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1.
Biochem Pharmacol ; 170: 113667, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31622577

RESUMO

Pathological cardiac hypertrophy (PCH) can be triggered by epidermal growth factor receptor (EGFR) transactivation. Progression of PCH can be prevented by inhibition of hyperactive Na+/H+ exchanger isoform 1 (NHE1). We first aimed, to limit PCH of spontaneously hypertensive rats (SHR) by specific and localized silencing of cardiac EGFR, and second to study the connection of its activation pathway with cardiac NHE1 activity. Short hairpin RNA (shRNA) against EGFR was delivered with a lentivirus (l-shEGFR) in the cardiac left ventricle (LV) wall. Protein expression was analyzed by immunoblots, and NHE1 activity was indirectly measured in isolated papillary muscles by rate of pHi recovery from transient acidification. EGFR protein expression in the LV was reduced compared to the group injected with l-shSCR (Scrambled sequence) without changes in ErbB2 or ErbB4. Hypertrophic parameters together with cardiomyocytes cross sectional area were reduced in animals injected with l-shEGFR. Echocardiographic analysis exhibited a reduced fractional shortening in the l-shSCR group 30 days following treatment that was not observed in l-shEGFR group. l-shEGFR treated rats presented a reduced basal production of reactive oxygen species and decreased lipid peroxidation. NHE1 activity was significantly diminished in hearts with a partial EGFR silencing, without modification of its protein expression. We conclude that specifically silencing cardiac EGFR expression prevents progression of PCH through a pathway that involves a decrease in the NHE1 activity. Lentiviral vectors prove to be a valuable tool for long term expression of shRNA, bringing the possibility to extend its use in clinical area.

2.
J Microbiol ; 57(6): 485-497, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31134579

RESUMO

Fusarium solani has drawn phytopathogenic, biotechnological, and medical interest. In humans, it is associated with localized infections, such as onychomycosis and keratomycosis, as well as invasive infections in immunocompromised patients. One pathogenicity factor of filamentous fungi is biofilm formation. There is still only scarce information about the in vitro mechanism of the formation and composition of F. solani biofilm. In this work, we describe the biofilm formed by a clinical keratomycosis isolate in terms of its development, composition and susceptibility to different antifungals and ultraviolet light (UV) at different biofilm formation stages. We found five biofilm formation stages using scanning electron microscopy: adherence, germination, hyphal development, maturation, and cell detachment. Using epifluorescence microscopy with specific fluorochromes, it was elucidated that the extracellular matrix consists of carbohydrates, proteins, and extracellular DNA. Specific inhibitors for these molecules showed significant biofilm reductions. The antifungal susceptibility against natamycin, voriconazole, caspofungin, and amphotericin B was evaluated by metabolic activity and crystal violet assay, with the F. solani biofilm preformation to 24 h increased in resistance to natamycin, voriconazole, and caspofungin, while the biofilm preformation to 48 h increased in resistance to amphotericin B. The preformed biofilm at 24 h protected and reduced UV light mortality. F. solani isolate could produce a highly structured extra biofilm; its cellular matrix consists of carbohydrate polymers, proteins, and eDNA. Biofilm confers antifungal resistance and decreases its susceptibility to UV light. The fungal biofilm functions as a survival strategy against antifungals and environmental factors.


Assuntos
Antifúngicos/farmacologia , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Biofilmes/efeitos da radiação , Infecções Oculares Fúngicas/microbiologia , Fusarium/efeitos dos fármacos , Fusarium/efeitos da radiação , Ceratite/microbiologia , Farmacorresistência Fúngica/efeitos dos fármacos , Farmacorresistência Fúngica/efeitos da radiação , Fungos/efeitos dos fármacos , Fungos/efeitos da radiação , Fusarium/patogenicidade , Humanos , Hifas/efeitos dos fármacos , Hifas/efeitos da radiação , México , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Viabilidade Microbiana/efeitos da radiação , Microscopia Eletrônica de Varredura
3.
Cell Physiol Biochem ; 52(2): 172-185, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30816666

