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1.
Sensors (Basel) ; 21(17)2021 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-34502738

RESUMO

In the field of computer vision, object detection consists of automatically finding objects in images by giving their positions. The most common fields of application are safety systems (pedestrian detection, identification of behavior) and control systems. Another important application is head/person detection, which is the primary material for road safety, rescue, surveillance, etc. In this study, we developed a new approach based on two parallel Deeplapv3+ to improve the performance of the person detection system. For the implementation of our semantic segmentation model, a working methodology with two types of ground truths extracted from the bounding boxes given by the original ground truths was established. The approach has been implemented in our two private datasets as well as in a public dataset. To show the performance of the proposed system, a comparative analysis was carried out on two deep learning semantic segmentation state-of-art models: SegNet and U-Net. By achieving 99.14% of global accuracy, the result demonstrated that the developed strategy could be an efficient way to build a deep neural network model for semantic segmentation. This strategy can be used, not only for the detection of the human head but also be applied in several semantic segmentation applications.


Assuntos
Pedestres , Semântica , Humanos , Processamento de Imagem Assistida por Computador , Redes Neurais de Computação
2.
Pharm Dev Technol ; 26(1): 48-59, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33121318

RESUMO

The pharmaceutical industry has to tackle the explosion of high amounts of poorly soluble APIs. This phenomenon leads to numerous sophisticated solutions. These include the use of multifactorial data analysis identifying correlations between the components and dosage form properties, laboratory and production process parameters with respect to the API liberation Example of such API is bicalutamide. Improved liberation is achieved by particle size reduction. Laboratory batches, with different PSD of API, were filled into gelatinous capsules and consequently granulated for tablet compression. Comparative dissolution profiles with Casodex 150 mg (Astra Zeneca) were performed. The component analysis was used for the statistical evaluation of f1 and f2 factors and D(v,0.9) and D[4,3] parameters of PSD to identify optimal PSD values. Suitable PSD limits for API were statistically confirmed in laboratory and in commercial scale with respect to optimized tablet properties. The tablets were bioequivalent with originator (n = 20; 90% CI for ln AUC0-120: 99.8-111.9%; 90% CI for ln cmax: 101.1-112.9%). In conclusion, the micronisation of the API is still an efficient and inexpensive method improving the bioavailability, although there are more complicated and expensive methods available. Statistical multifactorial methods improved the safety and reproducibility of production.


Assuntos
Anilidas/síntese química , Anilidas/metabolismo , Química Farmacêutica/métodos , Nitrilas/síntese química , Nitrilas/metabolismo , Compostos de Tosil/síntese química , Compostos de Tosil/metabolismo , Disponibilidade Biológica , Análise Multivariada , Comprimidos , Equivalência Terapêutica
3.
Biomolecules ; 10(1)2020 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-31947839

RESUMO

Lysosomal sequestration of anticancer therapeutics lowers their cytotoxic potential, reduces drug availability at target sites, and contributes to cancer resistance. Only recently has it been shown that lysosomal sequestration of weak base drugs induces lysosomal biogenesis mediated by activation of transcription factor EB (TFEB) which, in turn, enhances their accumulation capacity, thereby increasing resistance to these drugs. Here, we addressed the question of whether lysosomal biogenesis is the only mechanism that increases lysosomal sequestration capacity. We found that lysosomal sequestration of some tyrosine kinase inhibitors (TKIs), gefitinib (GF) and imatinib (IM), induced expansion of the lysosomal compartment. However, an expression analysis of lysosomal genes, including lysosome-associated membrane proteins 1, 2 (LAMP1, LAMP2), vacuolar ATPase subunit B2 (ATP6V1B2), acid phosphatase (ACP), and galactosidase beta (GLB) controlled by TFEB, did not reveal increased expression. Instead, we found that both studied TKIs, GF and IM, induced lysosomal fusion which was dependent on nicotinic acid adenine dinucleotide phosphate (NAADP) mediated Ca2+signaling. A theoretical analysis revealed that lysosomal fusion is sufficient to explain the enlargement of lysosomal sequestration capacity. In conclusion, we demonstrated that extracellular TKIs, GF and IM, induced NAADP/Ca2+ mediated lysosomal fusion, leading to enlargement of the lysosomal compartment with significantly increased sequestration capacity for these drugs without apparent lysosomal biogenesis.


