RESUMO
In recent years, C2ORF40 has been identified as a tumor suppressor gene with multiple functions, including roles in cell proliferation, migration, and senescence. To explore the role of the C2ORF40 gene in different tumors, we used multiple databases for analysis. Compared to adjacent normal tissues, C2ORF40 is downregulated in a variety of malignant tumors, including tumors such as breast cancer, colorectal cancer, bladder cancer, hepatocellular carcinoma and prostate cancer. Notably, low expression of the gene is significantly associated with poor overall survival and relapse-free survival rates. In specific cancers including colon cancer and prostate cancer, the expression of C2ORF40 is correlated with the infiltration of CAFs. C2ORF40 is also involved in biological processes such as cell apoptosis and regulation of protein stability. In conclusion, C2ORF40 can hold promise as a prognostic marker for pan-cancer analysis.
Assuntos
Regulação Neoplásica da Expressão Gênica , Neoplasias , Humanos , Prognóstico , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Neoplasias/mortalidade , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Masculino , FemininoRESUMO
This investigation aimed to use CRISPR-Cas9 gene-editing to knock down P-glycoprotein (P-gp) expression and then establish a feasible cell line to evaluate the potential pharmacoresistance of therapeutic agents mediated by efflux. A cationic liposome was prepared as a "smart bomb" by conjugating with a peptide-based targeting ligand (THRPPMWSPVWP), specifically binding to transferrin receptors at the blood-brain barrier (BBB), and then formed a nanocomplex with P-gp knockdown CRISPR/Cas9 plasmid. Higher uptakes of targeted and stable liposomes in bEND.3 cells were observed compared to non-peptide conjugated ones (p < 0.05). The P-gp transporters were successfully knocked down by the cell-nontoxic CRISPR/Cas9 targeted liposomes and P-gp associated ATP activities were higher in the transfected cells (p < 0.05). Functional studies of knocked down cells were evaluated by using prototypical P-gp substrates rhodamine 123 and doxorubicin. More accumulation of rhodamine 123 and higher cytotoxic sensitivity of doxorubicin was observed in the transfected cells as compared with those in the wild-type cells.
Assuntos
Células Endoteliais , Lipossomos , Animais , Camundongos , Células Endoteliais/metabolismo , Sistemas CRISPR-Cas/genética , Rodamina 123 , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Encéfalo/metabolismo , Doxorrubicina/farmacologiaRESUMO
A Cu-catalyzed three-component cascade cyclization among 2-formylbenzonitrile, cyclopropyl ketones, and diaryliodonium salts for the construction of fused isoindolin-1-one compounds is achieved. Pentacyclic isoindolinone derivatives could be obtained in moderate to good yields. The proposed mechanism involved a ring expansion of cyclopropyl ketones/formation of N-acyliminium/hetero-[4 + 2]-cycloaddition process.
RESUMO
Riluzole is currently one of two approved medications for the treatment of amyotrophic lateral sclerosis (ALS). However, brain disposition of riluzole, as a substrate of P-glycoprotein (P-gp), is limited by the efflux transporters at the blood-brain barrier (BBB). We propose to develop a liposomal co-delivery system that could effectively transport riluzole to brain cells by reducing efflux pumps with a P-gp inhibitor, verapamil. Riluzole and verapamil cocktail liposomes were prepared by lipid film hydration. The average particle size of cocktail liposomes was 194.3⯱â¯6.0â¯nm and their polydispersity index (PDI) was 0.272⯱â¯0.017. The encapsulation efficiencies of verapamil and riluzole in the cocktail liposomes were 86.0⯱â¯1.4% and 85.6⯱â¯1.1%, respectively. The drug release from cocktail liposomes after 8â¯h in PBS at 37⯰C was 78.4⯱â¯6.2% of riluzole and 76.7⯱â¯3.8% of verapamil. The average particle size of liposomes did not show significant changes at 4⯰C after three months. Verapamil cocktail liposomes inhibited P-gp levels measured by western blotting in dose and time-dependent manners in brain endothelial bEND.3 cells. Increased drug efflux transporters were detected in bEND.3 and astrocytes C8D1A cells, promoted by tumor necrosis factor (TNF-α) or hydrogen peroxide (H2O2). Restored accumulations of riluzole and fluorescent dye rhodamine 123 were observed in bEND.3 cells after treatments with cocktail liposomes. It indicated that inhibitory potential of co-delivery liposome system towards P-gp could mediate the transport of both P-gp substrates. Verapamil and riluzole co-loaded liposomes may be used to overcome pharmacoresistance of riluzole for improving ALS therapy.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/antagonistas & inibidores , Esclerose Lateral Amiotrófica/tratamento farmacológico , Astrócitos/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Resistência a Medicamentos/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Riluzol/farmacologia , Verapamil/farmacologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Animais , Astrócitos/metabolismo , Astrócitos/patologia , Encéfalo/metabolismo , Encéfalo/patologia , Linhagem Celular , Relação Dose-Resposta a Droga , Combinação de Medicamentos , Liberação Controlada de Fármacos , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Cinética , Lipossomos , Camundongos , Fármacos Neuroprotetores/administração & dosagem , Fármacos Neuroprotetores/metabolismo , Tamanho da Partícula , Riluzol/administração & dosagem , Riluzol/metabolismo , Solubilidade , Verapamil/administração & dosagemRESUMO