RESUMO

BACKGROUND/AIMS: Myocardial stretch increases cardiac force in two consecutive phases: The first one due to Frank-Starling mechanism, followed by the gradually developed slow force response (SFR). The latter is the mechanical counterpart of an autocrine/paracrine mechanism involving the release of angiotensin II (Ang II) and endothelin (ET) leading to Na⁺/H⁺ exchanger 1 (NHE-1) phosphorylation and activation. Since previous evidence indicates that p38-MAP kinase (p38-MAPK) negatively regulates the Ang II-induced NHE1 activation in vascular smooth muscle and the positive inotropic effect of ET in the heart, we hypothesized that this kinase might modulate the magnitude of the SFR to stretch. METHODS: Experiments were performed in isolated rat papillary muscles subjected to sudden stretch from 92 to 98% of its maximal length, in the absence or presence of the p38-MAPK inhibitor SB202190, or its inactive analogous SB202474. Western blot technique was used to determine phosphorylation level of p38-MAPK, ERK1/2, p90RSK and NHE-1 (previously immunoprecipitated with NHE-1 polyclonal antibody). Dual specificity phosphatase 6 (DUSP6) expression was evaluated by RT-PCR and western blot. Additionally, the Na⁺-dependent intracellular pH recovery from an ammonium prepulse-induced acid load was used to asses NHE-1 activity. RESULTS: The SFR was larger under p38-MAPK inhibition (SB202190), effect that was not observed in the presence of an inactive analogous (SB202474). Myocardial stretch activated p38-MAPK, while pre-treatment with SB202190 precluded this effect. Inhibition of p38-MAPK increased stretched-induced NHE-1 phosphorylation and activity, key event in the SFR development. Consistently, p38-MAPK inhibition promoted a greater increase in ERK1/2-p90RSK phosphorylation/activation after myocardial stretch, effect that may certainly be responsible for the observed increase in NHE-1 phosphorylation under this condition. Myocardial stretch induced up-regulation of the DUSP6, which specifically dephosphorylates ERK1/2, effect that was blunted by SB202190. CONCLUSION: Taken together, our data support the notion that p38-MAPK activation after myocardial stretch restricts the SFR by limiting ERK1/2-p90RSK phosphorylation, and consequently NHE-1 phosphorylation/activity, through a mechanism that involves DUSP6 up-regulation.


Assuntos
Fosfatase 6 de Especificidade Dupla/biossíntese , Regulação Enzimológica da Expressão Gênica , Sistema de Sinalização das MAP Quinases , Contração Miocárdica , Miocárdio/enzimologia , Regulação para Cima , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Imidazóis/farmacologia , Fosforilação/efeitos dos fármacos , Piridinas/farmacologia , Ratos , Ratos Wistar , Trocador 1 de Sódio-Hidrogênio/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores
4.
Eur J Pharmacol ; 849: 96-105, 2019 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-30721701

RESUMO

Since the original description as potent antianginal compounds, phosphodiesterase 5A inhibitors have continuously increased their possible therapeutic applications. In the heart, Sildenafil was shown to protect against an ischemic insult by decreasing cardiac Na+/H+ exchanger (NHE1) activity, action that was mediated by protein kinase G. p38 mitogen activated protein kinase (p38MAPK) activation was described in cardiac ischemia, but its precise role remains elusive. It has been shown that p38MAPK is activated by protein kinase G (PKG) in certain non-cardiac tissues, while in others modulates NHE1 activity. Current study was aimed to seek the role of p38MAPK in the Sildenafil-triggered pathway leading to NHE1 inhibition in myocardium. Rat isolated papillary muscles were used to evaluate NHE1 activity during intracellular pH recovery from an acidic load. Protein kinases phosphorylation (activation) was determined by western blot. Sustained acidosis promoted NHE1 hyperactivity by enhancing Ser703 phosphorylation, effect that was blunted by Sildenafil. p38MAPK inhibition reversed the effect of Sildenafil on NHE1. Activation of p38MAPK, by Sodium Arsenite or Anisomycin, mimicked the inhibitory effect of Sildenafil on the exchanger. Consistently, Sildenafil induced p38MAPK phosphorylation/activation during acidosis. Neither Sildenafil nor p38MAPK inhibition affected extracellular signal-regulated kinases 1/2 phosphorylation, kinases upstream NHE1. Furthermore, inhibition of NHE1 after p38MAPK activation was precluded by preventing the activation of protein phosphatase 2A with Okadaic Acid. Taken together, these results suggest that activation of p38MAPK is a necessary step to trigger the inhibitory effect of Sildenafil on cardiac NHE1 activity, thorough a mechanism that involves protein phosphatase 2A-mediated exchanger dephosphorylation.


