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1.
Int J Nanomedicine ; 14: 6231-6247, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31496683

RESUMO

Purpose: A palmitoylated prodrug of emtricitabine (FTC) was synthesized to extend the drug's half-life, antiretroviral activities and biodistribution. Methods: A modified FTC prodrug (MFTC) was synthesized by palmitoyl chloride esterification. MFTC's chemical structure was evaluated by nuclear magnetic resonance. The created hydrophobic prodrug nanocrystals were encased into a poloxamer surfactant and the pharmacokinetics (PK), biodistribution and antiretroviral activities of the nanoformulation (NMFTC) were assessed. The conversion of MFTC to FTC triphosphates was evaluated. Results: MFTC coated with poloxamer formed stable nanocrystals (NMFTC). NMFTC demonstrated an average particle size, polydispersity index and zeta potential of 350 nm, 0.24 and -20 mV, respectively. Drug encapsulation efficiency was 90%. NMFTC was readily taken up by human monocyte-derived macrophages yielding readily detected intracellular FTC triphosphates and an extended PK profile. Conclusion: NMFTC shows improved antiretroviral activities over native FTC. This is coordinate with its extended apparent half-life. The work represents an incremental advance in the development of a long-acting FTC formulation.


Assuntos
Composição de Medicamentos , Emtricitabina/farmacologia , Nanopartículas/química , Pró-Fármacos/farmacologia , Animais , Antirretrovirais/farmacologia , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Emtricitabina/sangue , Emtricitabina/síntese química , Emtricitabina/química , Humanos , Cinética , Macrófagos/efeitos dos fármacos , Masculino , Nanopartículas/ultraestrutura , Pró-Fármacos/síntese química , Pró-Fármacos/química , Espectroscopia de Prótons por Ressonância Magnética , Ratos Sprague-Dawley
2.
Spectrochim Acta A Mol Biomol Spectrosc ; 215: 266-275, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-30831397

RESUMO

Spectrophotometric analysis method based on artificial neural network (ANN), partial least squares regression (PLS) and principal component regression (PCR) models was proposed for the simultaneous determination of Emtricitabine (ETB) and Tenofovir alafenamide fumarate (TAF) in human immunodeficiency virus (HIV) drug. An artificial neural network consisting of two, five, and seven layers with 2,3,5,7, and 9 neurons was trained by applying a feed forward back-propagation learning. In this method, Levenberg-Marquardt (LM) and gradient descent with momentum and adaptive learning rate back propagation (GDX) algorithms were used. Statistical parameters indicated that the ability of LM was better than GDX algorithm. Also, root mean square error (RMSE) and recovery (%) of the PLS and PCR methods showed that PLS has worked better than PCR. The proposed models were compared to the high- performance liquid chromatography (HPLC) as a reference method. Furthermore, the obtained results of the one-way analysis of variance (ANOVA) test at the 95% confidence level represented that there was no significant difference between the proposed and reference methods.


Assuntos
Adenina/análogos & derivados , Fármacos Anti-HIV/análise , Emtricitabina/análise , Redes Neurais (Computação) , Espectrofotometria Ultravioleta/métodos , Adenina/análise , Adenina/química , Fármacos Anti-HIV/química , Calibragem , Emtricitabina/química , Análise dos Mínimos Quadrados , Análise Multivariada
3.
Nat Commun ; 10(1): 1413, 2019 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-30926773

RESUMO

The increasing global prevalence of human immunodeficiency virus (HIV) is estimated at 36.7 million people currently infected. Lifelong antiretroviral (ARV) drug combination dosing allows management as a chronic condition by suppressing circulating viral load to allow for a near-normal life; however, the daily burden of oral administration may lead to non-adherence and drug resistance development. Long-acting (LA) depot injections of nanomilled poorly water-soluble ARVs have shown highly promising clinical results with drug exposure largely maintained over months after a single injection. ARV oral combinations rely on water-soluble backbone drugs which are not compatible with nanomilling. Here, we evaluate a unique prodrug/nanoparticle formation strategy to facilitate semi-solid prodrug nanoparticles (SSPNs) of the highly water-soluble nucleoside reverse transcriptase inhibitor (NRTI) emtricitabine (FTC), and injectable aqueous nanodispersions; in vitro to in vivo extrapolation (IVIVE) modelling predicts sustained prodrug release, with activation in relevant biological environments, representing a first step towards complete injectable LA regimens containing NRTIs.


