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In this review article, we are going to present an overview on possible applications of light-addressable electrodes (LAE) as actuator/manipulation devices besides classical electrode structures. For LAEs, the electrode material consists of a semiconductor. Illumination with a light source with the appropiate wavelength leads to the generation of electron-hole pairs which can be utilized for further photoelectrochemical reaction. Due to recent progress in light-projection technologies, highly dynamic and flexible illumination patterns can be generated, opening new possibilities for light-addressable electrodes. A short introduction on semiconductor-electrolyte interfaces with light stimulation is given together with electrode-design approaches. Towards applications, the stimulation of cells with different electrode materials and fabrication designs is explained, followed by analyte-manipulation strategies and spatially resolved photoelectrochemical deposition of different material types.
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Técnicas Biosensibles , Técnicas Electroquímicas , Luz , Semiconductores , Técnicas Biosensibles/instrumentación , Técnicas Biosensibles/métodos , Materiales Biocompatibles Revestidos/síntesis química , Materiales Biocompatibles Revestidos/química , Materiales Biocompatibles Revestidos/provisión & distribución , Materiales Biocompatibles Revestidos/uso terapéutico , Técnicas Electroquímicas/instrumentación , Técnicas Electroquímicas/métodos , Electrodos , Galvanoplastia/instrumentación , Galvanoplastia/métodos , Diseño de Equipo , Humanos , Iluminación/instrumentación , Iluminación/métodos , Microtecnología/métodosRESUMEN
Monitoring the cellular metabolism of bacteria in (bio)fermentation processes is crucial to control and steer them, and to prevent undesired disturbances linked to metabolically inactive microorganisms. In this context, cell-based biosensors can play an important role to improve the quality and increase the yield of such processes. This work describes the simultaneous analysis of the metabolic behavior of three different types of bacteria by means of a differential light-addressable potentiometric sensor (LAPS) set-up. The study includes Lactobacillus brevis, Corynebacterium glutamicum, and Escherichia coli, which are often applied in fermentation processes in bioreactors. Differential measurements were carried out to compensate undesirable influences such as sensor signal drift, and pH value variation during the measurements. Furthermore, calibration curves of the cellular metabolism were established as a function of the glucose concentration or cell number variation with all three model microorganisms. In this context, simultaneous (bio)sensing with the multi-organism LAPS-based set-up can open new possibilities for a cost-effective, rapid detection of the extracellular acidification of bacteria on a single sensor chip. It can be applied to evaluate the metabolic response of bacteria populations in a (bio)fermentation process, for instance, in the biogas fermentation process.
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Bacterias/metabolismo , Técnicas Biosensibles , Luz , Potenciometría , Bacterias/efectos de los fármacos , Bacterias/crecimiento & desarrollo , Calibración , Recuento de Colonia Microbiana , Electrodos , Glucosa/farmacologíaRESUMEN
The miniaturization of microfluidic systems usually comes at the cost of more difficult integration of sensors and actuators inside the channel. As an alternative, this work demonstrates the embedding of semiconductor-based sensor and actuator technologies that can be spatially and temporally controlled from outside the channel using light. The first element is a light-addressable potentiometric sensor, consisting of an Al/Si/SiO2/Si3N4 structure, that can measure pH changes at the Si3N4/electrolyte interface. The pH value is a crucial factor in biological and chemical systems, and besides measuring, it is often important to bring the system out of equilibrium or to adjust and control precisely the surrounding medium. This can be done photoelectrocatalytically by utilizing light-addressable electrodes. These consist of a glass/SnO2:F/TiO2 structure, whereby direct charge transfer between the TiO2 and the electrolyte leads to a pH change upon irradiation. To complement the advantages of both, we integrated a light-addressable sensor with a pH sensitivity of 41.5 mV·pH-1 and a light-addressable electrode into a microfluidic setup. Here, we demonstrated a simultaneous operation with the ability to generate and record pH gradients inside a channel under static and dynamic flow conditions. The results show that dependent on the light-addressable electrode (LAE)-illumination conditions, pH changes up to ΔpH of 2.75 and of 3.52 under static and dynamic conditions, respectively, were spatially monitored by the light-addressable potentiometric sensor. After flushing with fresh buffer solution, the pH returned to its initial value. Depending on the LAE illumination, pH gradients with a maximum pH change of ΔpH of 1.42 were tailored perpendicular to the flow direction. In a final experiment, synchronous LAE illumination led to a stepwise increase in the pH inside the channel.