Assuntos
Coração/efeitos dos fármacos , Miocárdio/metabolismo , Citrato de Sildenafila/farmacologia , Trocadores de Sódio-Hidrogênio/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Acidose/enzimologia , Acidose/metabolismo , Acidose/patologia , Animais , Ativação Enzimática/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Miocárdio/citologia , Miocárdio/patologia , Fosforilação/efeitos dos fármacos , Proteína Fosfatase 2/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Wistar , Trocador 1 de Sódio-Hidrogênio/metabolismo
5.
Phys Rev Lett ; 120(24): 248004, 2018 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-29956967

RESUMO

Cluster morphology of spherical particles interacting with a short-range attraction has been extensively studied due to its relevance to many applications, such as the large-scale structure in amorphous materials, phase separation, protein aggregation, and organelle formation in cells. Although it was widely accepted that the range of the attraction solely controls the fractal dimension of clusters, recent experimental results challenged this concept by also showing the importance of the strength of attraction. Using Monte Carlo simulations, we conclusively demonstrate that it is possible to reduce the dependence of the cluster morphology to a single variable, namely, the reduced second virial coefficient, B_{2}^{*}, linking the local properties of colloidal systems to the extended law of corresponding states. Furthermore, the cluster size distribution exhibits two well-defined regimes: one identified for small clusters, whose fractal dimension, d_{f}, does not depend on the details of the attraction, i.e., small clusters have the same d_{f}, and another related to large clusters, whose morphology depends exclusively on B_{2}^{*}, i.e., d_{f} of large aggregates follows a master curve, which is only a function of B_{2}^{*}. This physical scenario is confirmed with the reanalysis of experimental results on colloidal-polymer mixtures.

7.
AAPS PharmSciTech ; 19(4): 1672-1680, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29520588

RESUMO

Silver nanoparticles (AgNPs) are known to interact with proteins, leading to modifications of the plasmonic absorption that can be used to monitor this interaction, entailing a promising application for sensing adsorption of therapeutic proteins in primary containers. First, transmission electron microscopy in combination with plasmonic absorption and light scattering responses were used to characterize AgNPs and protein-AgNP complexes, including its concentration dependence, using two therapeutic molecules as models: a monoclonal antibody (mAb) and a synthetic copolymer (SC). Upon interaction, a protein corona was formed around AgNPs with the consequent shifting and broadening of their characteristic surface plasmon resonance (SPR) band (400 nm) to 410 nm and longer wavelenghts. Additional studies revealed secondary and three-dimensional structure modifications of model proteins upon interaction with AgNPs by circular dichroism and fluorescence techniques, respectively. Based on the modification of the SPR condition of AgNPs upon interaction with proteins, we developed a novel protein-sensing application of AgNPs in primary containers. This strategy was used to conduct a compatibility assessment of model proteins towards five commercially available prefillable glass syringe (PFS) models. mAb- and SC-exposed PFSs showed that 74 and 94% of cases were positive for protein adsorption, respectively. Interestingly, protein adsorption on 15% of total tested PFSs was negligible (below the nanogram level). Our results highlight the need of a case-by-case compatibility assessment of therapeutic proteins and their primary containers. This strategy has the potential to be easily applied on other containers and implemented during early-stage product development by pharmaceutical companies and for routine use during batch release by packaging manufacturers.


Assuntos
Anticorpos Monoclonais/química , Dicroísmo Circular/métodos , Nanopartículas Metálicas/química , Prata/química , Adsorção , Anticorpos Monoclonais/análise , Anticorpos Monoclonais/metabolismo , Fluorescência , Humanos , Nanopartículas Metálicas/análise , Ligação Proteica/fisiologia , Proteínas Recombinantes/análise , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Prata/análise , Prata/metabolismo
8.
J Appl Physiol (1985) ; 125(2): 340-352, 2018 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-29357509

