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Spinocerebellar ataxia type 3 (SCA3) is a neurodegenerative disorder caused by mutant ataxin-3 with an abnormally expanded polyQ tract and is the most common dominantly inherited ataxia worldwide. There are no suitable therapeutic options for this disease. Autophagy, a defense mechanism against the toxic effects of aggregation-prone misfolded proteins, has been shown to have beneficial effects on neurodegenerative diseases. Thus, trehalose, which is an autophagy inducer, may have beneficial effects on SCA3. In the present study, we examined the effects of trehalose on an SCA3 cell model. After trehalose treatment, aggregate formation, soluble ataxin-3 protein levels and cell viability were evaluated in HEK293T cells overexpressing ataxin-3-15Q or ataxin-3-77Q. We also explored the mechanism by which trehalose affects autophagy and stress pathways. A filter trap assay showed that trehalose decreased the number of aggregates formed by mutant ataxin-3 containing an expanded polyQ tract. Western blot and Cell Counting Kit-8 (CCK-8) results demonstrated that trehalose also reduced the ataxin-3 protein levels and was safe for ataxin-3-expressing cells, respectively. Western blot and total antioxidant capacity assays suggested that trehalose had great therapeutic potential for treating SCA3, likely through its antioxidant activity. Our data indicate that trehalose plays a neuroprotective role in SCA3 by inhibiting the aggregation and reducing the protein level of ataxin-3, which is also known to protect against oxidative stress. These findings provide a new insight into the possibility of treating SCA3 with trehalose and highlight the importance of inducing autophagy in SCA3.
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Ataxina-3 , Doença de Machado-Joseph , Trealose , Trealose/farmacologia , Humanos , Ataxina-3/metabolismo , Ataxina-3/genética , Doença de Machado-Joseph/metabolismo , Doença de Machado-Joseph/tratamento farmacológico , Células HEK293 , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Autofagia/efeitos dos fármacos , Autofagia/fisiologia , Agregados Proteicos/efeitos dos fármacos , Mutação , Proteínas Repressoras/metabolismo , Proteínas Repressoras/genética , Agregação Patológica de Proteínas/metabolismo , PeptídeosRESUMO
Duchenne muscular dystrophy (DMD) is a rare X-linked recessive disorder characterized by loss-of-function mutations in the gene encoding dystrophin. These mutations lead to progressive functional deterioration including muscle weakness, respiratory insufficiency, and musculoskeletal deformities. Three-dimensional gait analysis (3DGA) has been used as a tool to analyze gait pathology through the quantification of altered joint kinematics, kinetics, and muscle activity patterns. Among 3DGA indices, the Gait Profile Score (GPS), has been used as a sensitive overall measure to detect clinically relevant changes in gait patterns in children with DMD. To enhance our understanding of the clinical translation of 3DGA, we report here the development of a population nonlinear mixed-effect model that jointly describes the disease progression of the 3DGA index, GPS, and the functional endpoint, North Star Ambulatory Assessment (NSAA). The final model consists of a quadratic structure for GPS progression and a linear structure for GPS-NSAA correlation. Our model was able to capture the improvement in function in GPS and NSAA in younger subjects, as well as the decline of function in older subjects. Furthermore, the model predicted NSAA (CFB) at 1 year reasonably well for DMD subjects ≤7 years old at baseline. The model tended to slightly underpredict the decline in NSAA after 1 year for those >7 years old at baseline, but the prediction summary statistics were well maintained within the standard deviation of observed data. Quantitative models such as this may help answer clinically relevant questions to facilitate the development of novel therapies in DMD.
