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1.
J Clin Pharmacol ; 55(7): 748-56, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25753401

RESUMO

P1066 is an open-label study of raltegravir in HIV positive youth, ages 4 weeks-18 years. Here we summarize P1066 pharmacokinetic (PK) data and a population PK model for the pediatric chewable tablet and oral granules. Raltegravir PK parameters were calculated using noncompartmental analysis. A 2-compartment model was developed using data from P1066 and an adult study of the pediatric formulations. Interindividual variability was described by an exponential error model, and residual variability was captured by an additive/proportional error model. Twelve-hour concentrations (C12h ) were calculated from the model-derived elimination rate constant and 8-hour observed concentration. Simulated steady-state concentrations were analyzed by noncompartmental analysis. Target area under the curve (AUC0-12h ) and C12h were achieved in each cohort. For the pediatric formulations, geometric mean AUC0-12h values were 18.0-22.6 µM-hr across cohorts, and C12h values were 71-130 nM, with lower coefficients of variation versus the film-coated tablet. A 2-compartment model with first-order absorption adequately described raltegravir plasma PK in pediatric and adult patients. Weight was a covariate on clearance and central volume and was incorporated using allometric scaling. Raltegravir chewable tablets and oral granules exhibited PK parameters consistent with those from prior adult studies and older children in P1066, as well as lower variability than the film-coated tablet.


Assuntos
Fármacos Anti-HIV/farmacocinética , Infecções por HIV/tratamento farmacológico , Modelos Biológicos , Raltegravir Potássico/farmacocinética , Adolescente , Adulto , Fatores Etários , Fármacos Anti-HIV/uso terapêutico , Área Sob a Curva , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Raltegravir Potássico/uso terapêutico , Comprimidos
2.
J Chromatogr B Analyt Technol Biomed Life Sci ; 945-946: 225-32, 2014 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-24361860

RESUMO

A sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) assay was developed and validated to facilitate the assessment of clinical pharmacokinetics of dolutegravir (DTG) in plasma samples. This work describes an assay system requiring only a 20µL aliquot of human plasma that is subjected to a simple acetonitrile protein precipitation containing a stably labeled isotope of DTG used as an internal standard. Chromatography was performed on an XBridge C18, 2.1mm×50mm, reversed phase analytical column, using a 60:40 acetonitrile/water mobile phase containing 0.1% formic acid. Detection of the analyte and internal standard was achieved by ESI positive ionization tandem mass spectrometry. The precursor/product transitions (m/z) monitored were 420.1/136.0 and 428.1/283.1 for DTG and DTG-IS, respectively. The dynamic range of this assay extends from 5 to 10,000ng/mL, with a mean coefficient of determination (r, mean±SD) of 0.9996±0.0003. The mean precision values for calibration standards ranged from 0.7 to 4.1%, while accuracy values were 98.3 to 102.0%. Validation results demonstrated high accuracy (≤6.5% deviation) and high precision (≤9.1% CV) for the quality control samples. This assay system provides an accurate, precise, and sensitive method for DTG quantitation and was successfully applied to clinical research samples as part of a phase I/II pediatric clinical trial.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Inibidores de Integrase de HIV/sangue , Compostos Heterocíclicos com 3 Anéis/sangue , Espectrometria de Massas em Tandem/métodos , Calibragem , Humanos , Limite de Detecção , Oxazinas , Piperazinas , Piridonas
3.
J Immunol ; 188(12): 6238-46, 2012 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-22586040