An efficient and mild synthesis of a variety of 3-(2-oxopropyl)-isoindolinone derivatives via a BF3·Et2O catalyzed cascade reaction among 3-hydroxyisoindolin-1-one and phenylacetylene was achieved. Various isoindolinone derivatives were obtained in good to excellent yields. The process, which avoided several drawbacks such as the requirement of concentrated protic acids and metal catalysts, protecting group of nitrogen, high temperature, and multistep synthesis, includes C(sp3)-OH cleavage, C-C coupling, and hydration of alkyne.
RESUMO
A three-component cascade cyclization was developed to synthesize 2,3-diarylisoindolin-1-one by using 2-formylbenzonitrile, arenes, and diaryliodonium salts. The process underwent copper-catalyzed tandem C-N/C-C bond formation, producing isoindolin-1-one derivatives in good to excellent yields.
RESUMO
Triple negative breast cancer (TNBC) typically exhibits rapid progression, high mortality and faster relapse rates relative to other breast cancer subtypes. In this report we examine the combination of taxanes (paclitaxel or docetaxel) with a breast cancer stem cell (CSC)-targeting agent sulforaphane for use against TNBC. We demonstrate that paclitaxel or docetaxel treatment induces IL-6 secretion and results in expansion of CSCs in TNBC cell lines. Conversely, sulforaphane is capable of preferentially eliminating CSCs, by inhibiting NF-κB p65 subunit translocation, downregulating p52 and consequent downstream transcriptional activity. Sulforaphane also reverses taxane-induced aldehyde dehydrogenase-positive (ALDH+) cell enrichment, and dramatically reduces the size and number of primary and secondary mammospheres formed. In vivo in an advanced treatment orthotopic mouse xenograft model together with extreme limiting dilution analysis (ELDA), the combination of docetaxel and sulforaphane exhibits a greater reduction in primary tumor volume and significantly reduces secondary tumor formation relative to either treatment alone. These results suggest that treatment of TNBCs with cytotoxic chemotherapy would be greatly benefited by the addition of sulforaphane to prevent expansion of and eliminate breast CSCs.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Isotiocianatos/farmacologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Paclitaxel/farmacologia , Taxoides/farmacologia , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Moduladores de Tubulina/farmacologia , Animais , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Docetaxel , Relação Dose-Resposta a Droga , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Mediadores da Inflamação/metabolismo , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Camundongos Endogâmicos NOD , Camundongos SCID , Subunidade p52 de NF-kappa B/genética , Subunidade p52 de NF-kappa B/metabolismo , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Fenótipo , Transdução de Sinais/efeitos dos fármacos , Sulfóxidos , Fatores de Tempo , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismo , Transcrição Gênica , Transfecção , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Although small interfering RNA (siRNA) holds great therapeutic promise, its delivery to the disease site remains a paramount obstacle. In this study, we tested whether brain endothelial cell-derived exosomes could deliver siRNA across the blood-brain barrier (BBB) in zebrafish. Natural exosomes were isolated from brain endothelial bEND.3 cell culture media and vascular endothelial growth factor (VEGF) siRNA was loaded in exosomes with the assistance of a transfection reagent. While fluorescence-activated cell flow cytometry and immunocytochemistry staining studies indicated that wild-type exosomes significantly increased the uptake of fluorescence-labeled siRNA in the autologous brain endothelial cells, decreased fluorescence intensity was observed in the cells treated with the tetraspanin CD63 antibody-blocked exosome-delivered formulation (p < 0.05). In the transport study, exosomes also enhanced the permeability of rhodamine 123 in a co-cultured monolayer of brain endothelial bEND.3 cell and astrocyte. Inhibition at the expression of VEGF RNA and protein levels was observed in glioblastoma-astrocytoma U-87 MG cells treated with exosome-delivered siRNAs. Imaging results showed that exosome delivered more siRNAs across the BBB in Tg(fli1:GFP) zebrafish. In a xenotransplanted brain tumor model, exosome-delivered VEGF siRNAs decreased the fluorescence intensity of labeled cancer cells in the brain of zebrafish. Brain endothelial cell-derived exosomes could be potentially used as a natural carrier for the brain delivery of exogenous siRNA.