Assuntos
Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Antineoplásicos/farmacologia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/efeitos dos fármacos , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Gefitinibe/farmacologia , Humanos , Mesilato de Imatinib/farmacologia , Células K562 , Biogênese de Organelas , Proteínas Tirosina Quinases/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos
4.
Biomolecules ; 9(11)2019 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-31683643

RESUMO

The Lysosomal sequestration of weak-base anticancer drugs is one putative mechanism for resistance to chemotherapy but it has never been directly proven. We addressed the question of whether the lysosomal sequestration of tyrosine kinase inhibitors (TKIs) itself contributes to the drug resistance in vitro. Our analysis indicates that lysosomal sequestration of an anticancer drug can significantly reduce the concentration at target sites, only when it simultaneously decreases its extracellular concentration due to equilibrium, since uncharged forms of weak-base drugs freely diffuse across cellular membranes. Even though the studied TKIs, including imatinib, nilotinib, and dasatinib, were extensively accumulated in the lysosomes of cancer cells, their sequestration was insufficient to substantially reduce the extracellular drug concentration. Lysosomal accumulation of TKIs also failed to affect the Bcr-Abl signaling. Cell pre-treatment with sunitinib significantly enhanced the lysosomal accumulation of the TKIs used; however, without apparent lysosomal biogenesis. Importantly, even increased lysosomal sequestration of TKIs neither decreased their extracellular concentrations nor affected the sensitivity of Bcr-Abl to TKIs. In conclusion, our results clearly show that the lysosomal sequestration of TKIs failed to change their concentrations at target sites, and thus, can hardly contribute to drug resistance in vitro.


Assuntos
Resistencia a Medicamentos Antineoplásicos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/metabolismo , Espaço Extracelular/efeitos dos fármacos , Espaço Extracelular/metabolismo , Humanos , Células K562 , Sunitinibe/farmacologia
5.
J Cell Biochem ; 120(10): 18406-18414, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31209929

RESUMO

The interaction between ABCB1 transporter and its substrates takes place in cell membranes but the available data precludes quantitative analysis of the interaction between transporter and substrate molecules. Further, the amount of transporter is usually expressed as a number of ABCB1 molecules per cell. In contrast, the substrate concentration in cell membranes is estimated by determination of substrate-lipid partition coefficient, as examples. In this study, we demonstrate an approach, which enables us to estimate the concentration of ABCB1 molecules within plasma membranes. For this purpose, human leukemia K562 cells with varying expression levels of ABCB1 were used: drug selected K562/Dox and K562/HHT cells with very high transporter expression, and K562/DoxDR2, K562/DoxDR1, and K562/DoxDR05 cells with gradually decreased expression of ABCB1 derived from K562/Dox cells using RNA interference technology. First, we determined the absolute amount of ABCB1 in cell lysates using immunoblotting and recombinant ABCB1 as a standard. We then determined the relative portion of transporter residing in the plasma membrane using immunohistochemistry in nonpermeabilized and permeabilized cells. These results enabled us to estimate the concentration of ABCB1 in the plasma membrane in resistant cells. The ABCB1 concentrations in the plasma membrane of drug selected K562/Dox and K562/HHT cells containing the highest amount of transporter reached millimolar levels. Concentrations of ABCB1 in the plasma membrane of resistant K562/DoxDR2, K562/DoxDR1, and K562/DoxDR05 cells with lower transporter expression were proportionally decreased.