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Progressão da Doença , Marcha , Distrofia Muscular de Duchenne , Humanos , Distrofia Muscular de Duchenne/fisiopatologia , Distrofia Muscular de Duchenne/genética , Criança , Estudos Longitudinais , Pré-Escolar , Masculino , Adolescente , Análise da Marcha/métodosRESUMO
Warfarin has a long record of safe and effective clinical use, and it remains one of the most commonly prescribed drugs for the prevention and treatment of thromboembolic conditions even in the era of direct oral anticoagulants. To address its large interindividual variability and narrow therapeutic window, the Clinical Pharmacogenetics Implementation Consortium has recommended using pharmacogenetic dosing algorithms, such as the ones developed by the International Warfarin Pharmacogenetics Consortium (IWPC) and by Gage et al, to dose warfarin when genotype information is available. In China, dosing algorithms based on local patient populations have been developed and evaluated for predictive accuracy of warfarin maintenance doses. In this study, percentage deviations of doses predicted by 15 Chinese dosing algorithms from that by IWPC and Gage algorithms were systematically evaluated to understand the differences between Chinese and Western algorithms. In general, dose predictions by Chinese dosing algorithms tended to be lower than those predicted by IWPC or Gage algorithms for the most prevalent VKORC1 and CYP2C9 genotypes in the Chinese population. The extent of negative prediction deviation appeared to be largest in the younger age group with smaller body weight. Our findings are consistent with previous reports that Asians have a higher sensitivity to warfarin and require lower doses than Western populations.
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Anticoagulantes , Varfarina , Humanos , Algoritmos , Anticoagulantes/administração & dosagem , Citocromo P-450 CYP2C9/genética , Relação Dose-Resposta a Droga , População do Leste Asiático , Genótipo , Farmacogenética , Vitamina K Epóxido Redutases/genética , Varfarina/administração & dosagemRESUMO
Duchenne muscular dystrophy (DMD) is a severe, progressive, and incurable X-linked disorder caused by mutations in the dystrophin gene. Patients with DMD have an absence of functional dystrophin protein, which results in chronic damage of muscle fibers during contraction, thus leading to deterioration of muscle quality and loss of muscle mass over time. Although there is currently no cure for DMD, improvements in treatment care and management could delay disease progression and improve quality of life, thereby prolonging life expectancy for these patients. Furthermore, active research efforts are ongoing to develop therapeutic strategies that target dystrophin deficiency, such as gene replacement therapies, exon skipping, and readthrough therapy, as well as strategies that target secondary pathology of DMD, such as novel anti-inflammatory compounds, myostatin inhibitors, and cardioprotective compounds. Furthermore, longitudinal modeling approaches have been used to characterize the progression of MRI and functional endpoints for predictive purposes to inform Go/No Go decisions in drug development. This review showcases approved drugs or drug candidates along their development paths and also provides information on primary endpoints and enrollment size of Ph2/3 and Ph3 trials in the DMD space.
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Synapses are key structures in neural networks, and are involved in learning and memory in the central nervous system. Investigating synaptogenesis and synaptic aging is important in understanding neural development and neural degeneration in diseases such as Alzheimer disease and Parkinson's disease. Our previous study found that synaptogenesis and synaptic maturation were harmonized with brain development and maturation. However, synaptic damage and loss in the aging cerebellum are not well understood. This study was designed to investigate the occurrence of synaptic aging in the cerebellum by observing the ultrastructural changes of dendritic spines and synapses in cerebellar Purkinje cells of aging mice. Immunocytochemistry, DiI diolistic assays, and transmission electron microscopy were used to visualize the morphological characteristics of synaptic buttons, dendritic spines and synapses of Purkinje cells in mice at various ages. With synaptic aging in the cerebellum, dendritic spines and synaptic buttons were lost, and the synaptic ultrastructure was altered, including a reduction in the number of synaptic vesicles and mitochondria in presynaptic termini and smaller thin specialized zones in pre- and post-synaptic membranes. These findings confirm that synaptic morphology and function is disrupted in aging synapses, which may be an important pathological cause of neurodegenerative diseases.