Assuntos
Neoplasias Encefálicas/terapia , Técnicas de Transferência de Genes , RNA Interferente Pequeno/administração & dosagem , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Astrócitos/metabolismo , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Encéfalo/patologia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Técnicas de Cocultura , Células Endoteliais/metabolismo , Exossomos/metabolismo , Humanos , Transfecção , Fator A de Crescimento do Endotélio Vascular/genética , Peixe-ZebraRESUMO
PURPOSE: The blood-brain barrier (BBB) essentially restricts therapeutic drugs from entering into the brain. This study tests the hypothesis that brain endothelial cell derived exosomes can deliver anticancer drug across the BBB for the treatment of brain cancer in a zebrafish (Danio rerio) model. MATERIALS AND METHODS: Four types of exosomes were isolated from brain cell culture media and characterized by particle size, morphology, total protein, and transmembrane protein markers. Transport mechanism, cell uptake, and cytotoxicity of optimized exosome delivery system were tested. Brain distribution of exosome delivered anticancer drugs was evaluated using transgenic zebrafish TG (fli1: GFP) embryos and efficacies of optimized formations were examined in a xenotransplanted zebrafish model of brain cancer model. RESULTS: Four exosomes in 30-100 diameters showed different morphologies and exosomes derived from brain endothelial cells expressed more CD63 tetraspanins transmembrane proteins. Optimized exosomes increased the uptake of fluorescent marker via receptor mediated endocytosis and cytotoxicity of anticancer drugs in cancer cells. Images of the zebrafish showed exosome delivered anticancer drugs crossed the BBB and entered into the brain. In the brain cancer model, exosome delivered anticancer drugs significantly decreased fluorescent intensity of xenotransplanted cancer cells and tumor growth marker. CONCLUSIONS: Brain endothelial cell derived exosomes could be potentially used as a carrier for brain delivery of anticancer drug for the treatment of brain cancer.
Assuntos
Antineoplásicos/farmacologia , Barreira Hematoencefálica/metabolismo , Neoplasias Encefálicas/tratamento farmacológico , Doxorrubicina/farmacologia , Sistemas de Liberação de Medicamentos/métodos , Células Endoteliais/metabolismo , Exossomos/metabolismo , Paclitaxel/farmacologia , Animais , Animais Geneticamente Modificados , Antineoplásicos/química , Antineoplásicos/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Permeabilidade Capilar , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Química Farmacêutica , Modelos Animais de Doenças , Doxorrubicina/química , Doxorrubicina/metabolismo , Endocitose , Genes Reporter , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Xenoenxertos , Humanos , Transplante de Neoplasias , Paclitaxel/química , Paclitaxel/metabolismo , Tamanho da Partícula , Tecnologia Farmacêutica/métodos , Fatores de Tempo , Carga Tumoral/efeitos dos fármacos , Peixe-ZebraRESUMO
Because adhesion of leukocytes to endothelial cells is the first step of vascular-neuronal inflammation, inhibition of adhesion and recruitment of leukocytes to vascular endothelial cells will have a beneficial effect on neuroinflammatory diseases. In this study, we used the pRNA of bacteriophage phi29 DNA packaging motor to construct a novel RNA nanoparticle for specific targeting to transferrin receptor (TfR) on the murine brain-derived endothelial cells (bEND5) to deliver ICAM-1 siRNA. This RNA nanoparticle (FRS-NPs) contained a FB4 aptamer targeting to TfR and a siRNA moiety for silencing the intercellular adhesion molecule-1 (ICAM-1). Our data indicated that this RNA nanoparticle was delivered into murine brain-derived endothelial cells. Furthermore, the siRNA was released from the FRS-NPs in the cells and knocked down ICAM-1 expression in the TNF-α-stimulated cells and in the cells under oxygen-glucose deprivation/reoxygenation (OGD/R) condition. The functional end points of the study indicated that FRS-NPs significantly inhibited monocyte adhesion to the bEND5 cells induced by TNF-α and OGD/R. In conclusion, our approach using RNA nanotechnology for siRNA delivery could be potentially applied for inhibition of inflammation in ischemic stroke and other neuroinflammatory diseases, or diseases affecting endothelium of vasculature.