Assuntos
Membrana Celular/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Western Blotting , Sobrevivência Celular/genética , Sobrevivência Celular/fisiologia , Imunofluorescência , Humanos , Células K562 , Interferência de RNA
6.
Int J Mol Sci ; 18(11)2017 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-29088066

RESUMO

The synthetic curcumin analogue, 3,5-bis[(2-fluorophenyl)methylene]-4-piperidinone (EF-24), suppresses NF-κB activity and exhibits antiproliferative effects against a variety of cancer cells in vitro. Recently, it was reported that EF-24-induced apoptosis was mediated by a redox-dependent mechanism. Here, we studied the effects of N-acetylcysteine (NAC) on EF-24-induced cell death. We also addressed the question of whether the main drug transporters, ABCB1 and ABCG2, affect the cytotoxic of EF-24. We observed that EF-24 induced cell death with apoptotic hallmarks in human leukemia K562 cells. Importantly, the loss of cell viability was preceded by production of reactive oxygen species (ROS), and by a decrease of reduced glutathione (GSH). However, neither ROS production nor the decrease in GSH predominantly contributed to the EF-24-induced cell death. We found that EF-24 formed an adduct with GSH, which is likely the mechanism contributing to the decrease of GSH. Although NAC abrogated ROS production, decreased GSH and prevented cell death, its protective effect was mainly due to a rapid conversion of intra- and extra-cellular EF-24 into the EF-24-NAC adduct without cytotoxic effects. Furthermore, we found that neither overexpression of ABCB1 nor ABCG2 reduced the antiproliferative effects of EF-24. In conclusion, a redox-dependent-mediated mechanism only marginally contributes to the EF-24-induced apoptosis in K562 cells. The main mechanism of NAC protection against EF-24-induced apoptosis is conversion of cytotoxic EF-24 into the noncytotoxic EF-24-NAC adduct. Neither ABCB1 nor ABCG2 mediated resistance to EF-24.


Assuntos
Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Compostos de Benzilideno/farmacologia , Proteínas de Neoplasias/metabolismo , Estresse Oxidativo , Piperidonas/farmacologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Acetilcisteína/metabolismo , Linhagem Celular Tumoral , Glutationa/metabolismo , Humanos , Leucemia/metabolismo , Proteínas de Neoplasias/genética , Espécies Reativas de Oxigênio/metabolismo
7.
Chem Biol Interact ; 273: 171-179, 2017 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-28623111

RESUMO

Recently, it has been suggested that imatinib (IM) and nilotinib (NIL) could be studied beyond their original application, as inhibitors of the drug efflux pump ABCB1 (P-glycoprotein, MDR1). Since the reversal of ABCB1-mediated resistance has never been successfully demonstrated in the clinic, we addressed the question of whether IM and NIL may actually serve as efficient inhibitors of ABCB1. Here we define an efficient inhibitor as a compound that achieves full (90-100%) reversal of drug efflux at a concentration that does not exhibit significant off-target toxicity in vitro. In this study, human leukemia K562 cells expressing various levels of ABCB1 were used. We observed that cells expressing higher ABCB1 levels required higher concentrations of IM and NIL to achieve full reversal of drug efflux. Among the well-known ABCB1 inhibitors, a similar effect was found for cyclosporin A (CsA) but not for zosuquidar. IM was efficient only in cells with the low and moderate ABCB1 expression at high concentrations that were cytotoxic in the absence of Bcr-Abl. In contrast, NIL was as efficient an inhibitor of ABCB1 as CsA. Low and moderate expression levels of ABCB1 could be efficiently inhibited by NIL concentrations without cytotoxic effects in the absence of Bcr-Abl. However, high expression levels of ABCB1 required higher NIL concentrations with off-target cytotoxic effects. In conclusion, application of NIL, but not of IM, in clinics is promising, however, only in cells with low ABCB1 expression levels. We hypothesize that some patients may benefit from an inhibitor exhibiting an ABCB1 expression-dependent effect.