Assuntos
Antirretrovirais/uso terapêutico , Sistemas de Liberação de Medicamentos , Infecções por HIV/tratamento farmacológico , Nanopartículas/uso terapêutico , Pró-Fármacos/uso terapêutico , Água/química , Antirretrovirais/química , Antirretrovirais/farmacocinética , Simulação por Computador , Quimioterapia Combinada , Emtricitabina/sangue , Emtricitabina/química , Emtricitabina/metabolismo , Emtricitabina/farmacocinética , Emulsões , Humanos , Pró-Fármacos/química , Solubilidade
4.
Int J Pharm ; 557: 390-401, 2019 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-30529658

RESUMO

Hydrophobic and hydrophilic thermoplastic poly(urethane) (TPU) mixtures offer the opportunity to tune water swelling capacity and diffusion rate for drugs exhibiting broadly different properties. We sought to (1) assess the range of drug diffusion rates achieved by varying hydrophilic-to-hydrophobic TPU ratio relative to varying ethylene vinyl acetate (EVA) crystallinity; (2) investigate the effect of mixture ratio on permeability of emtricitabine; and (3) investigate the impact of the extrusion process on mixing of the two TPUs and the resulting impact on drug diffusion. The permeability of water-soluble emtricitabine exhibited a 736-fold range across the blends of TPU, but only a 3.4-fold range across the EVA grades investigated. Varying hydrophilic content of the TPU mixture from 0% to 25% (w/w) led to a negligible permeability change, while changing hydrophilic content from 55% to 100% resulted in a linear 3-fold increase in drug permeability. Interestingly, an 123-fold permeability change occurred between 50% and 55% hydrophilic polymer. Extrusion process parameters exhibited minimal impact on homogeneity and drug diffusion. These findings suggest that hydrophilic polymer domains form a continuous network at levels above 55% hydrophilic TPU, thus facilitating a water-filled porous network when exposed to water that provides a mechanism for accelerated drug diffusion.


Assuntos
Fármacos Anti-HIV/química , Implantes de Medicamento , Emtricitabina/química , Poliuretanos/química , Polivinil/química , Inibidores da Transcriptase Reversa/química , Preparações de Ação Retardada/química , Liberação Controlada de Fármacos , Interações Hidrofóbicas e Hidrofílicas
5.
Expert Opin Pharmacother ; 19(8): 929-934, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29767543

RESUMO

INTRODUCTION: HIV eradication is not feasible and lifelong treatment is warranted to manage HIV infection. In this scenario, the advent of single-tablet, once-daily, fixed-dose co-formulations is important for reducing pill burden and maximize long-term drug adherence. Cobicistat-boosted darunavir along with emtricitabine and tenofovir alafenamide co-formulation (DRV/c/FTC/TAF or the trade name Symtuza®) is the first marketed protease inhibitor-based fixed-dose combination regimen for the treatment of HIV infection. It was approved in late 2017 by the European Medical Agency both for naïve patients and treatment-experienced patients with viral suppression. Areas covered: PubMed, ClinicalTrials.gov and presentations at scientific meetings were searched with the terms 'darunavir/cobicistat' and 'tenofovir alafenamide and emtricitabine' for clinical trials either conducted to date or ongoing as well as a review of abstracts from major HIV/AIDS and infectious diseases conferences from 2015 to up to date. Expert opinion: DRV/c/FTC/TAF is a novel unique antiretroviral drug co-formulation that exhibits a convenient dosing, satisfactory safety profile, and high antiviral efficacy, even in patients harboring viruses with resistance to antivirals other than darunavir in the short-midterm. It represents the first fixed-dose combination therapy including a protease inhibitor given as one single pill once daily for drug-naïve patients and as second-line antiretroviral therapy.