RESUMO
One of the biggest challenges for small interfering RNAs (siRNAs) as therapeutic agents is their insufficient cellular delivery efficiency. We developed long circulating and cationic liposomes to improve the cell uptake and inhibitory effectiveness of siRNA on the expression of vascular endothelial growth factor (VEGF) in cancer cells. SiRNA liposomes were obtained by polyelectrolyte complexation between negatively charged siRNA and positively charged liposome prepared by a hydration method. Gel electrophoresis was used to evaluate the loading efficiency of siRNA on the cationic liposome. The optimized siRNA liposomes were observed to be spherical in shape and had smooth surfaces with particle sizes of 167.7 ± 2.0 nm and zeta potentials of 4.03 ± 0.69 mV, which had no significant change when stored at 4 °C for three months. Fluorescence-activated cell sorting studies and confocal laser scanning images indicated that the cationic liposomes significantly increased the uptake of fluorescence-labeled siRNA in cancer cells. Effects of the siRNA on the inhibition of VEGF were tested by measuring concentrations of VEGF in cell culture media via an enzyme-linked immunosorbent assay and intracellular VEGF levels using a western blotting method. The liposomal siRNA was significantly effective at inhibiting the expression of VEGF in lung, liver and breast cancer cells. Optimal liposomes could effectively deliver siRNA into cancer cells and inhibit VEGF as a therapy agent.
Assuntos
Inativação Gênica , Proteínas de Neoplasias/antagonistas & inibidores , RNA Interferente Pequeno/administração & dosagem , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Transporte Biológico , Linhagem Celular Tumoral , Sobrevivência Celular , Humanos , Lipossomos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Tamanho da Partícula , RNA Interferente Pequeno/química , RNA Interferente Pequeno/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Novel aptamer-functionalized polyethylene glycol-polylactic acid (PEG-PLA) (APP) micelles were developed with the objective to target the transferrin receptor on brain endothelial cells. Flurbiprofen, a potential drug for therapeutic management of Alzheimer's disease (AD), was loaded into the APP micelles using the co-solvent evaporation method. Results indicated that 9.03% (w/w) of flurbiprofen was entrapped in APP with good retention capacity in vitro. Targeting potential of APPs was investigated using the transferring receptor-expressing murine brain endothelial bEND5 cell line. APPs significantly enhanced surface association of micelles to bEND5 cells as quantified by fluorescence spectroscopy. Most importantly, APPs significantly enhanced intracellular flurbiprofen delivery when compared to unmodified micelles. These results suggest that APP micelles may offer an effective strategy to deliver therapeutically effective flurbiprofen concentrations into the brain for AD patients.
Assuntos
Anti-Inflamatórios não Esteroides/administração & dosagem , Aptâmeros de Nucleotídeos/química , Encéfalo/metabolismo , Sistemas de Liberação de Medicamentos , Flurbiprofeno/administração & dosagem , Micelas , Polietilenoglicóis/química , Doença de Alzheimer/tratamento farmacológico , Animais , Anti-Inflamatórios não Esteroides/farmacocinética , Aptâmeros de Nucleotídeos/metabolismo , Sequência de Bases , Encéfalo/citologia , Linhagem Celular , Portadores de Fármacos/química , Portadores de Fármacos/metabolismo , Células Endoteliais/metabolismo , Flurbiprofeno/farmacocinética , Camundongos , Polietilenoglicóis/metabolismo , Receptores da Transferrina/metabolismoRESUMO
This study was performed to test the feasibility of chitosan and polylactic-co-glycolic acid (PLGA) incorporated nanoparticles as sustained-release carriers for the delivery of negatively charged low molecular weight heparin (LMWH). Fourier transform infrared (FTIR) spectrometry was used to evaluate the interactions between chitosan and LMWH. The shifts, intensity, and broadening of the characteristic peaks for the functional groups in the FTIR spectra indicated that strong interactions occur between the positively charged chitosans and the negatively charged LMWHs. Three types of LMWH nanoparticles (NP-1, NP-2, and NP-3) were prepared using chitosan with or without PLGA: NP-1 nanoparticles were formed by polyelectrolyte complexation after single mixing, NP-2 nanoparticles were prepared by polyelectrolyte complexation after single emulsion-diffusion-evaporation, and NP-3 nanoparticles were optimized by double emulsion-diffusion-evaporation. NP-3 nanoparticles of LMWH prepared by the emulsion-diffusion-evaporation method showed significant differences in particle morphology, size, zeta potential, and drug release profile compared to NP-1 nanoparticles formed by polyelectrolyte complexation. Another ionic complex of LMWH with chitosan-incorporated PLGA nanoparticles (NP-2) showed lower drug entrapment efficiency than that of NP-1 and NP-3. The drug release rate of NP-3 was slower than the release rates of NP-1 and NP-2, although particle morphology of NP-3 was similar to that of NP-2. Cell viability was not adversely affected when cells were treated with all three types of nanoparticles. The data presented in this study demonstrate that nanoparticles formulated with chitosan-PLGA could be a safe sustained-release carrier for the delivery of LMWH.