Assuntos
Mesilato de Imatinib/farmacologia , Pirimidinas/farmacologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/antagonistas & inibidores , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Relação Estrutura-Atividade , Células Tumorais Cultivadas
8.
Comput Intell Neurosci ; 2017: 3478602, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28607551

RESUMO

Detection of grapes in real-life images is a serious task solved by researchers dealing with precision viticulture. In the case of white wine varieties, grape detectors based on SVMs classifiers, in combination with a HOG descriptor, have proven to be very efficient. Simplified versions of the detectors seem to be the best solution for practical applications. They offer the best known performance versus time-complexity ratio. As our research showed, a conversion of RGB images to grayscale format, which is implemented at an image preprocessing level, is ideal means for further improvement of performance of the detectors. In order to enhance the ratio, we explored relevance of the conversion in a context of a detector potential sensitivity to a rotation of berries. For this purpose, we proposed a modification of the conversion, and we designed an appropriate method for a tuning of such modified detectors. To evaluate the effect of the new parameter space on their performance, we developed a specialized visualization method. In order to provide accurate results, we formed new datasets for both tuning and evaluation of the detectors. Our effort resulted in a robust grape detector which is less sensitive to image distortion.


Assuntos
Máquina de Vetores de Suporte , Vitis , Vinho/análise , Rotação
9.
J Oral Pathol Med ; 46(4): 301-306, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28199744

RESUMO

BACKGROUND: Buccal flexible films in the form of solid, thin, mucoadhesive patches can be used as dressings separating aphthous lesions from the environment of the oral cavity, which can in turn shorten the treatment period and reduce the pain perception. METHODS: The clinical study was performed on 36 volunteers suffering from aphthous lesions. The first group was treated using standard means-by application of an oral gel containing cholin salicylate (Mundisal) on the aphthous lesion. The second group was treated with the same preparation; however, the lesion was covered with a mucoadhesive film following the application of the gel. The criteria for statistical evaluation were the size of lesions in relation to the length of the treatment and the subjective perception of the treatment results. RESULTS AND CONCLUSIONS: The application of buccal films covering aphthous lesions during the treatment significantly increased the rate of healing when compared with the standard methods of treatment. While the pain improvement was statistically significant as soon as Day 3 in the experimental group, it was only apparent on Day 5 in the control group, and the number of successfully treated patients (pain perception improving to visual analogue scale 2 or less) was at all time points higher in the experimental group than in the control group. The results imply that the use of buccal films for treatment of aphthous lesions is very promising and can lead to a significant reduction in the duration of patients' discomfort.


Assuntos
Bandagens , Estomatite Aftosa/terapia , Adesivos/uso terapêutico , Administração Bucal , Adolescente , Adulto , Idoso , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem
10.
Pharm Dev Technol ; 22(2): 206-217, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-28058866

RESUMO

Hypromellose matrices exhibit extended burst effect immediately after contact with aqueous medium, especially when a water-soluble drug is incorporated. The objective of this study was to reduce burst effect and maintain complete dissolution of a very soluble levetiracetam over 12 h period from hypromellose K4M matrices to obtain zero-order kinetics. Desired changes were achieved by applying water dispersions of insoluble Eudragits® (NE, NM, RL, RS) as a granulation liquid to the drug/microcrystalline cellulose mixture during high-shear granulation (non-thermal treated set) and consequently by thermally treating granules or final tablets (TT), respectively. Applying Eudragit® water dispersions to the drug/microcrystalline cellulose mixture was recognized as an effective method of significantly reducing the burst release (25.4-33.7%) of levetiracetam in comparison with a reference sample without Eudragit®. Multivariate data analysis showed that the addition of Eudragit® reduced burst effect, increased fitting with zero-order kinetics, and supported matrix erosion as the supplementary mechanism to predominant diffusion. Moreover, resulting PCA sub-model revealed the addition of Eudragit® RL and thermal treatment of tablets to be the most suitable method of all. For a 12 h dissolution profile, characterized by low burst effect and drug release close to 100% at the 12th hour, sample RL_TT was the most suitable.


