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1.
Arzneimittelforschung ; 57(7): 462-6, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17803059

RESUMO

Two different tablets containing amlodipine besylate (CAS 111470-99-6) (Vazkor 10 mg tablet as test preparation and 10 mg tablet of the originator product as reference preparation) were investigated in 18 healthy male volunteers in order to compare the bioavailability and prove the bioequivalence between both treatments after oral single dose administration. The study was performed according to an open-label, randomized, two-period cross-over design with a wash-out phase of 21 days. Blood samples for pharmacokinetic profiling were taken up to 144 h post-dose, and amlodipine plasma concentrations were determined with a validated LC-MS/MS method. Maximum plasma concentrations (Cmax) of 6,183.7 pg/ml (test) and 5,366.7 pg/ml (reference) were achieved. Areas under the plasma concentration-time curve (AUC(0-infinity)) of 267,231.0 pg x h/ml (test) and 266,061.7 ng x h/ml (reference) were calculated. The median tmax was 5.6 h (test) and 6.1 h (reference). Plasma elimination half-lives (t 1/2) were 46.46 h (test) and 45.34 h (reference). Both primary target parameters AUC(0-infinity) and Cmax were tested parametrically by analysis of variance (ANOVA); 90% confidence intervals were between 93.20%-107.16% (AUC(0-infinity) and 103.36%-123.13% (Cmax). Bioequivalence between test and reference preparation was demonstrated since for both parameters AUC and Cmax the 90% confidence intervals of the T/R-ratios of logarithmically transformed data were in the generally accepted range of 80%-125%.


Assuntos
Anlodipino/farmacocinética , Bloqueadores dos Canais de Cálcio/farmacocinética , Adulto , Anlodipino/administração & dosagem , Anlodipino/efeitos adversos , Área Sob a Curva , Disponibilidade Biológica , Bloqueadores dos Canais de Cálcio/administração & dosagem , Bloqueadores dos Canais de Cálcio/efeitos adversos , Calibragem , Química Farmacêutica , Cromatografia Líquida de Alta Pressão , Estudos Cross-Over , Método Duplo-Cego , Meia-Vida , Humanos , Masculino , Controle de Qualidade
2.
Arzneimittelforschung ; 57(4): 227-31, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17515293

RESUMO

The aim of the present study was to compare the bioavailability of amoxicillin (CAS 26787-78-0) from two different amoxicillin tablets (Demoksil 1 g tablet as test preparation and 1 g tablet of the originator product as reference preparation). The study was conducted according to an open-label, randomised two-period cross-over design with a wash-out phase of 4-7 days. Blood samples for pharmacokinetic profiling were taken up to 10 h post-dose, and amoxicillin plasma concentrations were determined with a validated LC-MS/ MS method. Maximum plasma concentrations (C(max)) of 13,296.4 ng/ml (test) and 12,797.7 ng/ml (reference) were achieved. Areas under the plasma concentration-time curve (AUC(0-infinity)) of 39,556.7 ng x h/ml (test) and 38,599.1 ng x h/ml (reference) were calculated. The median t(max) was 1.62 h (test) and 1.54 h (reference). Plasma elimination half-lives (t(1/2)) of 1.64 h (test) and 1.65 h (reference) were determined. Both primary target parameters, AUC(0-infinity) and C(max) were tested parametrically by analysis of variance (ANOVA) and the 90% confidence intervals were between 96.76%-108.46% (AUC(0-infinity)) and 97.80%-111.98% (C(max)). Bioequivalence between test and reference preparation was demonstrated since for both parameters, AUC and C(max) the 90% confidence intervals of the T/R-ratios of logarithmically transformed data were in the generally accepted range of 80%-125%.


Assuntos
Amoxicilina/farmacocinética , Antibacterianos/farmacocinética , Adolescente , Adulto , Amoxicilina/administração & dosagem , Antibacterianos/administração & dosagem , Área Sob a Curva , Calibragem , Química Farmacêutica , Estudos Cross-Over , Método Duplo-Cego , Feminino , Meia-Vida , Humanos , Masculino , Controle de Qualidade , Espectrometria de Massas por Ionização por Electrospray , Equivalência Terapêutica
3.
Arzneimittelforschung ; 57(4): 232-7, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17515294

