledge, this is the first study to apply both p19Arf and nutlin-3 for the stimulation of p53 activity. These results support the notion that a p53 responsive vector may prove to be an interesting gene transfer tool, especially when combined with p53- activating agents, for the treatment of tumors that retain wild-type p53.

Leukotriene B(4) amplifies NF-kappa B activation in mouse macrophages by reducing SOCS1 inhibition of MyD88 expression

Activation of NF-kappa B and 5-lipoxygenase-mediated (5-LO-mediated) biosynthesis of the lipid mediator leukotriene B(4) (LTB(4)) are pivotal components of host defense and inflammatory responses. However, the role of LTB(4) in mediating innate immune responses elicited by specific TLR ligands and cytokines is unknown. Here we have shown that responses dependent on MyD88 (an adaptor protein that mediates signaling through all of the known TLRs, except TLR3, as well as IL-1 beta and IL-18) are reduced in mice lacking either 5-LO or the LTB(4) receptor BTL1, and that macrophages from these mice are impaired in MyD88-dependent activation of NF-kappa B. This macrophage defect was associated with lower basal and inducible expression of MyD88 and reflected impaired activation of STAT1 and overexpression of the STAT1 inhibitor SOCS1. Expression of MyD88 and responsiveness to the TLR4 ligand LPS were decreased by Stat1 siRNA silencing in WT macrophages and restored by Socs1 siRNA in 5-LO-deficient macrophages. These results uncover a pivotal role in macrophages for the GPCR BLT1 in regulating activation of NF-kappa B through Stat1-dependent expression of MyD88.

Increasing the symmetry of drug crystals: a monoclinic conformational polymorph of the platelet antiaggregating agent ticlopidine hydrochloride

Ticlopidine hydrochloride (TICLID (R)) is a platelet antiaggregating agent whose use as a potent antithrombotic pharmaceutical ingredient is widespread, even though this drug has not been well characterized in the solid state. Only the crystal phase used for drug product manufacturing is known. Here, a new polymorph of ticlopidine hydrochloride was discovered and its structure was determined. While the antecedent polymorph crystallizes in the triclinic space group P (1) over bar, the new crystal phase was solved in the monoclinic space group P2(1)/c. Both polymorphs crystallize as racemic mixtures of enantiomeric (ticlopidine)(+) cations. Detailed geometrical and packing comparisons between the crystal structures of the two polymorphs have allowed us to understand how different supramolecular architectures are assembled. It was feasible to conclude that the main difference between the two polymorphs is a rotation of about 120 degrees on the bridging bond between the thienopyridine and o-chlorobenzyl moieties. The differential o-chlorobenzyl conformation is related to changeable patterns of weak intermolecular contacts involving this moiety, such as edge-to-face Cl center dot center dot center dot pi and C-H center dot center dot center dot pi interactions in the new polymorph and face-to-face pi center dot center dot center dot pi contacts in the triclinic crystal phase, leading to a symmetry increase in the ticlopidine hydrochloride solid state form described for the first time in this study. Other conformational features are slightly different between the two polymorphs, such as the thienopyridine puckerings and the o-chlorophenyl orientations. These conformational characteristics were also correlated to the crystal packing patterns.

Molecular determinants of improved cathepsin B inhibition by new cystatins obtained by DNA shuffling

Background: Cystatins are inhibitors of cysteine proteases. The majority are only weak inhibitors of human cathepsin B, which has been associated with cancer, Alzheimer's disease and arthritis. Results: Starting from the sequences of oryzacystatin-1 and canecystatin-1, a shuffling library was designed and a hybrid clone obtained, which presented higher inhibitory activity towards cathepsin B. This clone presented two unanticipated point mutations as well as an N-terminal deletion. Reversing each point mutation independently or both simultaneously abolishes the inhibitory activity towards cathepsin B. Homology modeling together with experimental studies of the reverse mutants revealed the likely molecular determinants of the improved inhibitory activity to be related to decreased protein stability. Conclusion: A combination of experimental approaches including gene shuffling, enzyme assays and reverse mutation allied to molecular modeling has shed light upon the unexpected inhibitory properties of certain cystatin mutants against Cathepsin B. We conclude that mutations disrupting the hydrophobic core of phytocystatins increase the flexibility of the N-terminus, leading to an increase in inhibitory activity. Such mutations need not affect the inhibitory site directly but may be observed distant from it and manifest their effects via an uncoupling of its three components as a result of increased protein flexibility.