Assuntos
Anticonvulsivantes/administração & dosagem , Preparações de Ação Retardada/química , Derivados da Hipromelose/química , Piracetam/análogos & derivados , Ácidos Polimetacrílicos/química , Anticonvulsivantes/química , Celulose/química , Composição de Medicamentos/métodos , Liberação Controlada de Fármacos , Levetiracetam , Análise Multivariada , Piracetam/administração & dosagem , Piracetam/química , Solubilidade , Comprimidos , Temperatura
11.
Pharm Dev Technol ; 22(2): 138-147, 2017 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26401959

RESUMO

CONTEXT: The preparation of liquisolid systems (LSS) represents a promising method for enhancing a dissolution rate and bioavailability of poorly soluble drugs. The release of the drug from LSS tablets is affected by many factors, including the disintegration time. OBJECTIVE: The evaluation of differences among LSS containing varying amounts and types of commercially used superdisintegrants (Kollidon® CL-F, Vivasol® and Explotab®). MATERIALS AND METHODS: LSS were prepared by spraying rosuvastatin solution onto Neusilin® US2 and further processing into tablets. Varying amounts of superdisintegrants were used and the differences among LSS were evaluated. The multiple scatter plot method was used to visualize the relationships within the obtained data. RESULTS AND DISCUSSION: All disintegrants do not showed negative effect on the flow properties of powder blends. The type and concentration of superdisintegrant had an impact on the disintegration time and dissolution profiles of tablets. Tablets with Explotab® showed the longest disintegration time and the smallest amount of released drug. Fastest disintegration and dissolution rate were observed in tablets containing Kollidon® CL-F (≥2.5% w/w). Also tablets with Vivasol® (2.5-4.0% w/w) showed fast disintegration and complete drug release. CONCLUSION: Kollidon® CL-F and Vivasol® in concentration ≥2.5% are suitable superdisintegrants for LSS with enhanced release of drug.


Assuntos
Compostos de Alumínio/química , Anticolesterolemiantes/administração & dosagem , Compostos de Magnésio/química , Excipientes Farmacêuticos/química , Povidona/química , Rosuvastatina Cálcica/administração & dosagem , Silicatos/química , Amido/análogos & derivados , Anticolesterolemiantes/química , Composição de Medicamentos , Liberação Controlada de Fármacos , Rosuvastatina Cálcica/química , Solubilidade , Amido/química , Comprimidos/química
12.
Pharm Dev Technol ; 22(7): 881-888, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26763623

RESUMO

The aim of this study was to prepare tablets containing ground fruits of cornelian cherry (Cornus mas L.) with high antioxidant capacity. The experiment was planned and evaluated on Design of Experiment (DoE) principle using Multivariate Data Analysis (MVA) as modern tools used in Quality by Design (QbD) approach. Various tableting mixtures with three different particle sizes of the plant material (up to 800 µm, more than 800 µm and their mixture) and percentage of silicon dioxide (1, 3 and 5%) were prepared. Tablets with a diameter of 10 mm and mass of 400 mg were subsequently produced from these mixtures using two compression forces (C1=7 kN and C2=14 kN). Principal Component Analysis (PCA) and Multiple Linear Regression (MLR) with response surface methodology were used to find the influential process-formulation parameters and describe their optimal settings. Finally, it is possible to say that the increasing level of silicon dioxide and the decreasing particle size of ground cornelian cherry lead to prolongation of disintegration time and increase of radial hardness and abrasion loss. Maximal antioxidant activity was obtained using 5% amount of silicon dioxide, the largest particle size and the low compression force.


Assuntos
Antioxidantes , Cornus , Comprimidos , Frutas , Oxirredução
13.
Pharm Dev Technol ; 21(2): 214-21, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25495857

RESUMO

The aim of the study was to prepare PLGA microparticles for prolonged release of mirtazapine by o/w solvent evaporation method and to evaluate effects of PVA concentration and organic solvent choice on microparticles characteristics (encapsulation efficiency, drug loading, burst effect, microparticle morphology). Also in vitro drug release tests were performed and the results were correlated with kinetic model equations to approximate drug release mechanism. It was found that dichloromethane provided microparticles with better qualities (encapsulation efficiency 64.2%, yield 79.7%). Interaction between organic solvent effect and effect of PVA concentration was revealed. The prepared samples released the drug for 5 days with kinetics very close to that of zero order (R(2 )= 0.9549 - 0.9816). According to the correlations, the drug was probably released by a combination of diffusion and surface erosion, enhanced by polymer swelling and chain relaxation.