RESUMO

During ischemia, increased anaerobic glycolysis results in intracellular acidosis. Activation of alkalinizing transport mechanisms associated with carbonic anhydrases (CAs) leads to myocardial intracellular Ca2+ increase. We characterize the effects of inhibition of CA with benzolamide (BZ) during cardiac ischemia-reperfusion (I/R). Langendorff-perfused isolated rat hearts were subjected to 30 min of global ischemia and 60 min of reperfusion. Other hearts were treated with BZ (5 µM) during the initial 10 min of reperfusion or perfused with acid solution (AR, pH 6.4) during the first 3 min of reperfusion. p38MAPK, a kinase linked to membrane transporters and involved in cardioprotection, was examined in hearts treated with BZ in presence of the p38MAPK inhibitor SB202190 (10 µM). Infarct size (IZ) and myocardial function were assessed, and phosphorylated forms of p38MAPK, Akt, and PKCε were evaluated by immunoblotting. We determined the rate of intracellular pH (pHi) normalization after transient acid loading in the absence and presence of BZ or BZ + SB202190 in heart papillary muscles (HPMs). Mitochondrial membrane potential (ΔΨm), Ca2+ retention capacity and Ca2+-mediated swelling after I/R were also measured. BZ, similarly to AR, reduced IZ, improved postischemic recovery of myocardial contractility, increased phosphorylation of Akt, PKCε, and p38MAPK, and normalized ΔΨm and Ca2+ homeostasis, effects abolished after p38MAPK inhibition. In HPMs, BZ slowed pHi recovery, an effect that was restored after p38MAPK inhibition. We conclude that prolongation of acidic conditions during reperfusion by BZ could be responsible for the cardioprotective benefits of reduced infarction and better myocontractile function, through p38MAPK-dependent pathways. NEW & NOTEWORTHY Carbonic anhydrase inhibition by benzolamide (BZ) maintains acidity, decreases infarct size, and improves postischemic myocardial dysfunction in ischemia-reperfusion (I/R) hearts. Protection afforded by BZ mimicked the beneficial effects elicited by an acidic solution (AR). Increased phosphorylation of p38MAPK occurs in I/R hearts reperfused with BZ or with AR. Mitochondria from I/R hearts possess abnormal Ca2+ handling and a more depolarized membrane potential compared with control hearts, and these changes were restored by treatment with BZ or AR.

9.
J Mol Model ; 24(1): 13, 2017 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-29248994

RESUMO

Signaling systems allow microorganisms to sense and respond to different stimuli through the modification of gene expression. The phosphorelay signal transduction system in eukaryotes involves three proteins: a sensor protein, an intermediate protein and a response regulator, and requires the transfer of a phosphate group between two histidine-aspartic residues. The SLN1-YPD1-SSK1 system enables yeast to adapt to hyperosmotic stress through the activation of the HOG1-MAPK pathway. The genetic sequences available from Saccharomyces cerevisiae were used to identify orthologous sequences in Candida glabrata, and putative genes were identified and characterized by in silico assays. An interactome analysis was carried out with the complete genome of C. glabrata and the putative proteins of the phosphorelay signal transduction system. Next, we modeled the complex formed between the sensor protein CgSln1p and the intermediate CgYpd1p. Finally, phosphate transfer was examined by a molecular dynamic assay. Our in silico analysis showed that the putative proteins of the C. glabrata phosphorelay signal transduction system present the functional domains of histidine kinase, a downstream response regulator protein, and an intermediate histidine phosphotransfer protein. All the sequences are phylogenetically more related to S. cerevisiae than to C. albicans. The interactome suggests that the C. glabrata phosphorelay signal transduction system interacts with different proteins that regulate cell wall biosynthesis and responds to oxidative and osmotic stress the same way as similar systems in S. cerevisiae and C. albicans. Molecular dynamics simulations showed complex formation between the response regulator domain of histidine kinase CgSln1 and intermediate protein CgYpd1 in the presence of a phosphate group and interactions between the aspartic residue and the histidine residue. Overall, our research showed that C. glabrata harbors a functional SLN1-YPD1-SSK1 phosphorelay system.