RESUMO

Sultamicillin (CAS 76497-13-7) is a prodrug combination of ampicillin (CAS 69-53-4) and sulbactam (CAS 68373-14-8), with the antibiotic ampicillin and the beta-lactamase inhibitor sulbactam chemically linked as double ester. The present study was performed to investigate the relative bioavailability and to assess the bioequivalence of two different sultamicillin suspensions (Devasid 250 mg/5 ml as test preparation and 375 mg/7.5 ml of the originator product as reference preparation). Twenty-four healthy male volunteers received equal doses of the sultamicillin preparations according to an open, randomised, single-dose, two-period cross-over design with a wash-out phase of 7 days. Blood samples for pharmacokinetic profiling were taken up to 8 h post-dose, and ampicillin and sulbactam plasma concentrations were determined with a validated LC-MS/MS method. Maximum plasma concentrations (C(max)) of 11,267.4 ng/ml (ampicillin, test), 10,864.4 ng/ml (ampicillin, reference), 6,360.6 ng/ml (sulbactam, test and 6,410.7 ng/ml (sulbactam, reference) were achieved. Areas under the plasma concentration-time curve (AUC(0-infinity) of 17,512.9 ng x h/ml (ampicillin, test), 18,388.0 ng x h/ml (ampicillin, reference), 10,971.7 ng ng x h/ml (sulbactam, test) and 11,181.2 ng x h/ml (sulbactam, reference) were calculated. The median t(max) was 0.69 h (ampicillin, test), 0.85 h (ampicillin, reference), 0.72 h (sulbactam, Devasid) and 0.83 h (sulbactam, reference). Plasma elimination half-lives (t(1/2)) of 1.04 h (ampicillin, test), 1.03 h (ampicillin, reference), 1.26 h (sulbactam, Devasid) and 1.00 h (sulbactam, reference) were determined. Both primary target parameters AUC(0-infinity) and C(max) of ampicillin and sulbactam were tested parametrically by analysis of variance (ANOVA) and the 90% confidence intervals were between 84.58%-117.80% (AUC(0-infinity), ampicillin), 92.37%-119.93% (C(max), ampicillin), 85.81%-120.50% (AUC(0-infinity), sulbactam) and 88.41%-117.57% (C(max), sulbactam). Bioequivalence between test and reference preparation was demonstrated since for both parameters AUC and C(max) the 90% confidence intervals of the T/R-ratios of logarithmically transformed data were in the generally accepted range of 80%-125%.


Assuntos
Antibacterianos/farmacocinética , Adulto , Ampicilina/administração & dosagem , Ampicilina/sangue , Ampicilina/farmacocinética , Antibacterianos/administração & dosagem , Calibragem , Estudos Cross-Over , Humanos , Masculino , Controle de Qualidade , Espectrometria de Massas por Ionização por Electrospray , Sulbactam/administração & dosagem , Sulbactam/farmacocinética , Suspensões , Equivalência Terapêutica
4.
Photochem Photobiol Sci ; 6(2): 145-51, 2007 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-17277837

RESUMO

A promising clinical application of 5-aminolevulinic acid (5-ALA)-induced protoporphyrin IX (PP IX) is fluorescence detection and photodynamic treatment of residual tumour tissue during surgical resection of high grade malignant glioma. U373 MG human glioblastoma cells were used as a model system to study the relation between intracellular location and photodynamic efficacy of 5-ALA-induced PP IX in more detail. Therefore, ultra-sensitive fluorescence microscopy, using either optical excitation of whole cells or selective excitation of the plasma membrane by an evanescent electromagnetic field, was combined with quantitative measurements of intracellular porphyrin amount and phototoxicity. Glioblastoma cells accumulated PP IX to a moderate extent as compared to T47D breast cancer cells (high accumulation) or OV2774 ovarian cancer cells (low accumulation). Although photodynamic inactivation of the different cell lines (decreasing in the order T47D > U373 MG > OV2774) seemed to be directly related to PP IX accumulation, examination of the data in more detail revealed that photodynamic efficacy per photosensitizer molecule (PE) was about two times higher in glioblastoma and ovarian cancer cells as compared to breast cancer cells. The different photodynamic efficacy of PP IX was related to the different intracellular location. In contrast to breast cancer cells where PP IX fluorescence was localized in small granules, PP IX fluorescence in glioblastoma cells and ovarian cancer cells originated mainly from cellular membranes. Thus, the intracellular location of PP IX in a predominantly lipophilic environment, characterized by a comparably high photostability (probed by photobleaching and photoproduct formation) and a lower degree of porphyrin aggregation (probed previously by fluorescence decay kinetics), seems to be the key factor for high photodynamic efficacy of 5-ALA-induced PP IX. In the case of OV2774 ovarian cancer cells, however, a low PP IX accumulation limited cell inactivation upon irradiation, whereas the results obtained for glioblastoma cells are encouraging to develop PDT to an additional therapeutic option for the treatment of tumour margins in patients who underwent fluorescence-guided resection of high grade malignant glioma after 5-ALA administration.