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The aim of the present study was to establish a cell model of Alzheimer's disease (AD) and investigate the neurotoxic effects of ß-amyloid (Aß) on the cytoskeleton. PC12 cells were cultured and treated with Aß2535, and cell survival was analyzed with the MTT assay. Cell apoptosis was visualized using 4',6-diamidino-2-phenylindole staining and the terminal deoxynucleotidyl transferase dUTP nick-end labeling assay. Immunocytochemistry and phalloidin staining were used to label the cytoskeleton of PC12 cells. Aß25-35 was found to induce PC12 cell apoptosis in a dose-dependent manner (P<0.05). Moreover, Aß25-35 also caused dose-dependent disintegration of the cytoskeleton (P<0.05). Therefore, the PC12 cell cytoskeleton was found to be sensitive to Aß25-35 neurotoxicity. The disintegration of the cytoskeleton is likely an important pathological alteration in AD, and Aß is a key molecule involved in AD pathogenesis.
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Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Citoesqueleto/patologia , Fragmentos de Peptídeos/metabolismo , Doença de Alzheimer/metabolismo , Animais , Apoptose , Sobrevivência Celular , Citoesqueleto/metabolismo , Células PC12 , RatosRESUMO
There has been controversy regarding whether bioavailability of certain oral oncology drugs should be maximized by taking these medications with food, irrespective of label instructions in the dosing and administration section. To provide insight into this controversy, we conducted an in-depth analysis for oral antineoplastic drugs approved by the Food and Drug Administration in 2000-2016 and identified important issues influencing food labeling decisions. Furthermore, a case study involving sonidegib, a drug approved for locally advanced basal cell carcinoma with a significant food effect on exposure, was used to demonstrate the consequences of failure to adhere to food label recommendations using drug-specific population pharmacokinetic and exposure-toxicity models. In 2000-2009, 80% (4 out of 5) of all approved oral antineoplastics with increased bioavailability in the fed state were labeled as "take on empty stomach." In contrast, we found that in 2010-2016 there is a greater diversity in food recommendations for drugs with increased bioavailability in the fed state. Currently, many oral oncology drugs are given with food to maximize their bioavailability; however, as seen from our case study of sonidegib, failure to fully adhere to label recommendations to either take with food or not could lead to adverse consequences in terms of safety and efficacy.
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Adenocarcinoma/tratamento farmacológico , Antineoplásicos/administração & dosagem , Compostos de Bifenilo/administração & dosagem , Refeições , Piridinas/administração & dosagem , Administração Oral , Antineoplásicos/efeitos adversos , Antineoplásicos/farmacocinética , Disponibilidade Biológica , Compostos de Bifenilo/efeitos adversos , Compostos de Bifenilo/farmacocinética , Simulação por Computador , Rotulagem de Alimentos , Interações Alimento-Droga , Humanos , Cooperação do Paciente/estatística & dados numéricos , Piridinas/efeitos adversos , Piridinas/farmacocinética , Estados Unidos , United States Food and Drug AdministrationRESUMO
Autophagy is a highly conserved process of self-digestion to promote cell survival in response to nutrient starvation and other metabolic stresses. However, whether ischemic-hypoxic (IH) injury-induced autophagy acts as a neuroprotective mechanism or leads to neuroinjury is a subject of debate. It is known that autophagy is regulated by signaling pathways, including the mammalian target of rapamycin pathway. However, in neural IH injury, whether other signaling pathways are involved in the regulation of autophagy remains to be fully elucidated. In the present study, using the autophagy agonist (rampycin), autophagy antagonist [3-methyl adenine (3-MA)] and lysosome antagonist (MHY1485), autophagy was intervened with at oxygen-glucose deprivation (OGD) 6 h, in order to elucidate the regulatory mechanisms of autophagy. Using immunocytochemistry and western blot analysis, the expression levels of stress-related proteins, such as hypoxia-inducible factor-1α (HIF-1α) (a key regulator in hypoxia) and cyclooxygenase 2 (COX2; inflammatory indicator), were analyzed. In addition, the upstream proteins (Wnt1 and Wnt3a), downstream proteins (Dvl2, ß-catenin) and target proteins (C-myc and cyclin D) in the Wnt/ß-catenin signaling pathway were examined by immunocytochemistry and western blot analysis. The present study revealed that autophagy was activated with the upregulation of autophagic flux in IH injury; it was demonstrated that autophagy had a protective role in IH injury. The Wnt/ß-catenin pathway was involved in IH injury regulation, and the upstream proteins in the Wnt/ß-catenin signaling pathway were upregulated, whereas downstream proteins were downregulated by the activity of autophagy accordingly.