Assuntos
Antidepressivos/química , Preparações de Ação Retardada/química , Ácido Láctico/química , Mianserina/análogos & derivados , Ácido Poliglicólico/química , Liberação Controlada de Fármacos , Cinética , Cloreto de Metileno/química , Mianserina/química , Microesferas , Mirtazapina , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Solventes/química
14.
Biomed Res Int ; 2015: 580146, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26064926

RESUMO

Infectious stomatitis represents the most common oral cavity ailments. Current therapy is insufficiently effective because of the short residence time of topical liquid or semisolid medical formulations. An innovative application form based on bioadhesive polymers featuring prolonged residence time on the oral mucosa may be a solution to this challenge. This formulation consists of a mucoadhesive oral film with incorporated nanocomposite biomaterial that is able to release the drug directly at the target area. This study describes the unique approach of preparing mucoadhesive oral films from carmellose with incorporating a nanotechnologically modified clay mineral intercalated with chlorhexidine. The multivariate data analysis was employed to evaluate the influence of the formulation and process variables on the properties of the medical preparation. This evaluation was complemented by testing the antimicrobial and antimycotic activity of prepared films with the aim of finding the most suitable composition for clinical application. Generally, the best results were obtained with sample containing 20 mg of chlorhexidine diacetate carried by vermiculite, with carmellose in the form of nonwoven textile in its structure. In addition to its promising physicomechanical, chemical, and mucoadhesive properties, the formulation inhibited the growth of Staphylococcus and Candida; the effect was prolonged for tens of hours.


Assuntos
Anti-Infecciosos/administração & dosagem , Carboximetilcelulose Sódica/administração & dosagem , Sistemas de Liberação de Medicamentos , Nanocompostos/administração & dosagem , Estomatite/tratamento farmacológico , Anti-Infecciosos/química , Materiais Biocompatíveis/administração & dosagem , Materiais Biocompatíveis/química , Carboximetilcelulose Sódica/química , Química Farmacêutica , Quitosana/química , Clorexidina/administração & dosagem , Clorexidina/química , Humanos , Boca/efeitos dos fármacos , Boca/microbiologia , Nanocompostos/química , Polímeros/administração & dosagem , Polímeros/química , Estomatite/microbiologia
15.
Chem Biol Interact ; 239: 100-10, 2015 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-26115783

RESUMO

Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), an uncoupler of mitochondrial oxidative phosphorylation, inhibits cell proliferation and induces cell death with apoptotic features. It was reported that the cytotoxic effects of FCCP are preceded by a rapid glutathione (GSH) depletion with a subsequent loss of mitochondrial transmembrane potential (ΔΨ). The GSH depletion was suggested as the cause of apoptosis in FCCP treated cells. This conclusion was further supported by the finding that all adverse effects of FCCP including cell death can be prevented by N-acetylcysteine (NAC) a precursor of GSH synthesis (Han and Park, 2011). Here, we argue that neither loss of ΔΨ nor GSH depletion is sufficient to account for induction of apoptosis in FCCP treated leukemia K562 cells. Indeed, the lowest concentration of FCCP that brings about the permanent loss of ΔΨ and the extensive decrease in GSH level induces cell death in minor population of cells. Only much higher concentrations of FCCP, that exceed the range to achieve permanent collapse of ΔΨ, induce extensive apoptosis. The low proapoptotic activity of FCCP could be explained by hyperactivation of protein kinase B/Akt. A detailed LC/MS/MS analysis of cell extracts revealed extensive formation of FCCP adducts with GSH. This effect could explain the mechanism of GSH depletion, which is currently unknown. Although NAC induces an increase in the GSH pool, this effect is not crucial for abrogation of FCCP cytotoxicity. Indeed, the presence of NAC in the growth medium causes a rapid clearance of FCCP due to its quantitative conversion into the FCCP-NAC adduct, which is the real cause of abrogated FCCP cytotoxicity.