RESUMO

MicroRNAs (miRNAs) are ∼22-nt small RNAs that are important regulators of mRNA turnover and translation. Recent studies have shown the importance of the miRNA pathway in HIV-1 infection, particularly in maintaining latency. Our initial in vitro studies demonstrated that HIV-1-infected HUT78 cells expressed significantly higher IL-10 levels compared with uninfected cultures. IL-10 plays an important role in the dysregulated cytotoxic T cell response to HIV-1, and in silico algorithms suggested that let-7 miRNAs target IL10 mRNA. In a time course experiment, we demonstrated that let-7 miRNAs fall rapidly following HIV-1 infection in HUT78 cells with concomitant rises in IL-10. To show a direct link between let-7 and IL-10, forced overexpression of let-7 miRNAs resulted in significantly reduced IL-10 levels, whereas inhibition of the function of these miRNAs increased IL-10. To demonstrate the relevance of these results, we focused our attention on CD4(+) T cells from uninfected healthy controls, chronic HIV-1-infected patients, and long-term nonprogressors. We characterized miRNA changes in CD4(+) T cells from these three groups and demonstrated that let-7 miRNAs were highly expressed in CD4(+) T cells from healthy controls and let-7 miRNAs were significantly decreased in chronic HIV-1 infected compared with both healthy controls and long-term nonprogressors. We describe a novel mechanism whereby IL-10 levels can be potentially modulated by changes to let-7 miRNAs. In HIV-1 infection, the decrease in let-7 miRNAs may result in an increase in IL-10 from CD4(+) T cells and provide the virus with an important survival advantage by manipulating the host immune response.


Assuntos
Linfócitos T CD4-Positivos/metabolismo , Regulação da Expressão Gênica/genética , Infecções por HIV/genética , Interleucina-10/biossíntese , MicroRNAs/metabolismo , Adulto , Linfócitos T CD4-Positivos/virologia , Citometria de Fluxo , Regulação da Expressão Gênica/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Humanos , Interleucina-10/análise , Interleucina-10/imunologia , Masculino , MicroRNAs/genética , MicroRNAs/imunologia , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/análise , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa
4.
J Acquir Immune Defic Syndr ; 56(5): e130-2, 2011 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-22046602
5.
J Virol ; 84(6): 2972-82, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20053739

RESUMO

Ebola virus (EBOV) cellular attachment and entry is initiated by the envelope glycoprotein (GP) on the virion surface. Entry of this virus is pH dependent and associated with the cleavage of GP by proteases, including cathepsin L (CatL) and/or CatB, in the endosome or cell membrane. Here, we characterize the product of CatL cleavage of Zaire EBOV GP (ZEBOV-GP) and evaluate its relevance to entry. A stabilized recombinant form of the EBOV GP trimer was generated using a trimerization domain linked to a cleavable histidine tag. This trimer was purified to homogeneity and cleaved with CatL. Characterization of the trimeric product by N-terminal sequencing and mass spectrometry revealed three cleavage fragments, with masses of 23, 19, and 4 kDa. Structure-assisted modeling of the cathepsin L-cleaved ZEBOV-GP revealed that cleavage removes a glycosylated glycan cap and mucin-like domain (MUC domain) and exposes the conserved core residues implicated in receptor binding. The CatL-cleaved ZEBOV-GP intermediate bound with high affinity to a neutralizing antibody, KZ52, and also elicited neutralizing antibodies, supporting the notion that the processed intermediate is required for viral entry. Together, these data suggest that CatL cleavage of EBOV GP exposes its receptor-binding domain, thereby facilitating access to a putative cellular receptor in steps that lead to membrane fusion.


Assuntos
Catepsina L/metabolismo , Ebolavirus , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/metabolismo , Internalização do Vírus , Sequência de Aminoácidos , Animais , Anticorpos Neutralizantes/imunologia , Linhagem Celular , Ebolavirus/imunologia , Ebolavirus/fisiologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Dados de Sequência Molecular , Multimerização Proteica , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Proteínas do Envelope Viral/genética
6.
Biomol Concepts ; 1(3-4): 285-96, 2010 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-25962003

RESUMO

Transcriptional regulation by small RNA molecules, including small interfering RNA and microRNA, has emerged as an important gene expression modulator. The regulatory pathways controlling gene expression, post-transcriptional gene silencing and transcriptional gene silencing (TGS) have been demonstrated in yeast, plants and more recently in human cells. In this review, we discuss the currents models of transcriptional regulation and the main components of the RNA-induced silencing complex and RNA-induced transcriptional silencing complex machinery, as well as confounding off-target effects and gene activation. We also discuss RNA-mediated TGS within the NF-κB motif of the human immunodeficiency virus type 1 5' long tandem repeat promoter region and the associated epigenetic modifications. Finally, we outline the current RNA interference (RNAi) delivery methods and describe the current status of human trials investigating potential RNAi therapeutics for several human diseases.