Interference with Hemozoin Formation Represents an Important Mechanism of Schistosomicidal Action of Antimalarial Quinoline Methanols

Background: The parasitic trematode Schistosoma mansoni is one of the major causative agents of human schistosomiasis, which afflicts 200 million people worldwide. Praziquantel remains the main drug used for schistosomiasis treatment, and reliance on the single therapy has been prompting the search for new therapeutic compounds against this disease. Our group has demonstrated that heme crystallization into hemozoin (Hz) within the S. mansoni gut is a major heme detoxification route with lipid droplets involved in this process and acting as a potential chemotherapeutical target. In the present work, we investigated the effects of three antimalarial compounds, quinine (QN), quinidine (QND) and quinacrine (QCR) in a murine schistosomiasis model by using a combination of biochemical, cell biology and molecular biology approaches. Methodology/Principal Findings: Treatment of S. mansoni-infected female Swiss mice with daily intraperitoneal injections of QN, and QND (75 mg/kg/day) from the 11(th) to 17(th) day after infection caused significant decreases in worm burden (39%-61%) and egg production (42%-98%). Hz formation was significantly inhibited (40%-65%) in female worms recovered from QN- and QND-treated mice and correlated with reduction in the female worm burden. We also observed that QN treatment promoted remarkable ultrastructural changes in male and female worms, particularly in the gut epithelium and reduced the granulomatous reaction to parasite eggs trapped in the liver. Microarray gene expression analysis indicated that QN treatment increased the expression of transcripts related to musculature, protein synthesis and repair mechanisms. Conclusions: The overall significant reduction in several disease burden parameters by the antimalarial quinoline methanols indicates that interference with Hz formation in S. mansoni represents an important mechanism of schistosomicidal action of these compounds and points out the heme crystallization process as a valid chemotherapeutic target to treat schistosomiasis.

Recombinant human prolactin promotes human beta cell survival via inhibition of extrinsic and intrinsic apoptosis pathways

Transplantation of pancreatic islets constitutes a promising alternative treatment for type 1 diabetes. However, it is limited by the shortage of organ donors. Previous results from our laboratory have demonstrated beneficial effects of recombinant human prolactin (rhPRL) treatment on beta cell cultures. We therefore investigated the role of rhPRL action in human beta cell survival, focusing on the molecular mechanisms involved in this process. Human pancreatic islets were isolated using an automated method. Islet cultures were pre-treated in the absence or presence of rhPRL and then subjected to serum starvation or cytokine treatment. Beta cells were labelled with Newport green and apoptosis was evaluated using flow cytometry analysis. Levels of BCL2 gene family members were studied by quantitative RT-PCR and western blot. Caspase-8, -9 and -3 activity, as well as nitric oxide production, were evaluated by fluorimetric assays. The proportion of apoptotic beta cells was significantly lowered in the presence of rhPRL under both cell death-induced conditions. We also demonstrated that cytoprotection may involve an increase of BCL2/BAX ratio, as well as inhibition of caspase-8, -9 and -3. Our study provides relevant evidence for a protective effect of lactogens on human beta cell apoptosis. The results also suggest that the improvement of cell survival may involve, at least in part, inhibition of cell death pathways controlled by the BCL2 gene family members. These findings are highly relevant for improvement of the islet isolation procedure and for clinical islet transplantation.

LEUCINE ATTENUATES SKELETAL MUSCLE WASTING VIA INHIBITION OF UBIQUITIN LIGASES

The aim of this study was to assess the effect of leucine supplementation on elements of the ubiquitin proteasome system (UPS) in rat skeletal muscle during immobilization. This effect was evaluated by submitting the animals to a leucine supplementation protocol during hindlimb immobilization, after which different parameters were determined, including: muscle mass; cross-sectional area (CSA); gene expression of E3 ligases/deubiquitinating enzymes; content of ubiquitinated proteins; and rate of protein synthesis. Our results show that leucine supplementation attenuates soleus muscle mass loss driven by immobilization. In addition, the marked decrease in the CSA in soleus muscle type I fibers, but not type II fibers, induced by immobilization was minimized by leucine feeding. Interestingly, leucine supplementation severely minimized the early transient increase in E3 ligase [muscle ring finger 1 (MuRF1) and muscle atrophy F-box (MAFbx)/atrogin-1] gene expression observed during immobilization. The reduced peak of E3 ligase gene expression was paralleled by a decreased content of ubiquitinated proteins during leucine feeding. The protein synthesis rate decreased by immobilization and was not affected by leucine supplementation. Our results strongly suggest that leucine supplementation attenuates muscle wasting induced by immobilization via minimizing gene expression of E3 ligases, which consequently could downregulate UPS-driven protein degradation. It is notable that leucine supplementation does not restore decreased protein synthesis driven by immobilization. Muscle Nerve 41: 800-808, 2010