Assuntos
Ácido Aminolevulínico/farmacologia , Glioblastoma/metabolismo , Fármacos Fotossensibilizantes/farmacologia , Protoporfirinas/metabolismo , Ácido Aminolevulínico/química , Ácido Aminolevulínico/efeitos da radiação , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta à Radiação , Humanos , Lasers , Fotoquímica , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/efeitos da radiação , Protoporfirinas/química , Protoporfirinas/efeitos da radiação , Sensibilidade e Especificidade , Espectrometria de Fluorescência/instrumentação , Espectrometria de Fluorescência/métodos , Relação Estrutura-Atividade , Células Tumorais Cultivadas
5.
J Biomed Opt ; 11(3): 34011, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16822061

RESUMO

A novel setup for fluorescence measurements of surfaces of biological samples, in particular the plasma membrane of living cells, is described. The method is based on splitting of a laser beam and multiple total internal reflections (TIR) within the bottom of a microtiter plate (cell substrate), such that up to 96 individual samples are illuminated simultaneously by an evanescent electromagnetic field. Main prerequisites are an appropriate thickness and a high transmission of the glass bottom, which is attached to the 96-well cell culture plate by a noncytotoxic adhesive. Glass rods of rectangular cross sections are optically coupled to this bottom for TIR illumination. Fluorescence arising from the plasma membrane of living cells is detected simultaneously from all samples using an integrating charge-coupled device (CCD) camera. The TIR fluorescence reader is validated using cultivated cells incubated with different fluorescent markers, as well as stably transfected cells expressing a fluorescent membrane-associating protein. In addition, particularly with regard to potential pharmaceutical applications, the kinetics of the intracellular translocation of a fluorescent protein kinase c fusion protein upon stimulation of the cells is determined.


Assuntos
Técnicas de Cultura de Células/instrumentação , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Proteínas de Membrana/metabolismo , Análise em Microsséries/instrumentação , Microscopia de Fluorescência/instrumentação , Refratometria/instrumentação , Animais , Técnicas de Cultura de Células/métodos , Desenho de Equipamento , Análise de Falha de Equipamento , Humanos , Cinética , Taxa de Depuração Metabólica , Análise em Microsséries/métodos , Microscopia de Fluorescência/métodos , Refratometria/métodos , Células Tumorais Cultivadas
6.
J Fluoresc ; 14(5): 649-54, 2004 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-15617271

RESUMO

Lifetime images of autofluorescence of cultivated endothelial cells were recorded using a novel picosecond laser diode in the near ultraviolet range (375 nm). In contrast to existing picosecond light sources this wavelength permits efficient excitation of the free and protein bound coenzyme NADH with fluorescence lifetimes of 0.4-0.5 ns and 2.0-2.5 ns, respectively. The effective fluorescence lifetime tau(eff) (depending on both lifetimes) was homogenously distributed over the cells with some shortening in the perinuclear region, possibly close to mitochondria. A slight decrease of tau(eff) was observed after inhibition of the mitochondrial respiratory chain, whereas a slight increase was observed after inhibition of the glycolytic pathway, thus indicating variations of the ratio of free and protein bound NADH. Although present applications are still limited by their low pulse energy (< or = 5 pJ), uv picosecond laser diodes have a large potential in high resolution fluorescence microscopy and fluorescence lifetime endoscopy.


Assuntos
Diagnóstico por Imagem/métodos , Células Endoteliais/metabolismo , Lasers , Microscopia de Fluorescência/métodos , Raios Ultravioleta , Trifosfato de Adenosina/metabolismo , Animais , Bovinos , Linhagem Celular , Desoxiglucose/farmacologia , Diagnóstico por Imagem/instrumentação , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Microscopia de Fluorescência/instrumentação , NAD/química , NAD/metabolismo , NADP/química , NADP/metabolismo , Rotenona/farmacologia , Espectrometria de Fluorescência
7.
Photochem Photobiol Sci ; 3(8): 817-22, 2004 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15295640

RESUMO

Membranes of living cells are characterized by laser-assisted fluorescence microscopy, in particular a combination of microspectrofluorometry, total internal reflection fluorescence microscopy (TIRFM), fluorescence lifetime imaging (FLIM) and Forster resonance energy transfer (FRET) spectroscopy. The generalized polarization (GP, characterizing a spectral shift which depends on the phase of membrane lipids) as well as the effective fluorescence lifetime (tau(eff)) of the membrane marker laurdan were revealed to be appropriate parameters for membrane stiffness and fluidity. GP decreased with temperature, but increased during cell growth and was always higher for the plasma membrane than for intracellular membranes. Microdomains of different fluorescence lifetimes tau(eff) were observed at temperatures above 30 degree C and disappeared during cell aging. Non-radiative energy transfer was used to detect laurdan selectively in close proximity to a molecular acceptor (DiI) and may present a possibility for measuring membrane dynamics in specific microenvironments.