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Autofagia/fisiologia , Hipóxia-Isquemia Encefálica/metabolismo , Via de Sinalização Wnt/fisiologia , beta Catenina/metabolismo , Adenina/análogos & derivados , Adenina/farmacologia , Animais , Autofagia/efeitos dos fármacos , Modelos Biológicos , Morfolinas/farmacologia , Células PC12 , Ratos , Sirolimo/farmacologia , Triazinas/farmacologia , Via de Sinalização Wnt/efeitos dos fármacosRESUMO
Cold exposure is an external stress factor that causes skin frostbite as well as a variety of diseases. Estrogen might participate in neuroprotection after cold exposure, but its precise mechanism remains unclear. In this study, mice were exposed to 10°C for 7 days and 0-4°C for 30 days to induce a model of chronic cold exposure. Results showed that oxidative stress-related c-fos and cyclooxygenase 2 expressions, MAP1LC3-labeled autophagic cells, Iba1-labeled activated microglia, and interleukin-1ß-positive pyramidal cells were increased in the hippocampal CA1 area. Chronic cold exposure markedly elevated the levels of estrogen in the blood and the estrogen receptor, G protein-coupled receptor 30. These results indicate that neuroimmunoreactivity is involved in chronic cold exposure-induced pathological alterations, including oxidative stress, neuronal autophagy, and neuroimmunoreactivity. Moreover, estrogen exerts a neuroprotective effect on cold exposure.
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Neuronal apoptosis mediated by the mitochondrial apoptosis pathway is an important pathological process in cerebral ischemia-reperfusion injury. 14,15-EET, an intermediate metabolite of arachidonic acid, can promote cell survival during ischemia/reperfusion. However, whether the mitochondrial apoptotic pathway is involved this survival mechanism is not fully understood. In this study, we observed that infarct size in ischemia-reperfusion injury was reduced in sEH gene knockout mice. In addition, Caspase 3 activation, cytochrome C release and AIF nuclear translocation were also inhibited. In this study, 14,15-EET pretreatment reduced neuronal apoptosis in the oxygen-glucose deprivation and re-oxygenation group in vitro. The mitochondrial apoptosis pathway was also inhibited, as evidenced by AIF translocation from the mitochondria to nucleus and the reduction in the expressions of cleaved-caspase 3 and cytochrome C in the cytoplasm. 14,15-EET could reduce neuronal apoptosis through upregulation of the ratio of Bcl-2 (anti-apoptotic protein) to Bax (apoptosis protein) and inhibition of Bax aggregation onto mitochondria. PI3K/AKT pathway is also probably involved in the reduction of neuronal apoptosis by EET. Our study suggests that 14,15-EET could suppress neuronal apoptosis and reduce infarct volume through the mitochondrial apoptotic pathway. Furthermore, the PI3K/AKT pathway also appears to be involved in the neuroprotection against ischemia-reperfusion by 14,15-EET.
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Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Apoptose/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Traumatismo por Reperfusão/tratamento farmacológico , Ácido 8,11,14-Eicosatrienoico/farmacologia , Animais , Apoptose/fisiologia , Citocromos c/efeitos dos fármacos , Citocromos c/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Traumatismo por Reperfusão/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Current genotype-guided algorithms for warfarin dosing fail to deliver optimal performance in two aspects: 1) these algorithms are not able to achieve the same level of benefits in non-white populations, since they were developed based on multivariate regression analysis with mostly European/White data and did not include genetic variants found frequently in non-white populations; 2) these algorithms do not account for the dynamic dose/response relationship and were limited in their usefulness to guide dosing during the initiation phase, as the possession of variant VKORC1 and/or CYP2C9 polymorphisms has been associated with a more rapid attainment of target international normalized ratio (INR) and higher risk of over-anticoagulation even in genotype-guided patients. To address these shortcomings, we report on the novel use of a previously published kinetic/pharmacodynamic (K/PD) model to develop a warfarin dosing nomogram to be used across genotypes and ethnicities. Our approach leverages data from both ethnically diverse and European patients, while accounting for the differential dose/response behaviors due to VKORC1 and CYP2C9 genotypes. According to simulations, the utilization of our dosing nomogram could enable effective attainment of therapeutic INR within one week in both ethnically diverse and European populations, while maintaining uniform INR response profiles across genotypes. Furthermore, in silico clinical trial simulations using the K/PD model could be a feasible approach to help to further refine our dosing nomogram to be more applicable in the clinical setting and explore possible outcomes even before prospective clinical trials are initiated.