PKC beta II inhibition attenuates myocardial infarction induced heart failure and is associated with a reduction of fibrosis and pro-inflammatory responses

Protein kinase C beta II (PKC beta II) levels increase in the myocardium of patients with end-stage heart failure (HF). Also targeted overexpression of PKC beta II in the myocardium of mice leads to dilated cardiomyopathy associated with inflammation, fibrosis and myocardial dysfunction. These reports suggest a deleterious role of PKC beta II in HF development. Using a post-myocardial infarction (MI) model of HF in rats, we determined the benefit of chronic inhibition of PKC beta II on the progression of HF over a period of 6 weeks after the onset of symptoms and the cellular basis for these effects. Four weeks after MI, rats with HF signs that were treated for 6 weeks with the PKC beta II selective inhibitor (beta IIV5-3 conjugated to TAT(47-57) carrier peptide) (3 mg/kg/day) showed improved fractional shortening (from 21% to 35%) compared to control (TAT(47-57) carrier peptide alone). Formalin-fixed mid-ventricle tissue sections stained with picrosirius red, haematoxylin and eosin and toluidine blue dyes exhibited a 150% decrease in collagen deposition, a two-fold decrease in inflammation and a 30% reduction in mast cell degranulation, respectively, in rat hearts treated with the selective PKC beta II inhibitor. Further, a 90% decrease in active TGF beta 1 and a significant reduction in SMAD2/3 phosphorylation indicated that the selective inhibition of PKC beta II attenuates cardiac remodelling mediated by the TGF-SMAD signalling pathway. Therefore, sustained selective inhibition of PKC beta II in a post-MI HF rat model improves cardiac function and is associated with inhibition of pathological myocardial remodelling.

The process of drug dispensing and distribution at four Brazilian hospitals: A multicenter descriptive study

A multicenter descriptive study was carried out in two steps: an interview with providers involved in the medication processes, and then non-participating observation of their environment and practices. Only one hospital was found to have a bar-coding, dispensing system connected to a computerized prescription system. fit all participating hospitals at least 90% of the drugs were dispensed and distributed as unit doses, but in none of them did pharmacists assess prescriptions. The study findings showed that the processes of drug dispensing and distribution in Brazilian hospitals encounter several problems, mostly associated to work environment conditions and inadequacy in drug ordering and requests.

Adverse drug events in an intensive care unit of a university hospital

Purpose Adverse drug events (ADEs) are harmful and occur with alarming frequency in critically ill patients. Complex pharmacotherapy with multiple medications increases the probability of a drug interaction (DI) and ADEs in patients in intensive care units (ICUs). The objective of the study is to determine the frequency of ADEs among patients in the ICU of a university hospital and the drugs implicated. Also, factors associated with ADEs are investigated. Methods This cross-sectional study investigated 299 medical records of patients hospitalized for 5 or more days in an ICU. ADEs were identified through intensive monitoring adopted in hospital pharmacovigilance and also ADE triggers. Adverse drug reactions (ADR) causality was classified using the Naranjo algorithm. Data were analyzed through descriptive analysis, and through univariate and multiple logistic regression. Results The most frequent ADEs were ADRs type A, of possible causality and moderate severity. The most frequent ADR was drug-induced acute kidney injury. Patients with ADEs related to DIs corresponded to 7% of the sample. The multiple logistic regression showed that length of hospitalization (OR = 1.06) and administration of cardiovascular drugs (OR = 2.2) were associated with the occurrence of ADEs. Conclusion Adverse drug reactions of clinical significance were the most frequent ADEs in the ICU studied, which reduces patient safety. The number of ADEs related to drug interactions was small, suggesting that clinical manifestations of drug interactions that harm patients are not frequent in ICUs.