Assuntos
Fármacos Anti-HIV/uso terapêutico , Infecções por HIV/tratamento farmacológico , Comprimidos/química , Adenina/efeitos adversos , Adenina/análogos & derivados , Adenina/química , Adenina/uso terapêutico , Fármacos Anti-HIV/efeitos adversos , Fármacos Anti-HIV/química , Ensaios Clínicos como Assunto , Cobicistat/efeitos adversos , Cobicistat/química , Cobicistat/uso terapêutico , Darunavir/efeitos adversos , Darunavir/química , Darunavir/uso terapêutico , Farmacorresistência Viral , Quimioterapia Combinada , Emtricitabina/efeitos adversos , Emtricitabina/química , Emtricitabina/uso terapêutico , Humanos , Nefropatias/etiologia
6.
Biomed Chromatogr ; 32(4)2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29216682

RESUMO

A combination of antiretroviral agents is frequently used in effective treatment of the human immunodeficiency virus infection. In this study, two different separation methods are presented for the simultaneous determination of emtricitabine, rilpivirine and tenofovir from raw materials and urine samples. Developed liquid chromatography and capillary electrophoresis methods were thoroughly optimized for high analytical performances. Optimization of multiple variables at the same time by performing a minimum number of experiments was achieved by the Box-Behnken design, which is an experimental design in response surface methodology, in capillary electrophoresis. The results of the experimental design ensure minimum analysis time with well-separated analytes. Separation conditions, such as different stationary phases, pH level, organic modifiers and temperatures in liquid chromatography method, were also optimized. In particular, among stationary phases, the core-shell column especially enhanced the effectiveness of separation in liquid chromatography. Both methods were fully validated and applied to real samples. The main advantage of the developed methods is the separation of the drug combination in a short time with high efficiency and without any time-consuming steps.


Assuntos
Antirretrovirais/urina , Cromatografia Líquida/métodos , Eletroforese Capilar/métodos , Emtricitabina/urina , Rilpivirina/urina , Tenofovir/urina , Antirretrovirais/química , Antirretrovirais/isolamento & purificação , Emtricitabina/química , Emtricitabina/isolamento & purificação , Humanos , Limite de Detecção , Modelos Lineares , Modelos Estatísticos , Reprodutibilidade dos Testes , Rilpivirina/química , Rilpivirina/isolamento & purificação , Tenofovir/química , Tenofovir/isolamento & purificação
8.
Artigo em Inglês | MEDLINE | ID: mdl-28651173

RESUMO

OBJECTIVES: To present the validation and clinical application of a LC-MS/MS method for the quantification of lamivudine (3TC), emtricitabine (FTC) and tenofovir (TFV) in dried blood spots (DBS) and dried breast milk spots (DBMS). METHODS: DBS and DBMS were prepared from 50 and 30µL of drug-spiked whole blood and human breast milk, respectively. Following extraction with acetonitrile and water, chromatographic separation utilised a Synergi polar column with a gradient mobile phase program consisting of 0.1% formic acid in water and 0.1% formic acid in acetonitrile. Detection and quantification was performed using a TSQ Quantum Ultra triple quadrupole mass spectrometer. The analytical method was used to evaluate NRTI drug levels in HIV-positive nursing mothers-infant pairs. RESULTS: The assay was validated over the concentration range of 16.6-5000ng/mL for 3TC, FTC and TFV in DBS and DBMS except for TFV in DBMS where linearity was established from 4.2-1250ng/mL. Intra and inter-day precision (%CV) ranged from 3.5-8.7 and accuracy was within 15% for all analytes in both matrices. The mean recovery in DBS was >61% and in DBMS >43% for all three analytes. Matrix effect was insignificant. Median AUC0-8 values in maternal DBS and DBMS, respectively, were 4683 (4165-6057) and 6050 (5217-6417)ngh/mL for 3TC, 3312 (2259-4312) and 4853 (4124-6691)ngh/mL for FTC and 1559 (930-1915) and 56 (45-80)ngh/mL for TFV. 3TC and FTC were quantifiable (>16.6ng/mL) in DBS from 2/6 and 1/6 infants respectively whereas TFV was undetectable in all infants. CONCLUSIONS: DBS and DBMS sampling for bioanalysis of 3TC, FTC and TFV is straightforward, robust, accurate and precise, and ideal for use in low-resource settings.