7.
HIV Clin Trials ; 10(3): 193-9, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19632958

RESUMO

PURPOSE: Challenges exist regarding antiretroviral quantitation in the female genital tract. Endocervical wicking using sterile tear flow test strips is an alternative to conventional methods due to the consistent sample volume obtained. METHODS: A novel method for measuring antiretrovirals in cervicovaginal secretions using Sno-strip wicking was developed and tested by spiking Sno-strips with known concentrations of tenofovir, nevirapine, atazanavir, lopinavir, and ritonavir in blank cervicovaginal lavage fluid. Drug concentrations were determined by high-performance liquid chromatography with ultraviolet or mass spectrometry detection. RESULTS: Mean extraction recoveries were 91% for tenofovir, 89% for nevirapine, 63% for atazanavir, 60% for lopinavir, and 61% for ritonavir relative to controls. Freezing spiked samples for 24 hours at -80 degrees C had no effect on recovery. CONCLUSIONS: Results suggest that the antiretrovirals tested can be efficiently extracted from Sno-strips, although a greater percentage of tenofovir and nevirapine was recovered. Storage of Sno-strip samples up to 24 hours before analysis showed no difference in the percentage of drug recovered compared with immediate analysis. Quantitating antiretroviral penetration into the female genital tract may assist in determining optimal therapeutic antiretroviral regimens to both decrease the risk of HIV transmission and prevent development of HIV drug resistance.


Assuntos
Antirretrovirais/análise , Colo do Útero/química , Inibidores da Protease de HIV/análise , HIV-1/efeitos dos fármacos , Vagina/química , Esfregaço Vaginal/métodos , Antirretrovirais/farmacocinética , Sulfato de Atazanavir , Colo do Útero/virologia , Cromatografia Líquida de Alta Pressão , Feminino , Infecções por HIV/prevenção & controle , Inibidores da Protease de HIV/farmacocinética , Humanos , Lopinavir , Oligopeptídeos/análise , Oligopeptídeos/farmacocinética , Piridinas/análise , Piridinas/farmacocinética , Pirimidinonas/análise , Pirimidinonas/farmacocinética , Ritonavir/análise , Ritonavir/farmacocinética , Vagina/virologia , Esfregaço Vaginal/instrumentação
8.
HIV Clin Trials ; 10(1): 41-7, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19362995

RESUMO

PURPOSE: The objective of this study was to examine lamivudine (3TC), zidovudine (ZDV), nelfinavir (NFV), and its active nelfinavir metabolite (M8) concentrations in paired maternal plasma and amniotic fluid samples to determine antiretroviral penetration or accumulation in the fetal compartment. METHOD: Ten paired amniotic fluid and maternal plasma samples were obtained during caesarian section for pharmacokinetic analysis. Antiretroviral concentrations were measured in both matrices using high-performance liquid chromatography (HPLC) and mass spectrometry (LC/MS) methodologies. RESULTS: Median maternal plasma concentrations for NFV, M8, 3TC, and ZDV were 456, 244, 176, and 794 ng/mL, respectively, while median amniotic fluid concentrations were 118, 21, 2537, and 1483 ng/mL, respectively. The median NFV amniotic fluid to maternal plasma ratio was 0.44; the median M8 ratio was 0.11. Median 3TC and ZDV amniotic fluid to plasma ratios were 11.9 and 1.5, respectively. CONCLUSIONS: NFV and M8 exhibited partial drug transfer and/or accumulation in the amniotic compartment, whereas ZDV and 3TC concentrations mostly exceeded that in maternal plasma. Overall, all drugs achieved exposures in the amniotic fluid in excess of their wild-type viral susceptibilities. Amniotic fluid is an important compartment in the prevention of mother-to-child transmission; a further understanding of protease inhibitor and other antiretroviral drug penetration into amniotic fluid is warranted.