Assuntos
Candida glabrata/metabolismo , Simulação por Computador , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Modelos Moleculares , Proteínas Quinases/metabolismo , Transdução de Sinais , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Fosforilação , Filogenia , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Quinases/genética , Processamento de Proteína Pós-Traducional , Saccharomycetales/metabolismo
10.
Sci Rep ; 7(1): 12125, 2017 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-28935954

RESUMO

Glatiramer Acetate (GA) is an immunomodulatory medicine approved for the treatment of multiple sclerosis, whose mechanisms of action are yet to be fully elucidated. GA is comprised of a complex mixture of polypeptides with different amino acid sequences and structures. The lack of sensible information about physicochemical characteristics of GA has contributed to its comprehensiveness complexity. Consequently, an unambiguous determination of distinctive attributes that define GA is of highest relevance towards dissecting its identity. Herein we conducted a study of characteristic GA heterogeneities throughout its manufacturing process (process signatures), revealing a strong impact of critical process parameters (CPPs) on the reactivity of amino acid precursors; reaction initiation and polymerization velocities; and peptide solubility, susceptibility to hydrolysis, and size-exclusion properties. Further, distinctive GA heterogeneities were correlated to defined immunological and toxicological profiles, revealing that GA possesses a unique repertoire of active constituents (epitopes) responsible of its immunological responses, whose modification lead to altered profiles. This novel approach established CPPs influence on intact GA peptide mixture, whose physicochemical identity cannot longer rely on reduced properties (based on complete or partial GA degradation), providing advanced knowledge on GA structural and functional relationships to ensure a consistent manufacturing of safe and effective products.

12.
Rev Esp Enferm Dig ; 109(8): 609, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28715902

RESUMO

Within the functional tests, the High Resolution Manometry has been a great improvement in the diagnosis of esophageal motor disorders, especially those that cause obstruction or difficulty for the normal progress of the alimentary content. We present the case of a patient with dysphagia to which a manometry is performed in the diagnostic process, where the presence of two types of Acalasia is present in the same tracing.


Assuntos
Acalasia Esofágica/diagnóstico , Adulto , Deglutição , Transtornos de Deglutição/diagnóstico , Transtornos de Deglutição/etiologia , Acalasia Esofágica/complicações , Acalasia Esofágica/fisiopatologia , Transtornos da Motilidade Esofágica , Feminino , Humanos , Manometria
13.
J Vis Exp ; (119)2017 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-28117815

RESUMO

Biotherapeutic proteins, such as monoclonal antibodies (mAbs), are feasible alternatives for the treatment of chronic-degenerative diseases. The biological activity of these proteins depends on their physicochemical properties. The use of high-performance techniques like chromatography and capillary electrophoresis has been described for the analysis of physicochemical heterogeneity of mAbs. Nowadays, capillary zone electrophoresis (CZE) technique constitutes one of the most resolutive and sensitive assays for the analysis of biomolecules. Besides, the electro-driven separation in CZE is governed by extensive properties of matter and offers the advantage of analyzing proteins close to their native state. However, the successful implementation of this technique for routine analysis depends on the skills of the analyst at the critical steps during sample and system preparation. The purpose of this tutorial is to detail the steps to succeed in the CZE analysis of mAbs. Further, this protocol can be used for the development and improvement of skills of the personnel involved in protein analytical chemistry laboratories.


Assuntos
Anticorpos Monoclonais/isolamento & purificação , Eletroforese Capilar/métodos , Isoformas de Proteínas/isolamento & purificação
14.
J Pharm Biomed Anal ; 132: 133-140, 2017 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-27721069

RESUMO

Complex pharmaceuticals are in demand of competent analytical methods able to analyze charge heterogeneity as a critical quality attribute (CQA), in compliance with current regulatory expectations. A notorious example is glatiramer acetate (GA), a complex polypeptide mixture useful for the treatment of relapsing-remitting multiple sclerosis. This pharmaceutical challenges the current state of analytical technology in terms of the capacity to study their constituent species. Thus, a strong cation exchange methodology was designed under the lifecycle approach to support the establishment of GA identity, trough the evaluation of its chromatographic profile, which acts as a charge heterogeneity fingerprint. In this regard, a maximum relative margin of error of 5% for relative retention time and symmetry factor were proposed for the analytical target profile. The methodology met the proposed requirements after precision and specificity tests results, the former comprised of sensitivity and selectivity. Subsequently, method validation was conducted and showed that the method is able to differentiate between intact GA and heterogeneity profiles coming from stressed, fractioned or process-modified samples. In summary, these results provide evidence that the method is adequate to assess charge heterogeneity as a CQA of this complex pharmaceutical.