Assuntos
Membrana Celular/química , Fluidez de Membrana , Microscopia de Fluorescência/métodos , Animais , Células CHO , Divisão Celular , Cricetinae , Meia-Vida , Lasers , Microdomínios da Membrana , Microscopia de Fluorescência/instrumentação , Espectrometria de Fluorescência , Temperatura
8.
Bioconjug Chem ; 15(2): 359-65, 2004.
Artigo em Inglês | MEDLINE | ID: mdl-15025532

RESUMO

The present paper describes the chemical synthesis and in vitro characterization of a novel, high-affinity, fluorescent progesterone receptor (PR) antagonist. The three-step synthesis was carried out starting from mifepristone. After demethylation with calcium oxide, the methylamino group was alkylated with 6-bromohexanol, and the resulting compound was reacted with fluorescein 5-isothiocyanate, yielding the fluorescein-mifepristone conjugate. Interaction of the conjugate as well as of its precursors with PR was determined in cell culture (alkaline phosphatase assay and transactivation assay). Antiprogestagenic activity of the intermediates were comparable to that of the parent compound. Even after attachment of the bulky fluorescein moiety, considerable antiprogestagenic activity was maintained. Microscopic studies revealed that fluorescence of the conjugate was almost confined to the nuclei of steroid hormone receptor-positive cells, whereas the nuclei of steroid hormone receptor-negative cells remained unstained. To our knowledge, this is the first report on a fluorescent ligand for PR suitable for studies in living cells. It is proposed that the present fluorescent PR antagonist might serve as a lead compound for the development of contrast agents for PR imaging, e.g., by near-infrared optical imaging.


Assuntos
Fluoresceína/síntese química , Mifepristona/síntese química , Receptores de Progesterona/antagonistas & inibidores , Linhagem Celular Tumoral , Fluoresceína/metabolismo , Humanos , Microscopia de Fluorescência , Mifepristona/metabolismo , Receptores de Progesterona/metabolismo , Frações Subcelulares/química , Frações Subcelulares/metabolismo
9.
Photochem Photobiol Sci ; 3(1): 127-31, 2004 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-14768629

RESUMO

A novel setup for fluorescence intensity and lifetime imaging (FLIM) of living cells is reported. Time-resolving techniques are combined with total internal reflection fluorescence microscopy (TIRFM), which permits optical excitation of either plasma membranes or whole cells depending on whether the angle of incidence of the excitation light is greater or smaller than the critical angle for total internal reflection. The method is applied to BKEz-7 endothelial cells incubated with various concentrations of the well established mitochondrial marker rhodamine 123(R123). Measurements show that only at low concentrations this dye is mainly located within the mitochondria, whereas at higher concentrations an accumulation within the plasma membrane occurs as well. Concomitantly, fluorescence quenching in the mitochondria is observed at high concentrations, probably due to aggregation of the R123 molecules. Therefore, for diagnostic applications the concentration of R123 in the incubation medium should not be above 25 microM.


Assuntos
Endotélio Vascular/citologia , Células Epiteliais/citologia , Corantes Fluorescentes , Microscopia de Fluorescência/métodos , Rodamina 123 , Animais , Aorta , Bovinos , Células Cultivadas , Microscopia de Fluorescência/instrumentação
10.
J Biomed Opt ; 7(3): 410-6, 2002 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12175291

RESUMO

The plasma membrane of Chinese hamster ovary cells was made permeable using the focused beam of an argon ion laser (488 nm) and phenol red as a light absorbing dye. Small circular dark spots on the cell surface appeared immediately after laser irradiation and disappeared within about 5 min. They were related to transient changes in membrane properties, which could be visualized using the fluorescent marker laurdan, and were probably due to a local increase in temperature. According to a colony forming assay, cell viability was maintained by using light doses up to 2.5 MJ/cm(2) applied for 1 s. In addition to measurements of the efflux of the cytoplasmic marker calcein, cell transfection using a green fluorescent protein (GFP) coding plasmid was studied: brightly fluorescent GFP with an emission maximum around 510 nm was observed within part of the cells after 24 h. The transfection rates after laser irradiation were around 30% for younger subcultures and less than 10% for aging cells. This may be due to age dependent changes in the phase transition of membrane lipids from gel phase to liquid crystalline phase. High transfection rates, visual control and universality towards various cell lines are possibly the main advantages of laser-assisted optoporation in comparison with presently existing methods of cell transfection.


Assuntos
Permeabilidade da Membrana Celular , Animais , Células CHO , Sobrevivência Celular , Cricetinae , Proteínas de Fluorescência Verde , Lasers , Proteínas Luminescentes/genética , Microscopia de Fluorescência , Óptica e Fotônica , Plasmídeos/genética , Proteínas Recombinantes/genética , Espectrometria de Fluorescência , Transfecção
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