Assuntos
Líquido Amniótico/metabolismo , Infecções por HIV/tratamento farmacológico , Inibidores da Protease de HIV/farmacocinética , Transmissão Vertical de Doenças Infecciosas/prevenção & controle , Lamivudina/farmacocinética , Nelfinavir/farmacocinética , Complicações Infecciosas na Gravidez/tratamento farmacológico , Zidovudina/farmacocinética , Cromatografia Líquida de Alta Pressão , Feminino , Infecções por HIV/sangue , Infecções por HIV/transmissão , Inibidores da Protease de HIV/sangue , Inibidores da Protease de HIV/uso terapêutico , Humanos , Lamivudina/sangue , Lamivudina/uso terapêutico , Espectrometria de Massas , Nelfinavir/sangue , Nelfinavir/uso terapêutico , Gravidez , Complicações Infecciosas na Gravidez/sangue , Zidovudina/sangue , Zidovudina/uso terapêutico
9.
Artigo em Inglês | MEDLINE | ID: mdl-18430616

RESUMO

This work describes an assay system that has been developed to quantify raltegravir concentrations in human plasma using a liquid-liquid extraction technique paired with HPLC separation and MS-MS detection. The dynamic range of this assay extends from 1 to 3000 ng/mL, with a coefficient of determination (r(2), mean+/-SD) of 0.9992+/-0.0002. The mean precision values for calibration standards ranged from 0.6% to 3.0%, while accuracy values were 96.5-104.3%. This procedure is an accurate, precise, and sensitive method for raltegravir quantitation and was successfully validated using external proficiency testing.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Inibidores de Integrase de HIV/sangue , Pirrolidinonas/sangue , Espectrometria de Massas em Tandem/métodos , Calibragem , Humanos , Controle de Qualidade , Raltegravir Potássico , Sensibilidade e Especificidade
10.
Clin Infect Dis ; 45(3): 391-4, 2007 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-17599320

RESUMO

The objective of this study was to serially quantitate the concentration of nevirapine in breast milk after discontinuation of treatment. Samples were collected from both breasts of a human immunodeficiency virus-infected patient for 3 weeks. Nevirapine was quantifiable for up to 17 days after discontinuation of therapy; total nevirapine concentrations remained above the 90% inhibitory concentration for 6 days, and no differences were observed between breasts.


Assuntos
Fármacos Anti-HIV/isolamento & purificação , Infecções por HIV/tratamento farmacológico , Leite Humano/química , Nevirapina/isolamento & purificação , Nevirapina/uso terapêutico , Fármacos Anti-HIV/uso terapêutico , Feminino , Lateralidade Funcional , Humanos , Gravidez , Complicações Infecciosas na Gravidez/tratamento farmacológico , Complicações Infecciosas na Gravidez/virologia
11.
Rapid Commun Mass Spectrom ; 21(13): 2087-94, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17546653

RESUMO

A sensitive and specific method for the quantitation of tenofovir (TFV) in human plasma by liquid chromatography/electrospray ionization mass spectrometry was developed and validated. Plasma samples were prepared by solid-phase extraction performed on Waters Oasis cation-exchange cartridges (30 mg). Chromatographic separation was performed isocratically on a reversed-phase Waters Atlantis dC18 column (2.0x100 mm, 3 microm). The mobile phase consisted of a hydroxylamine/acetic acid buffer (pH 6.75) and methanol (93:7, v/v). The acquisition was performed in selected ion monitoring mode for the protonated molecular ions [M+H]+ of m/z 288.2 for TFV and 212.2 for the internal standard, zalcitibine. The method was fully validated to determine its specificity, recovery, linearity and sensitivity, accuracy and precision. The analytical range was set at 1-750 ng/mL using a 200 microL plasma sample, with a mean coefficient of determination (r2) of 0.9969. The mean accuracies for the calibration standards ranged from -5.0 to 4.3%, while the precisions were within 1.2 and 6.4%. Intra-assay and inter-assay mean accuracies for three quality control concentrations (2, 60, and 600 ng/mL) ranged from -6.1 to 10.7%, while the precisions were within 1.3 and 9.1%. TFV was shown to be stable under normal storage and assay conditions; no degradation was seen when stored at -20 degrees C or -80 degrees C for up to 6 months, and after 16 h at room temperature in the injection matrix. The present method provides an accurate, precise, and sensitive tool for TFV quantitation and was successfully applied to an external proficiency-testing program and pharmacokinetic analysis.