Assuntos
Fármacos Anti-HIV/análise , Teste em Amostras de Sangue Seco/métodos , Emtricitabina/análise , Lamivudina/análise , Leite Humano/química , Tenofovir/análise , Adulto , Fármacos Anti-HIV/sangue , Fármacos Anti-HIV/química , Área Sob a Curva , Cromatografia Líquida/métodos , Estudos de Coortes , Emtricitabina/sangue , Emtricitabina/química , Feminino , Humanos , Lactente , Lamivudina/sangue , Lamivudina/química , Modelos Lineares , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Espectrometria de Massas em Tandem/métodos , Tenofovir/sangue , Tenofovir/química , Adulto Jovem
9.
Nucleic Acids Res ; 45(10): 6228-6237, 2017 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-28402499

RESUMO

Nucleoside reverse transcriptase inhibitors (NRTIs) with L-stereochemistry have long been an effective treatment for viral infections because of the strong D-stereoselectivity exhibited by human DNA polymerases relative to viral reverse transcriptases. The D-stereoselectivity of DNA polymerases has only recently been explored structurally and all three DNA polymerases studied to date have demonstrated unique stereochemical selection mechanisms. Here, we have solved structures of human DNA polymerase ß (hPolß), in complex with single-nucleotide gapped DNA and L-nucleotides and performed pre-steady-state kinetic analysis to determine the D-stereoselectivity mechanism of hPolß. Beyond a similar 180° rotation of the L-nucleotide ribose ring seen in other studies, the pre-catalytic ternary crystal structures of hPolß, DNA and L-dCTP or the triphosphate forms of antiviral drugs lamivudine ((-)3TC-TP) and emtricitabine ((-)FTC-TP) provide little structural evidence to suggest that hPolß follows the previously characterized mechanisms of D-stereoselectivity. Instead, hPolß discriminates against L-stereochemistry through accumulation of several active site rearrangements that lead to a decreased nucleotide binding affinity and incorporation rate. The two NRTIs escape some of the active site selection through the base and sugar modifications but are selected against through the inability of hPolß to complete thumb domain closure.


Assuntos
DNA Polimerase beta/metabolismo , Inibidores da Transcriptase Reversa/metabolismo , Catálise , Domínio Catalítico , Cristalografia por Raios X , DNA Polimerase beta/química , DNA Polimerase beta/genética , Nucleotídeos de Desoxicitosina/metabolismo , Emtricitabina/química , Emtricitabina/metabolismo , Humanos , Cinética , Lamivudina/química , Lamivudina/metabolismo , Modelos Moleculares , Conformação Molecular , Mutação de Sentido Incorreto , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Estereoisomerismo , Relação Estrutura-Atividade , Especificidade por Substrato
10.
J Am Chem Soc ; 139(1): 465-471, 2017 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-27959534