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Anticoagulantes/administração & dosagem , Varfarina/administração & dosagem , Algoritmos , Relação Dose-Resposta a Droga , Genótipo , HumanosRESUMO
In this study, we investigated the causal relationship between chronic cold exposure and insulin resistance and the mechanisms of how DNA methylation and histone deacetylation regulate cold-reduced insulin resistance. 46 adult male mice from postnatal day 90-180 were randomly assigned to control group and cold-exposure group. Mice in cold-exposure group were placed at temperature from -1 to 4 °C for 30 days to mimic chronic cold environment. Then, fasting blood glucose, blood insulin level and insulin resistance index were measured with enzymatic methods. Immunofluorescent labeling was carried out to visualize the insulin receptor substrate 2 (IRS2), Obese receptor (Ob-R, a leptin receptor), voltage-dependent anion channel protein 1 (VDAC1), cytochrome C (cytC), 5-methylcytosine (5-mC) positive cells in hippocampal CA1 area. Furthermore, the expressions of some proteins mentioned above were detected with Western blot. The results showed: â Chronic cold exposure could reduce the insulin resistance index (P < 0.01) and increase the number of IRS2 positive cells and Ob-R positive cells in hippocampus (P < 0.01). â¡ The expressions of mitochondrial energy-relative proteins, VDAC1 and cytC, were higher in cold-exposure group than in control group with both immunohistochemical staining and Western blot (P < 0.01). ⢠Chronic cold exposure increased DNA methylation and histone deacetylation in the pyramidal cells of CA1 area and led to an increase in the expression of histone deacetylase 1 (HDAC1) and DNA methylation relative enzymes (P < 0.01). In conclusion, chronic cold exposure can improve insulin sensitivity, with the involvement of DNA methylation, histone deacetylation and the regulation of mitochondrial energy metabolism. These epigenetic modifications probably form the basic mechanism of cold-reduced insulin resistance.
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Temperatura Baixa , Metilação de DNA/genética , Metabolismo Energético/fisiologia , Histonas/metabolismo , Resistência à Insulina/fisiologia , Mitocôndrias/metabolismo , 5-Metilcitosina/metabolismo , Acetilação , Animais , Glicemia/análise , Citocromos c/metabolismo , Epigênese Genética , Insulina/sangue , Insulina/metabolismo , Proteínas Substratos do Receptor de Insulina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Processamento de Proteína Pós-Traducional , Receptores para Leptina/metabolismo , Canal de Ânion 1 Dependente de Voltagem/metabolismoRESUMO
FDA recommendations to manage polymorphic CYP-mediated drug-drug interactions (DDIs) and gene-drug interactions (GDIs) are typically similar. However, DDIs may not always reliably predict GDIs because the victim drug may have multiple metabolic pathways and the perpetrator drug may affect multiple enzymes or transporters. Consequently, it is of great interest to both the pharmaceutical industry and regulatory agencies to determine if DDI studies can be leveraged to inform GDIs or vice versa for dose adjustment and labeling. The objective of this study was to investigate under what circumstances DDIs can be used to predict GDIs for prototypical CYP2C9, CYP2C19, and CYP2D6 substrates. We investigated model substrates for CYP2D6 (metoprolol, dextromethorphan, atomoxetine, and vortioxetine), CYP2C9 (warfarin, flurbiprofen, and celecoxib), and CYP2C19 (omeprazole and clopidogrel). Data on drug exposure for poor metabolizers (GDI) and for DDIs mediated by strong/moderate inhibitors in extensive metabolizers were collected. The impact of DDIs and GDIs on drug exposure was compared using: (1) a descriptive and (2) a physiologically based pharmacokinetic convergence analysis. Results from both approaches indicate that information on DDIs can be used to reliably predict GDIs for CYP2D6 substrates. The situation is more complex for CYP2C9 and CYP2C19 substrates because dose of the inhibitor (CYP2C9) and potency of the inhibitor (CYP2C19) impact the extent to which perpetrator drugs phenotypically convert extensive metabolizers to poor(er) metabolizers.