Assuntos
Cátions , Cromatografia por Troca Iônica/métodos , Acetato de Glatiramer/química , Trifosfato de Adenosina/química , Acetato de Glatiramer/análise , Peptídeos/química , Probabilidade , Controle de Qualidade , Padrões de Referência , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
15.
Soft Matter ; 12(46): 9303-9313, 2016 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-27801925

RESUMO

Colloidal gels formed by colloid-polymer mixtures with an intermediate volume fraction (ϕc ≈ 0.4) are investigated by confocal microscopy. In addition, we have performed Monte Carlo simulations based on a simple effective pair potential that includes a short-range attractive contribution representing depletion interactions, and a longer-range repulsive contribution describing the electrostatic interactions due to the presence of residual charges. Despite neglecting non-equilibrium effects, experiments and simulations yield similar gel structures, characterised by, e.g., the pair, angular and bond distribution functions. We find that the structure hardly depends on the strength of the attraction if the electrostatic contribution is fixed, but changes significantly if the electrostatic screening is changed. This delicate balance between attractions and repulsions, which we quantify by the second virial coefficient, also determines the location of the gelation boundary.

16.
Electron. j. biotechnol ; 19(6): 63-69, Nov. 2016. ilus
Artigo em Inglês | LILACS | ID: biblio-840315

RESUMO

Background: Developing countries have an estimate of ten times more approved biosimilars than developed countries. This disparity demands the need of an objective regulation that incorporates health policies according to the technological and economical capabilities of each country. One of the challenges lies on the establishment of comparability principles based on a physicochemical and biological characterization that should determine the extent of additional non-clinical and clinical studies. This is particularly relevant for licensed biosimilars in developing countries, which have an extensive clinical experience since their approval as generics' in some cases more than a decade. To exemplify the current status of biosimilars in Mexico' a characterization exercise was conducted on licensed filgrastim biosimilars using pharmacopeial and extended characterization methodologies. Results: Most of the evaluated products complied with the pharmacopeial criteria and showed comparability in their Critical Quality Attributes (CQAs) towards the reference product. These results were expected in accordance with their equivalent performance during their licensing as generics. Accordingly' a rational approval and registration renewal scheme for biosimilars is proposed, that considers the proper identification of CQAs and its thoroughly evaluation using selected techniques. Conclusions: This approach provides support to diminish uncertainty of exhibiting different pharmacological profiles and narrows or even avoids the necessity of comparative clinical studies. Ultimately, this proposal is intended to improve the accessibility to high quality biosimilars in Latin America and other developing countries.


Assuntos
Medicamentos Biossimilares , Medicamentos Genéricos , Países em Desenvolvimento , Controle de Medicamentos e Entorpecentes , Filgrastim , América Latina , Política Pública , Controle de Qualidade
17.
J Am Heart Assoc ; 5(10)2016 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-27744404

RESUMO

BACKGROUND: Myocardial stretch increases force biphasically: the Frank-Starling mechanism followed by the slow force response (SFR). Based on pharmacological strategies, we proposed that epidermal growth factor (EGF) receptor (EGFR or ErbB1) activation is crucial for SFR development. Pharmacological inhibitors could block ErbB4, a member of the ErbB family present in the adult heart. We aimed to specifically test the role of EGFR activation after stretch, with an interference RNA incorporated into a lentiviral vector (small hairpin RNA [shRNA]-EGFR). METHODS AND RESULTS: Silencing capability of p-shEGFR was assessed in EGFR-GFP transiently transfected HEK293T cells. Four weeks after lentivirus injection into the left ventricular wall of Wistar rats, shRNA-EGFR-injected hearts showed ≈60% reduction of EGFR protein expression compared with shRNA-SCR-injected hearts. ErbB2 and ErbB4 expression did not change. The SFR to stretch evaluated in isolated papillary muscles was ≈130% of initial rapid phase in the shRNA-SCR group, while it was blunted in shRNA-EGFR-expressing muscles. Angiotensin II (Ang II)-dependent Na+/H+ exchanger 1 activation was indirectly evaluated by intracellular pH measurements in bicarbonate-free medium, demonstrating an increase in shRNA-SCR-injected myocardium, an effect not observed in the silenced group. Ang II- or EGF-triggered reactive oxygen species production was significantly reduced in shRNA-EGFR-injected hearts compared with that in the shRNA-SCR group. Chronic lentivirus treatment affected neither the myocardial basal redox state (thiobarbituric acid reactive substances) nor NADPH oxidase activity or expression. Finally, Ang II or EGF triggered a redox-sensitive pathway, leading to p90RSK activation in shRNA-SCR-injected myocardium, an effect that was absent in the shRNA-EGFR group. CONCLUSIONS: Our results provide evidence that specific EGFR activation after myocardial stretch is a key factor in promoting the redox-sensitive kinase activation pathway, leading to SFR development.