RESUMO

DNA polymerases are essential enzymes that faithfully and efficiently replicate genomic information.1-3 The mechanism of nucleotide incorporation by DNA polymerases has been extensively studied structurally and kinetically, but several key steps following phosphodiester bond formation remain structurally uncharacterized due to utilization of natural nucleotides. It is thought that the release of pyrophosphate (PPi) triggers reverse conformational changes in a polymerase in order to complete a full catalytic cycle as well as prepare for DNA translocation and subsequent incorporation events. Here, by using the triphosphates of chain-terminating antiviral drugs lamivudine ((-)3TC-TP) and emtricitabine ((-)FTC-TP), we structurally reveal the correct sequence of post-chemistry steps during nucleotide incorporation by human DNA polymerase ß (hPolß) and provide a structural basis for PPi release. These post-catalytic structures reveal hPolß in an open conformation with PPi bound in the active site, thereby strongly suggesting that the reverse conformational changes occur prior to PPi release. The results also help to refine the role of the newly discovered third divalent metal ion for DNA polymerase-catalyzed nucleotide incorporation. Furthermore, a post-chemistry structure of hPolß in the open conformation, following incorporation of (-)3TC-MP, with a second (-)3TC-TP molecule bound to the active site in the absence of PPi, suggests that nucleotide binding stimulates PPi dissociation and occurs before polymerase translocation. Our structural characterization defines the order of the elusive post-chemistry steps in the canonical mechanism of a DNA polymerase.


Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , Nucleotídeos/metabolismo , Antivirais/química , Antivirais/metabolismo , Biocatálise , DNA Polimerase Dirigida por DNA/química , Emtricitabina/química , Emtricitabina/metabolismo , Humanos , Lamivudina/química , Lamivudina/metabolismo , Nucleotídeos/química , Conformação Proteica
11.
J Chromatogr B Analyt Technol Biomed Life Sci ; 1033-1034: 234-241, 2016 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-27571683

RESUMO

Accurate and sensitive liquid-chromatography tandem mass spectrometry method for the quantification of tenofovir and emtricitabine in seminal plasma has been developed and full validated. Molecules were separated by high-performance liquid chromatography on an Atlantis T3 C18 column using a gradient of deionized water and methanol, including 0.05% formic acid (250µl/min) and detected by electrospray ionisation/tandem mass spectrometry in positive ion mode. The method was validated over a clinical range of 3.13-1000ng/mL for tenofovir and 6.25-2000ng/mL for emtricitabine. Inter and intra-assay precisions were <9.37% for tenofovir and<10.88% for emtricitabine, and accuracies were between 0.48% and 8.43% for tenofovir, and between 0.64% and 13.87% for emtricitabine. The developed method was successfully applied for analysing tenofovir and emtricitabine concentrations in seminal plasma samples from a clinical study. The use of tandem mass spectrometry can be a suitable method for the analysis of this kind of matrices, providing high sensitivity and specificity to the analysis.


Assuntos
Cromatografia Líquida/métodos , Emtricitabina/análise , Sêmen/química , Espectrometria de Massas em Tandem/métodos , Tenofovir/análise , Estabilidade de Medicamentos , Emtricitabina/química , Humanos , Limite de Detecção , Masculino , Padrões de Referência , Reprodutibilidade dos Testes , Tenofovir/química
12.
J Pharm Biomed Anal ; 129: 473-481, 2016 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-27497648

RESUMO

Combination antiretroviral (cARV) treatment is more common in human immunodeficiency virus (HIV) infection. In many instances, treatment regimen includes two or more combination of drugs from six different classes. Some of the antiretroviral combination medications are under study at preclinical and clinical stages. A precise method is required to quantify the drug concentration in biological matrices to study pharmacokinetic behavior and tissue distribution profile in animals and/or humans. We have developed and validated a sensitive and precise liquid chromatography-tandem mass spectrometry method for simultaneous quantification of selected antiretroviral drugs, tenofovir (TNF), emtricitabine (FTC), rilpivirine (RPV), dolutegravir (DTG) and elvitegravir (EVG) in mouse biological matrices. This method involves a solid phase extraction, simple isocratic chromatographic separation using Restek Pinnacle DB BiPh column (50mm×2.1mm, 5µm) and mass spectrometric detection by an API 3200 Q Trap instrument. The total run time for each sample was 6min. The method was validated in the concentration range of 5-2000ng/mL for FTC, RPV, DTG, EVG and 10-4000ng/mL for TNF respectively with correlation coefficients (r(2)) higher than 0.9976. The results of intra and inter-run assay precision and accuracy were within acceptance limits for all the five analytes. This method was used to support the study of pharmacokinetics and tissue distribution profile of nanoformulated antiretroviral drugs in mice.