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Citocromos/genética , Citocromos/metabolismo , Interações Medicamentosas , Farmacogenética , Área Sob a Curva , Simulação por Computador , Citocromo P-450 CYP2C19/genética , Citocromo P-450 CYP2C9/genética , Citocromo P-450 CYP2D6/genética , Inibidores Enzimáticos/farmacologia , Humanos , Farmacocinética , Especificidade por SubstratoRESUMO
To investigate the pattern of neural differentiation and synaptogenesis in the mouse retina, immunolabeling, BrdU assay and transmission electron microscopy were used. We show that the neuroblastic cell layer is the germinal zone for neural differentiation and retinal lamination. Ganglion cells differentiated initially at embryonic day 13 (E13), and at E18 horizontal cells appeared in the neuroblastic cell layer. Neural stem cells in the outer neuroblastic cell layer differentiated into photoreceptor cells as early as postnatal day 0 (P0), and neural stem cells in the inner neuroblastic cell layer differentiated into bipolar cells at P7. Synapses in the retina were mainly located in the outer and inner plexiform layers. At P7, synaptophysin immunostaining appeared in presynaptic terminals in the outer and inner plexiform layers with button-like structures. After P14, presynaptic buttons were concentrated in outer and inner plexiform layers with strong staining. These data indicate that neural differentiation and synaptogenesis in the retina play important roles in the formation of retinal neural circuitry. Our study showed that the period before P14, especially between P0 and P14, represents a critical period during retinal development. Mouse eye opening occurs during that period, suggesting that cell differentiation and synaptic formation lead to the attainment of visual function.
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The objective of this study is to understand the impairment of learning and memory in mouse after chronic nitrite exposure. The animal model of nitrite exposure in mouse was created with the daily intubation of nitrite in adult healthy male mice for 3 months. Furthermore, the mouse's learning and memory abilities were tested with Morris water maze, and the expression of Synaptophysin and γ-Synuclein was visualized with immunocytochemistry and Western blot. Our results showed that nitrite exposure significantly prolonged the escape latency period (ELP) and decreased the values of the frequency across platform (FAP) as well as the accumulative time in target quadrant (ATITQ) compared to control, in dose-dependent manner. In addition, after nitrite exposure, synaptophysin (SYN) positive buttons in the visual cortex was reduced, in contrast the increase of γ-synuclein positive cells. The results above were supported by Western blot as well. We conclude that nitrite exposure could lead to a decline in mice's learning and memory. The overexpression of γ-synuclein contributed to the synaptic loss, which is most likely the cause of learning and memory impairment. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1720-1730, 2016.