Assuntos
Quitosana/química , Heparina de Baixo Peso Molecular/química , Ácido Láctico/química , Nanopartículas/química , Ácido Poliglicólico/química , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Preparações de Ação Retardada/química , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos/métodos , Emulsões/química , Células Epiteliais/efeitos dos fármacos , Estudos de Viabilidade , Humanos , Tamanho da Partícula , Copolímero de Ácido Poliláctico e Ácido PoliglicólicoRESUMO
Heart tissues from hibernating mammals, such as ground squirrels, are able to endure hypothermia, hypoxia and other extreme insulting factors that are fatal for human and nonhibernating mammals. This study was designed to understand adaptive mechanisms involved in intracellular Ca(2+) homeostasis in cardiomyocytes from the mammalian hibernator, ground squirrel, compared to rat. Electrophysiological and confocal imaging experiments showed that the voltage-dependence of L-type Ca(2+) current (I(Ca)) was shifted to higher potentials in ventricular myocytes from ground squirrels vs. rats. The elevated threshold of I(Ca) did not compromise the Ca(2+)-induced Ca(2+) release, because a higher depolarization rate and a longer duration of action potential compensated the voltage shift of I(Ca). Both the caffeine-sensitive and caffeine-resistant components of cytosolic Ca(2+) removal were more rapid in ground squirrels. Ca(2+) sparks in ground squirrels exhibited larger amplitude/size and much lower frequency than in rats. Due to the high I(Ca) threshold, low SR Ca(2+) leak and rapid cytosolic Ca(2+) clearance, heart cells from ground squirrels exhibited better capability in maintaining intracellular Ca(2+) homeostasis than those from rats and other nonhibernating mammals. These findings not only reveal adaptive mechanisms of hibernation, but also provide novel strategies against Ca(2+) overload-related heart diseases.
Assuntos
Cálcio/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Citosol/metabolismo , Eletrofisiologia , Homeostase , Potenciais da Membrana/fisiologia , Ratos , Sciuridae , TemperaturaRESUMO
This study tests the feasibility of inhalable pegylated liposomal formulations of low molecular weight heparin (LMWH) for treatment of two clinical manifestations of vascular thromboembolism: deep vein thrombosis (DVT) and pulmonary embolism (PE). Conventional distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) and long-circulating pegylated (DSPE-PEG-2000 and DSPE-PEG-5000) liposomes were prepared by hydration method. Formulations were evaluated for particle size, entrapment efficiency, stability, pulmonary absorption, anticoagulant, and thrombolytic effects in rats. Pulmonary absorption was monitored by measuring plasma antifactor Xa activity; anticoagulant and thrombolytic effects were studied by measuring reduction in thrombus weight and amount of dissolved radioactive clot in the blood, respectively. Pegylated liposomal were smaller and showed greater drug entrapment efficiency than conventional liposomes. All formulations produced an increase in pulmonary absorption and circulation time of LMWH upon first dosing. Three repeated dosings of conventional liposomes resulted in decreased half-life and bioavailability; no changes in these parameters were observed with pegylated liposomes. PEG-2000 liposomes were effective in reducing thrombus weight when administered every 48 h over 8 days. In terms of thrombolytic effects and dosing frequency, PEG-2000 liposomes administered via the pulmonary route at a dose of 100 U/kg were as effective as 50 U/kg LMWH administered subcutaneously. This paper suggests that inhalable pegylated liposomes of LMWH could be a potential noninvasive approach for DVT and PE treatment.