Assuntos
Emtricitabina/química , Compostos Heterocíclicos com 3 Anéis/química , Quinolonas/química , Rilpivirina/química , Tenofovir/química , Animais , Antirretrovirais/química , Antirretrovirais/farmacocinética , Cromatografia Líquida/métodos , Emtricitabina/farmacocinética , Feminino , Infecções por HIV/metabolismo , Compostos Heterocíclicos com 3 Anéis/farmacocinética , Camundongos , Quinolonas/farmacocinética , Rilpivirina/farmacocinética , Extração em Fase Sólida/métodos , Espectrometria de Massas em Tandem/métodos , Tenofovir/farmacocinética
13.
J Pharm Biomed Anal ; 128: 438-446, 2016 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-27344633

RESUMO

The present study investigated drug-drug interaction behaviour of emtricitabine (FTC) and tenofovir disoproxil fumarate (TDF) under solid state stability test conditions. Six interaction products were separated and detected by high performance liquid chromatography coupled to photodiode array detector (HPLC-PDA) using C18 column. The same were characterized using LC-high resolution mass spectrometry (LC-HRMS), LC-multi stage mass spectrometry (LC-MS(n)) and online hydrogen/deuterium (H/D) exchange studies. The interaction pathway among the two drugs was outlined based on the elucidated structures. Four of the six interaction products were also formed in marketed tablets containing FTC and TDF (along with efavirenz (EFV)) that were kept without packing under accelerated condition of 40°C/75% RH till 6 months.


Assuntos
Estabilidade de Medicamentos , Emtricitabina/química , Espectrometria de Massas , Tenofovir/química , Antirretrovirais/química , Benzoxazinas/química , Cromatografia Líquida de Alta Pressão , Interações de Medicamentos , Emtricitabina/análogos & derivados , Estrutura Molecular , Comprimidos/química , Tenofovir/análogos & derivados
14.
J Pharm Biomed Anal ; 122: 16-20, 2016 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-26829517

RESUMO

This communication describes the application of an existing intracellular methodology to the quantitation of tenofovir-diphosphate (TFV-DP) and emtricitabine-triphosphate (FTC-TP) from erythrocytes using dried blood spots (DBS). Concentrations were determined from a 3mm DBS punch extracted into a 70:30 methanol:water solution (lysed cellular matrix). This extraction solution was then subjected to a previously validated analytical procedure for lysed cellular matrix. Experiments for DBS validation used replicate samples from study participants to demonstrate acceptable reproducibility with spot volumes ranging from 10-50 µL and punch location either from the edge or center of the spot. Analysis of paired DBS with purified red blood cells showed that a 3mm DBS punch contained an average of 11.9 million cells for the observed hematocrit range of the participants (35-50%). Numerous stability tests were completed showing that whole blood in an EDTA vacutainer could sit for 24h at room temperature prior to spotting, and DBS could remain at room temperature for up to five days including shipment at ambient using 2-days delivery. DBS stability in storage was acceptable up to 18 months at -20°C or -80°C and DBS could undergo 4 Freeze/Thaw cycles. The described method was applied to HIV prophylaxis studies, demonstrating powerful associations with HIV acquisition through its ability to discriminate gradients of adherence.


Assuntos
Adenina/análogos & derivados , Bioensaio/métodos , Teste em Amostras de Sangue Seco/métodos , Emtricitabina/química , Eritrócitos/química , Organofosfatos/química , Polifosfatos/química , Adenina/química , Infecções por HIV/sangue , Hematócrito/métodos , Humanos , Adesão à Medicação , Metanol/química , Reprodutibilidade dos Testes , Soluções/química , Temperatura Ambiente , Água/química
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