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Aprendizagem/efeitos dos fármacos , Memória/efeitos dos fármacos , Nitritos/toxicidade , Sinapses/efeitos dos fármacos , Animais , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Sinapses/metabolismo , Sinapses/ultraestrutura , Sinaptofisina/metabolismo , Sinucleínas/metabolismo , Córtex Visual/efeitos dos fármacos , Córtex Visual/metabolismo , Córtex Visual/ultraestruturaRESUMO
Gefitinib (Iressa) is a selective and potent EGFR tyrosine kinase inhibitor. It received an accelerated FDA approval in 2003 for the treatment of patients with nonsmall cell lung cancer (NSCLC) and represents the first-line therapy for NSCLC with EGFR mutations. In the work presented herein, the disposition of gefitinib was investigated extensively in mouse in both plasma and 11 organs (liver, heart, lung, spleen, gut, brain, skin, fat, eye, kidney, and muscle) after a single IV dose of 20 mg/kg. Gefitinib demonstrated extensive distribution in most tissues, except for the brain, and tissue to plasma partition coefficients (K pt) ranged from 0.71 (brain) to 40.5 (liver). A comprehensive whole-body physiologically based pharmacokinetic (PBPK) model of gefitinib in mice was developed, which adequately captured gefitinib concentration-time profiles in plasma and various tissues. Predicted plasma and tissue AUC values agreed well with the values calculated using the noncompartmental analysis (<25% difference). The PBPK model was further extrapolated to humans after taking into account the interspecies differences in physiological parameters. The simulated concentrations in human plasma were in line with the observed concentrations in healthy volunteers and patients with solid malignant tumors after both IV infusion and oral administration. Considering the extensive tissue distribution of gefitinib, plasma concentration may not be an ideal surrogate marker for gefitinib exposure at the target site or organ of toxicity (such as the skin). Since our whole-body PBPK model can predict gefitinib concentrations not only in plasma but also in various organs, our model may have clinical applications in efficacy and safety assessment of gefitinib.
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Antineoplásicos/farmacocinética , Inibidores de Proteínas Quinases/farmacocinética , Quinazolinas/farmacocinética , Administração Oral , Animais , Área Sob a Curva , Proteínas Sanguíneas/metabolismo , Gefitinibe , Voluntários Saudáveis , Humanos , Infusões Intravenosas , Masculino , Camundongos , Modelos Estatísticos , Neoplasias/metabolismo , Farmacocinética , Ligação Proteica , Especificidade da Espécie , Distribuição TecidualRESUMO
In order to understand the mechanisms of alcohol-induced neuroapoptosis through the ceramide pathway, sphingomyelin synthase 2 knockout (SMS2-/-) mice were used to make the prenatal alcohol exposure model, and the role of ceramide regulation on alcohol-induced neuroapoptosis was studied in the offspring. Initially the levels of serum sphingomyelin (SM) were detected with enzymatic method in P0 pups after alcohol exposure in parents. Then the apoptosis of mossy cells in the offspring hippocampus was investigated after prenatal alcohol exposure with immunohistochemistry and TUNEL assay. Finally the expression of activated Caspase 8 and activated Caspase 3 in the offspring hippocampus was detected with Western blot analysis. Our results showed that SM levels were down-regulated in a dose-dependent manner (p<0.05) after prenatal alcohol exposure in wild-type (WT) and SMS2-/- pups. However, SM levels of serum in SMS2-/- pups were significantly lower than that in WT pups (p<0.01). Furthermore, we found that mossy cells were very sensitive to alcohol-induced neuroapoptosis. In both WT pups and SMS2-/- pups, the number of apoptotic mossy cells in the hippocampus increased after prenatal alcohol exposure in a dose dependent manner (p<0.05) and decreased with the growing age. Compared with WT pups, the number of apoptotic mossy cells in the hippocampus of SMS2-/- pups increased (p<0.05). Western blotting showed that the expression of activated Caspase 8 and activated Caspase 3 of hippocampal tissue in WT pups and SMS2-/- pups increases after prenatal alcohol exposure, consistent with results from TUNEL assay and immunocytochemistry. Our study suggests that mossy cells may be the easily attacked cells for fetal alcohol spectrum disorder (FASD), and ceramide is involved in the alcohol-induced neural apoptosis. The mechanism probably lies in the accumulated ceramide in SMS2 mice, and the increase of activated Caspase 8 and Caspase 3 promotes alcohol-induced neuroapoptosis.