Assuntos
Adenina/análogos & derivados , Cromatografia Líquida/métodos , Organofosfonatos/sangue , Inibidores da Transcriptase Reversa/sangue , Espectrometria de Massas por Ionização por Electrospray/métodos , Adenina/sangue , Adenina/química , Adenina/farmacocinética , Calibragem , Estabilidade de Medicamentos , Congelamento , Humanos , Estrutura Molecular , Organofosfonatos/química , Organofosfonatos/farmacocinética , Padrões de Referência , Reprodutibilidade dos Testes , Inibidores da Transcriptase Reversa/química , Inibidores da Transcriptase Reversa/farmacocinética , Sensibilidade e Especificidade , Temperatura , Tenofovir , Fatores de Tempo , Zalcitabina/química
12.
Proc Natl Acad Sci U S A ; 103(43): 15987-91, 2006 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-17043214

RESUMO

The remarkable infectivity and virulence of the 1918 influenza virus resulted in an unprecedented pandemic, raising the question of whether it is possible to develop protective immunity to this virus and whether immune evasion may have contributed to its spread. Here, we report that the highly lethal 1918 virus is susceptible to immune protection by a preventive vaccine, and we define its mechanism of action. Immunization with plasmid expression vectors encoding hemagglutinin (HA) elicited potent CD4 and CD8 cellular responses as well as neutralizing antibodies. Antibody specificity and titer were defined by a microneutralization and a pseudotype assay that could assess antibody specificity without the need for high-level biocontainment. This pseudotype inhibition assay can define evolving serotypes of influenza viruses and facilitate the development of immune sera and neutralizing monoclonal antibodies that may help contain pandemic influenza. Notably, mice vaccinated with 1918 HA plasmid DNAs showed complete protection to lethal challenge. T cell depletion had no effect on immunity, but passive transfer of purified IgG from anti-H1(1918) immunized mice provided protective immunity for naïve mice challenged with infectious 1918 virus. Thus, humoral immunity directed at the viral HA can protect against the 1918 pandemic virus.


Assuntos
Surtos de Doenças , Vírus da Influenza A/imunologia , Vacinas contra Influenza/imunologia , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Sequência de Aminoácidos , Animais , Peso Corporal/imunologia , Expressão Gênica , Genes Reporter/genética , Vetores Genéticos/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vírus da Influenza A/genética , Lentivirus/genética , Camundongos , Dados de Sequência Molecular , Infecções por Orthomyxoviridae/epidemiologia , Taxa de Sobrevida , Vacinas de DNA/genética , Vacinas de DNA/imunologia
13.
J Virol ; 80(2): 1025-31, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16379003

RESUMO

Virus-encoded modulation of apoptosis may serve as a mechanism to enhance cell survival and virus persistence. The impact of productive varicella-zoster virus (VZV) infection on apoptosis appears to be cell type specific, as infected human sensory neurons are resistant to apoptosis, yet human fibroblasts readily become apoptotic. We sought to identify the viral gene product(s) responsible for this antiapoptotic phenotype in primary human sensory neurons. Treatment with phosphonoacetic acid to inhibit viral DNA replication and late-phase gene expression did not alter the antiapoptotic phenotype, implicating immediate-early (IE) or early genes or a virion component. Compared to the parental VZV strain (rOKA), a recombinant virus unable to express one copy of the diploid IE gene ORF63 (rOka deltaORF63) demonstrated a significant induction of apoptosis in infected neurons, as determined by three methods: annexin V staining, deoxynucleotidyltransferase-mediated dUTP-biotin nick end label staining, and transmission electron microscopy. Furthermore, neurons transfected with a plasmid expressing ORF63 resisted apoptosis induced by nerve growth factor withdrawal. These results show that ORF63 can suppress apoptosis of neurons and provide the first identification of a VZV gene encoding an antiapoptotic function. As ORF63 is expressed in neurons during both productive and latent infection, it may play a significant role in viral pathogenesis by promoting neuron survival during primary and reactivated infections.


Assuntos
Apoptose , Varicela/virologia , Herpesvirus Humano 3/fisiologia , Proteínas Imediatamente Precoces/fisiologia , Proteínas do Envelope Viral/fisiologia , Células Cultivadas , Regulação para Baixo , Herpesvirus Humano 3/química , Humanos , Neurônios Aferentes/fisiologia , Neurônios Aferentes/virologia
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