Assuntos
Receptores ErbB/genética , Coração/fisiopatologia , Miocárdio/metabolismo , Angiotensina II/farmacologia , Animais , Receptores ErbB/metabolismo , Inativação Gênica , Proteínas de Fluorescência Verde , Células HEK293 , Coração/efeitos dos fármacos , Humanos , Concentração de Íons de Hidrogênio , Masculino , RNA Interferente Pequeno , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Receptor ErbB-2/metabolismo , Receptor ErbB-4/metabolismo , Trocador 1 de Sódio-Hidrogênio/metabolismo , Vasoconstritores/farmacologia
18.
Cardiovasc Pathol ; 25(6): 468-477, 2016 Nov - Dec.
Artigo em Inglês | MEDLINE | ID: mdl-27614168

RESUMO

BACKGROUND: Two potent carbonic anhydrase (CA) inhibitors with widely differing membrane permeability, poorly diffusible benzolamide (BZ), and highly diffusible ethoxzolamide (ETZ) were assessed to determine whether they can reduce cardiac dysfunction in rats subjected to coronary artery ligation (CAL)-induced myocardial infarction. METHODS AND RESULTS: Rats with evidence of heart failure (HF) at 32 weeks following a permanent left anterior coronary artery occlusion were treated with placebo, BZ, or ETZ (4 mg kgday-1) for 4 weeks at which time left ventricular function and structure were evaluated. Lung weight/body weight (LW/BW) ratio increased in CAL rats by 17±1% vs. control, suggesting pulmonary edema. There was a trend for BZ and ETZ to ameliorate the increase in LW/BW by almost 50% (9±5% and 9±8%, respectively, versus CAL) (P=.16, NS). Echocardiographic assessment showed decreased left ventricular midwall shortening in HF rats, 21±1% vs. control 32±1%, which was improved by BZ to 29±1% and ETZ to 27±1%, and reduced endocardial shortening in HF rats 38±3% vs. control 62±1%, partially restored by BZ and ETZ to ~50%. Expression of the hypoxia-inducible membrane-associated CAIX isoform increased by ~60% in HF rat hearts, and this effect was blocked by ETZ. CONCLUSIONS: We conclude that CAL-induced myocardial interstitial fibrosis and associated decline in left ventricular function were diminished with BZ or ETZ treatment. The reductions in cardiac remodeling in HF with both ETZ and BZ CA inhibitors suggest that inhibition of a membrane-bound CA appears to be the critical site for this protection.


Assuntos
Benzolamida/farmacologia , Inibidores da Anidrase Carbônica/farmacologia , Etoxzolamida/farmacologia , Coração/efeitos dos fármacos , Infarto do Miocárdio/patologia , Animais , Vasos Coronários/cirurgia , Modelos Animais de Doenças , Immunoblotting , Ligadura , Masculino , Ratos , Ratos Wistar , Função Ventricular Esquerda/efeitos dos fármacos , Remodelação Ventricular/efeitos dos fármacos
19.
J Immunol Res ; 2016: 9697080, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27382576