Assuntos
Fibrinolíticos/administração & dosagem , Fibrinolíticos/uso terapêutico , Heparina de Baixo Peso Molecular/administração & dosagem , Heparina de Baixo Peso Molecular/uso terapêutico , Lipossomos/química , Embolia Pulmonar/tratamento farmacológico , Trombose Venosa/tratamento farmacológico , Administração por Inalação , Animais , Masculino , Tamanho da Partícula , Polietilenoglicóis/química , Ratos , Ratos Sprague-Dawley , Tromboembolia Venosa/tratamento farmacológicoRESUMO
BACKGROUND: Currently low molecular weight heparin (LMWH) is administered as subcutaneous injection. This study sought to investigate the feasibility of LMWH as an inhalable dry powder (DPI) formulation and evaluate the interaction of the drug with lactose when used as a carrier. The study also compares the extent of pulmonary absorption of LMWH administered as a dry powder with that administered as an aerosolized aqueous solution. METHODS: The formulations were prepared by mixing LMWH in an aqueous solution of lactose followed by lyophilization of the resulting solution. The lyophilized preparation was then ground and sieved. Physical characterization of the formulations was performed by Fourier transform infrared spectroscopy (FTIR), X-ray powder diffraction (XRD), scanning electron microscopy (SEM), particle size analysis, and determination of aerodynamic diameter. For in vivo studies, formulations were administered to anesthetized rats, and drug absorption was monitored by measuring plasma antifactor Xa activity. RESULTS AND CONCLUSIONS: In the FTIR scan, all characteristic peaks of lactose and LMWH were observed, suggesting that there was no strong interaction between lactose and LMWH. Although the aerodynamic diameter of the formulation (DPI-2) that was sieved through 170- and 230-mesh screens was similar to that of the formulation (DPI-1) sieved through 120- and 170-mesh screens, the particle sizes of the two formulations were significantly different. Dry powder formulations of LMWH were better absorbed compared to an inhalable solution of LMWH. One of the dry powder formulations (DPI-2) produced an almost 1.5-fold increase in the relative bioavailability (41.6%) compared to the liquid formulation of LMWH (32.5%). Overall, the data presented here suggest that lactose does not adversely affect the physical-chemical characteristics of the drug, and that lactose can be used as a carrier for pulmonary delivery of LMWH.
Assuntos
Anticoagulantes/administração & dosagem , Excipientes/química , Heparina de Baixo Peso Molecular/administração & dosagem , Lactose/química , Administração por Inalação , Aerossóis , Animais , Anticoagulantes/farmacocinética , Disponibilidade Biológica , Química Farmacêutica/métodos , Heparina de Baixo Peso Molecular/farmacocinética , Masculino , Nebulizadores e Vaporizadores , Tamanho da Partícula , Pós , Ratos , Ratos Sprague-Dawley , Espectroscopia de Infravermelho com Transformada de Fourier , Difração de Raios XRESUMO
As the most prototypical G protein-coupled receptor, beta-adrenergic receptor (betaAR) regulates the pace and strength of heart beating by enhancing and synchronizing L-type channel (LCC) Ca(2+) influx, which in turn elicits greater sarcoplasmic reticulum (SR) Ca(2+) release flux via ryanodine receptors (RyRs). However, whether and how betaAR-protein kinase A (PKA) signaling directly modulates RyR function remains elusive and highly controversial. By using unique single-channel Ca(2+) imaging technology, we measured the response of a single RyR Ca(2+) release unit, in the form of a Ca(2+) spark, to its native trigger, the Ca(2+) sparklet from a single LCC. We found that acute application of the selective betaAR agonist isoproterenol (1 microM, < or = 20 min) increased triggered spark amplitude in an LCC unitary current-independent manner. The increased ratio of Ca(2+) release flux underlying a Ca(2+) spark to SR Ca(2+) content indicated that betaAR stimulation helps to recruit additional RyRs in synchrony. Quantification of sparklet-spark kinetics showed that betaAR stimulation synchronized the stochastic latency and increased the fidelity (i.e., chance of hit) of LCC-RyR intermolecular signaling. The RyR modulation was independent of the increased SR Ca(2+) content. The PKA antagonists Rp-8-CPT-cAMP (100 microM) and H89 (10 microM) both eliminated these effects, indicating that betaAR acutely modulates RyR activation via the PKA pathway. These results demonstrate unequivocally that RyR activation by a single LCC is accelerated and synchronized during betaAR stimulation. This molecular mechanism of sympathetic regulation will permit more fundamental studies of altered betaAR effects in cardiovascular diseases.