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Etanol/toxicidade , Transtornos do Espectro Alcoólico Fetal/fisiopatologia , Hipocampo/efeitos dos fármacos , Esfingomielinas/sangue , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Animais , Animais Recém-Nascidos , Caspase 3/metabolismo , Caspase 8/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Feminino , Transtornos do Espectro Alcoólico Fetal/sangue , Transtornos do Espectro Alcoólico Fetal/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Hipocampo/citologia , Hipocampo/enzimologia , Hipocampo/patologia , Camundongos , Camundongos Knockout , Gravidez , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismoRESUMO
Astrocytes are intimately involved in the formation and development of retinal vessels. Astrocyte dysfunction is a major cause of blood-retinal barrier injury and other retinal vascular diseases. In this study, the development of the retinal vascular system and the formation of the blood-retinal barrier in mice were investigated using immunofluorescence staining, gelatin-ink perfusion, and transmission electron microscopy. The results showed that the retinal vascular system of mice develops from the optic disc after birth, and radiates out gradually to cover the entire retina, taking the papilla optica as the center. First, the superficial vasculature is formed on the inner retinal layer; then, the vasculature extends into the inner and outer edges of the retinal inner nuclear layer, forming the deep vasculature that is parallel to the superficial vasculature. The blood-retinal barrier is mainly composed of endothelium, basal lamina and the end-feet of astrocytes, which become mature during mouse development. Initially, the naive endothelial cells were immature with few organelles and many microvilli. The basal lamina was uniform in thickness, and the glial end-feet surrounded the outer basal lamina incompletely. In the end, the blood-retinal barrier matures with smooth endothelia connected through tight junctions, relatively thin and even basal lamina, and relatively thin glial cell end-feet. These findings indicate that the development of the vasculature in the retina follows the rules of "center to periphery" and "superficial layer to deep layers". Its development and maturation are spatially and temporally consistent with the functional performance of retinal neurons and photosensitivity. The blood-retinal barrier gradually becomes mature via the process of interactions between astrocytes and blood vessel cells.
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Cajal-Retzius cells are reelin-secreting neurons in the marginal zone of the neocortex and hippocampus. The aim of this study was to investigate Cajal-Retzius cells in Alzheimer's disease pathology. Results revealed that the number of Cajal-Retzius cells markedly reduced with age in both wild type and in mice over-expressing the Swedish double mutant form of amyloid precursor protein 695 (transgenic (Tg) 2576 mice). Numerous reelin-positive neurons were positive for activated caspase 3 in Tg2576 mice, suggesting that Cajal-Retzius neuronal loss occurred via apoptosis in this Alzheimer's disease model. Compared with wild type, the number of Cajal-Retzius cells was significantly lower in Tg2576 mice. Western blot analysis confirmed that reelin levels were markedly lower in Tg2576 mice than in wild-type mice. The decline in Cajal-Retzius cells in Tg2576 mice was found to occur concomitantly with the onset of Alzheimer's disease amyloid pathology and related behavioral deficits. Overall, these data indicated that Cajal-Retzius cell loss occurred with the onset and development of Alzheimer's disease.
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The past decade has seen tremendous efforts in the research and development of new chemotherapeutic drugs using target-based approaches. These efforts have led to the discovery of small molecule tyrosine kinase inhibitors (TKIs). Following the initial approval of imatinib by the US FDA in 2001, more than 15 TKIs targeting different tyrosine kinases have been approved, and numerous others are in various phases of clinical evaluation. Unlike conventional chemotherapy that can cause non-discriminating damage to both normal and cancerous cells, TKIs attack cancer-specific targets and therefore have a more favorable safety profile. However, although TKIs have had outstanding success in cancer therapy, there has been increasing evidence of resistance to TKIs. The enhanced efflux of TKIs by ATP-binding cassette (ABC) transporters over-expressed in cancer cells has been found to be one such important resistance mechanism. Another major drawback of TKI therapies that has been increasingly recognized is the extensive inter-individual pharmacokinetic variability, in which ABC transporters seem to play a major role as well. This review covers recent findings on the interactions of small molecule TKIs with ABC transporters. The effects of ABC transporters on anticancer efficacy and the absorption, distribution, metabolism, excretion, and toxicity (ADME-Tox) of the small molecule TKIs are summarized in detail. Since TKIs have been found to not only serve as substrates of ABC transporters, but also as modulators of these proteins via inhibition or induction, their influence upon ABC transporters and potential role on TKI-drug interactions are discussed as well.