Assuntos
Apoptose/efeitos dos fármacos , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/farmacologia , Glutationa/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Acetilcisteína/química , Acetilcisteína/farmacologia , Carbonil Cianeto p-Trifluormetoxifenil Hidrazona/química , Glutationa/química , Humanos , Células K562/efeitos dos fármacos , Células K562/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo
16.
Biomed Res Int ; 2015: 892671, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26090454

RESUMO

Effective assessment and management of wound pain can facilitate both improvements in healing rates and overall quality of life. From a pharmacological perspective, topical application of nonsteroidal anti-inflammatory drugs in the form of film wound dressings may be a good choice. Thus, the aim of this work was to develop novel layered film wound dressings containing ibuprofen based on partially substituted fibrous sodium carboxymethylcellulose (nonwoven textile Hcel NaT). To this end, an innovative solvent casting method using a sequential coating technique has been applied. The concentration of ibuprofen which was incorporated as an acetone solution or as a suspension in a sodium carboxymethylcellulose dispersion was 0.5 mg/cm(2) and 1.0 mg/cm(2) of film. Results showed that developed films had adequate mechanical and swelling properties and an advantageous acidic surface pH for wound application. An in vitro drug release study implied that layered films retained the drug for a longer period of time and thus could minimize the frequency of changing the dressing. Films with suspended ibuprofen demonstrated higher drug content uniformity and superior in vitro drug release characteristics in comparison with ibuprofen incorporation as an acetone solution. Prepared films could be potential wound dressings for the effective treatment of wound pain in low exuding wounds.


Assuntos
Carboximetilcelulose Sódica/administração & dosagem , Ibuprofeno/administração & dosagem , Dor/tratamento farmacológico , Cicatrização/efeitos dos fármacos , Bandagens , Humanos , Dor/patologia
17.
Eur J Haematol ; 95(2): 150-9, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25323158

RESUMO

Increased expression of the ABCB1 gene in cancer cells is usually connected with occurrence of multidrug resistance (MDR) and poor prognosis. However, the correlation between ABCB1 expression and MDR phenotype is difficult to prove in clinical samples. Most of the researchers believe that these difficulties are due to the poor reliability and sensitivity of assays for detection of ABCB1 expression in clinical samples. However, the complexity of P-gp mediated resistance cannot be reduced to the methodical difficulties only. Here, we addressed the question how widely used methods for detection of ABCB1 expression levels could predict its functional activity and thus its contribution to drug resistance in defined conditions in vitro. The ABCB1 expression was assessed at the mRNA level by quantitative real-time polymerase chain reaction (qRT-PCR), and at the protein level by flow cytometry using UIC2 antibody. The ABCB1 function was monitored using a calcein AM accumulation assay. We observed that K562 cells have approximately 320 times higher level of ABCB1 mRNA than HL-60 cells without detectable function. In addition, resistant K562/Dox cells exhibited significantly higher ABCB1 mRNA expression than resistant K562/HHT cells. However, the functional tests clearly indicated opposite results. Flow cytometric assessment of P-gp, although suggested as a reliable method, contradicted the functional test in K562/Dox and K562/HHT cells. We further used a set of MDR cells expressing various levels of P-gp. Similarly here, flow cytometry not always corresponded to the functional analysis. Our results strongly suggest that an approach which exclusively relies on a simple correlation between ABCB1 expression, either at the mRNA level or protein level, and overall resistance may fail to predict actual contribution of P-gp to overall resistance as the data indicating transporter expression reflect its function only roughly even in well-defined in vitro conditions.


Assuntos
Expressão Gênica , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Linhagem Celular Tumoral , Humanos , Técnicas In Vitro
18.
Acta Pharm ; 64(4): 403-17, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25531782

RESUMO

The main objective of this study was to evaluate the influence of the formulation and process parameters on PLGA microparticles containing a practically insoluble model drug (ibuprofen) prepared by the o/w solvent evaporation method. Multivariate data analysis was used. The effects of altered stirring speed of a mechanical stirrer (600, 1000 rpm), emulsifier concentrations (PVA concentration 0.1 %, 1 %) and solvent selection (dichloromethane, ethyl acetate) on microparticle characteristics (encapsulation efficiency, drug loading, burst effect) were observed. It was found that with increased stirring speed, the PVA concentration or the use of ethyl acetate had a significantly negative effect on encapsulation efficiency. In addition, ethyl acetate had an adverse effect on the burst effect, while increased stirring speed had the opposite effect. Drug load was not affected by any particular variable, but rather by the interactions of evaluated variables.