RESUMO

Etanercept is a recombinant fusion protein approved for the treatment of TNF-α mediated diseases such as rheumatoid arthritis (RA), psoriasis, psoriatic arthritis, and ankylosing spondylitis. Herein, we present an evaluation of the physicochemical and biological properties of a biosimilar etanercept and its reference product followed by a clinical study in patients diagnosed with RA intended to demonstrate comparability of their immunomodulatory activity. Identity analyses showed a total correspondence of the primary and higher-order structure between the two products. In regard to intrinsic heterogeneity, both products showed to be highly heterogenous; however the biosimilar etanercept exhibited similar charge and glycan heterogeneity intervals compared to the reference product. Apoptosis inhibition assay also showed that, despite the high degree of heterogeneity exhibited by both products, no significant differences exist in their in vitro activity. Finally, the clinical assessment conducted in RA-diagnosed patients did not show significant differences in the evaluated pharmacodynamic markers of both products. Collectively, the results from the comparability exercise provide convincing evidence that the evaluated biosimilar etanercept can be considered an effective alternative for the treatment of RA.


Assuntos
Doenças Autoimunes/tratamento farmacológico , Doenças Autoimunes/imunologia , Medicamentos Biossimilares/farmacologia , Medicamentos Biossimilares/uso terapêutico , Etanercepte/farmacologia , Etanercepte/uso terapêutico , Sequência de Aminoácidos , Biomarcadores , Medicamentos Biossimilares/química , Linhagem Celular , Dicroísmo Circular , Relação Dose-Resposta a Droga , Etanercepte/química , Humanos , Fatores Imunológicos/farmacologia , Fatores Imunológicos/uso terapêutico , Imunomodulação/efeitos dos fármacos , Espectrometria de Massas , Resultado do Tratamento , Fator de Necrose Tumoral alfa/antagonistas & inibidores
20.
J Allergy Clin Immunol ; 138(1): 241-248.e3, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-26936803

RESUMO

BACKGROUND: Chronic granulomatous disease (CGD) is a rare primary immunodeficiency caused by inborn errors of the phagocyte nicotinamide adenine dinucleotide phosphate oxidase complex. From the first year of life onward, most affected patients display multiple, severe, and recurrent infections caused by bacteria and fungi. Mycobacterial infections have also been reported in some patients. OBJECTIVE: Our objective was to assess the effect of mycobacterial disease in patients with CGD. METHODS: We analyzed retrospectively the clinical features of mycobacterial disease in 71 patients with CGD. Tuberculosis and BCG disease were diagnosed on the basis of microbiological, pathological, and/or clinical criteria. RESULTS: Thirty-one (44%) patients had tuberculosis, and 53 (75%) presented with adverse effects of BCG vaccination; 13 (18%) had both tuberculosis and BCG infections. None of these patients displayed clinical disease caused by environmental mycobacteria, Mycobacterium leprae, or Mycobacterium ulcerans. Most patients (76%) also had other pyogenic and fungal infections, but 24% presented solely with mycobacterial disease. Most patients presented a single localized episode of mycobacterial disease (37%), but recurrence (18%), disseminated disease (27%), and even death (18%) were also observed. One common feature in these patients was an early age at presentation for BCG disease. Mycobacterial disease was the first clinical manifestation of CGD in 60% of these patients. CONCLUSION: Mycobacterial disease is relatively common in patients with CGD living in countries in which tuberculosis is endemic, BCG vaccine is mandatory, or both. Adverse reactions to BCG and severe forms of tuberculosis should lead to a suspicion of CGD. BCG vaccine is contraindicated in patients with CGD.


Assuntos
Doença Granulomatosa Crônica/complicações , Infecções por Mycobacterium/diagnóstico , Infecções por Mycobacterium/etiologia , Vacina BCG/administração & dosagem , Infecções Bacterianas/diagnóstico , Infecções Bacterianas/epidemiologia , Infecções Bacterianas/etiologia , Infecções Bacterianas/mortalidade , Criança , Pré-Escolar , Feminino , Doença Granulomatosa Crônica/epidemiologia , Doença Granulomatosa Crônica/mortalidade , Doença Granulomatosa Crônica/terapia , Humanos , Lactente , Masculino , Infecções por Mycobacterium/epidemiologia , Infecções por Mycobacterium/mortalidade , Micoses/diagnóstico , Micoses/epidemiologia , Micoses/etiologia , Micoses/mortalidade , Avaliação de Resultados da Assistência ao Paciente , Estudos Retrospectivos , Tuberculose/diagnóstico , Tuberculose/etiologia
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