Evaluation of three brands of drug interaction software for use in intensive care units

Objective To evaluate drug interaction software programs and determine their accuracy in identifying drug-drug interactions that may occur in intensive care units. Setting The study was developed in Brazil. Method Drug interaction software programs were identified through a bibliographic search in PUBMED and in LILACS (database related to the health sciences published in Latin American and Caribbean countries). The programs` sensitivity, specificity, and positive and negative predictive values were determined to assess their accuracy in detecting drug-drug interactions. The accuracy of the software programs identified was determined using 100 clinically important interactions and 100 clinically unimportant ones. Stockley`s Drug Interactions 8th edition was employed as the gold standard in the identification of drug-drug interaction. Main outcome Sensitivity, specificity, positive and negative predictive values. Results The programs studied were: Drug Interaction Checker (DIC), Drug-Reax (DR), and Lexi-Interact (LI). DR displayed the highest sensitivity (0.88) and DIC showed the lowest (0.69). A close similarity was observed among the programs regarding specificity (0.88-0.92) and positive predictive values (0.88-0.89). The DIC had the lowest negative predictive value (0.75) and DR the highest (0.91). Conclusion The DR and LI programs displayed appropriate sensitivity and specificity for identifying drug-drug interactions of interest in intensive care units. Drug interaction software programs help pharmacists and health care teams in the prevention and recognition of drug-drug interactions and optimize safety and quality of care delivered in intensive care units.

Antimicrobial drug administration errors identified in Brazilian multicentric study

Medication administration errors (MAE) are the most frequent kind of medication errors. Errors with antimicrobial drugs (AD) are relevant because they may interfere inpatient safety and in the development of microbial resistance. The aim of this study is to analyze the AD errors detected in a Brazilian multicentric study of MAE. It was a devcriptive and explorotory study carried out in clinical units in five Brazilian teaching hospitals. The hospitals were investigated during 30 days. MAE were detected by observation technique. MAE were classified in categories: wrong route(WR), wrong patient(WP), wrong dose(WD) wrong time (WT) and unordered drug (UD). AD with MA E were classified by Anatomical-Therapeutical-Chemical Classification System. AD with narrow therapeutic index (NTI) wet-e identified A descriptive statistical analysis was performed using SPSS version 11.5 software. A total of 1500 errors were observed, 277 (18.5%) of them were error with AD. The hopes of AD error were: WT87.7%, QD 6.9%, WR 1.5%, UD 3.2% and WP 0.7%. The number of AD found was 36. The mostly ATC class were fluoroquinolones 13.9%, combinations of penicillin 13.9%, macrolides 8.3% and third-generation cephalosporines 5.6%. The parenteral drug dosage form was associated with 55.6% of AD. 16.7% of AD were NTI. 47.4% of WD and 21.8% WT were with NTI drugs. This study shows that these errors should be considered potential areas for improvement in the medication process and patient safety plus there is requirement to develop rational drug use of AD.

Antiproliferative Effect of Benzophenones and their Influence on Cathepsin Activity

The antiproliferative activity of two prenylated benzophenones isolated from Rheedia brasiliensis. the tri-prenylated garciniaphenone and the tetraprenylated benzophenone 7-epiclusianone, was investigated against human cancer cell lines. The antiproliferative activity on melanoma (UACC-62), breast (MCF-7), drug-resistant breast (NCI-ADR), lung/non-small cells (NCI460), ovarian (OVCAR 03), prostate (PC03), kidney (786-0), lung (NCI-460) and tongue (CRL-1624 and CRL-1623) cancer cells was determined using spectrophotometric quantification of the cellular protein content. The effect of these benzophenones on the activity of cathepsins B and G was also investigated. Garciniaphenone displayed cytostatic activity in all cell lines, whereas 7-epiclusianone showed a dose-dependent cytotoxic effect. The IC(50) values for cell proliferation revealed that 7-epiclusianone is more active than garciniaphenone against most of the cell lines. Furthermore, the antiproliferative effects demonstrated by garciniaphenone and 7-epiclusianone were related to their cathepsin inhibiting properties. In conclusion, 7-epiclusianone is a promising naturally occurring agent which displays multiple inhibitory effects which may be working in concert to inhibit cancer cell proliferation in vitro. The putative pathway by which 7-epiclusianone affects cancer cell development may involve cathepsin inhibition. Copyright (C) 2009 John Wiley & Sons, Ltd.