Assuntos
Portadores de Fármacos/química , Ibuprofeno/administração & dosagem , Ácido Láctico/química , Microesferas , Ácido Poliglicólico/química , Acetatos/química , Química Farmacêutica/métodos , Composição de Medicamentos/métodos , Excipientes/química , Ibuprofeno/química , Cloreto de Metileno/química , Análise Multivariada , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Álcool de Polivinil/química , Solubilidade , Solventes/química
19.
Acta Pharm ; 64(3): 355-67, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25296681

RESUMO

The article describes the development and production of tablets using direct compression of powder mixtures. The aim was to describe the impact of filler particle size and the time of lubricant addition during mixing on content uniformity according to the Good Manufacturing Practice (GMP) process validation requirements. Processes are regulated by complex directives, forcing the producers to validate, using sophisticated methods, the content uniformity of intermediates as well as final products. Cutting down of production time and material, shortening of analyses, and fast and reliable statistic evaluation of results can reduce the final price without affecting product quality. The manufacturing process of directly compressed tablets containing the low dose active pharmaceutical ingredient (API) warfarin, with content uniformity passing validation criteria, is used as a model example. Statistic methods have proved that the manufacturing process is reproducible. Methods suitable for elucidation of various properties of the final blend, e.g., measurement of electrostatic charge by Faraday pail and evaluation of mutual influences of researched variables by partial least square (PLS) regression, were used. Using these methods, it was proved that the filler with higher particle size increased the content uniformity of both blends and the ensuing tablets. Addition of the lubricant, magnesium stearate, during the blending process improved the content uniformity of blends containing the filler with larger particles. This seems to be caused by reduced sampling error due to the suppression of electrostatic charge.


Assuntos
Tecnologia Farmacêutica/normas , Varfarina/química , Varfarina/normas , Química Farmacêutica , Fidelidade a Diretrizes , Guias como Assunto , Dureza , Análise dos Mínimos Quadrados , Lubrificantes/química , Lubrificantes/normas , Modelos Estatísticos , Tamanho da Partícula , Pós , Controle de Qualidade , Reprodutibilidade dos Testes , Eletricidade Estática , Ácidos Esteáricos/química , Ácidos Esteáricos/normas , Propriedades de Superfície , Comprimidos , Tecnologia Farmacêutica/métodos
20.
Biomed Res Int ; 2014: 179568, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25136560

RESUMO

Oral mucosa is an attractive region for the local and systemic application of many drugs. Oral mucoadhesive films are preferred for their prolonged time of residence, the improved bioavailability of the drug they contain, their painless application, their protection against lesions, and their nonirritating properties. This work was focused on preparation of nonmedicated carmellose-based films using both solvent casting and impregnation methods, respectively. Moreover, a modern approach to evaluation of mucoadhesive films applying analysis of texture and subsequent multivariate data analysis was used. In this experiment, puncture strength strongly correlated with tensile strength and could be used to obtain necessary information about the mechanical film characteristics in films prepared using both methods. Puncture work and tensile work were not correlated in films prepared using the solvent casting method, as increasing the amount of glycerol led to an increase in the puncture work in thinner films. All measured texture parameters in films prepared by impregnation were significantly smaller compared to films prepared by solvent casting. Moreover, a relationship between the amount of glycerol and film thickness was observed, and a greater recalculated tensile/puncture strength was needed for an increased thickness in films prepared by impregnation.


Assuntos
Sistemas de Liberação de Medicamentos/métodos , Teste de Materiais , Membranas Artificiais , Mucosa Bucal , Administração Oral , Avaliação Pré-Clínica de Medicamentos
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