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Técnicas Biosensibles , Luz , Dióxido de Silicio , Técnicas Biosensibles/métodos , Electrólitos , Dispositivos Laboratorio en un Chip , Concentración de Iones de HidrógenoRESUMEN
BACKGROUND: Cladribine tablets for the treatment of relapsing multiple sclerosis (RMS) are administered in two pulsed treatment courses in two consecutive years, totalling a maximum of 20 treatment days. Here we present data collected shortly after the launch of cladribine tablets, focusing on the patient's perspective. The objective was to investigate patients' perceived effectiveness, tolerability, and convenience, as well as global satisfaction of and with cladribine tablets. METHODS: CLEVER was a non-interventional multicentre study conducted in Germany from 12/2017 to 7/2020. Adult patients with RMS initiating therapy with cladribine tablets were included. Observation time per patient was 24 weeks, comprising 3 visits (baseline, week 4 and 24). The primary endpoint was overall treatment satisfaction at week 24, assessed by the Treatment Satisfaction Questionnaire for Medication 14 items (TSQM 1.4). Subgroup analyses included stratification by prior treatment. RESULTS: In total, 491 patients (69.2 % female; mean (±SD) age 40.3 (±11.5) years, 85.1 % pre-treated, median EDSS 2.5) initiated therapy with cladribine tablets and were included in the analysis. At week 24, the mean (±SD) global TSQM satisfaction score was 75.6 (±19.0). For patients switching from either injectables or oral medication, the change in therapy satisfaction from baseline to week 24 was positive in all TSQM domains with clinically meaningful effect sizes in the global satisfaction and side effects domains. Most patients (85.5 %) remained relapse-free over 24 weeks. Out of 491 patients, 187 (38.1 %) experienced at least one adverse event and 8 patients (1.6 %) one serious adverse event. CONCLUSION: Treatment satisfaction with cladribine tablets was high. The switch from prior injectables or oral medication translated into increased treatment satisfaction at week 24 with clinically meaningful effects in the global satisfaction and side effects domains. Effectiveness and safety were consistent with results from clinical studies.
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INTRODUCTION: Treatment adherence is a critical success factor in the disease-modifying therapy (DMT) of multiple sclerosis (MS). The REBISTART study prospectively evaluated adherence in patients using components of a patient support program (PSP). METHODS: The 12-month non-interventional multicenter study examined the real-world adherence to subcutaneously (sc) injected interferon beta-1a (Rebif®). Patient-assessed adherence was measured by a visual analog scale (VAS) and the Morisky Medication Adherence Scale (MMAS). Objective adherence data were obtained by readouts from the RebiSmart® injection device. RESULTS: Of 333 patients, 70.9% used the nursing service as the core component of the PSP. Self-assessed VAS-based adherence was stable over time at 94.0-96.3%. Similarly, MMAS score (maximum 4) was 3.8-3.9 at all visits, also reflecting high self-assessed adherence. In 269 patients using the RebiSmart® injection device, mean readout-based objective adherence was similarly high (93.0-98.4% throughout visits). At last available visit, VAS-based adherence was independent of participation in the PSP nursing service (93.1% with participation versus 91.7% without it). Adherence was also independent of injection method or disease-related measures, including fatigue, depression, cognition, and quality of life. The most frequent reason for the premature discontinuations (38.7% of patients) was "change of treatment" (10.0%). DISCUSSION: We suggest that subgroups that may specifically benefit from PSP include patients who live alone, use multiple comedications, and are affected by cognitive impairment, depression, and/or fatigue. Further studies should investigate the potential usefulness of PSPs in these populations. CONCLUSIONS: Very high adherence rates independent of the PSP nursing service over 1 year of treatment indicate that IFN beta-1a sc is an easy-to-use and well-tolerated disease-modifying drug. TRIAL REGISTRATION NUMBER: Vfa.de: No. 892. https://www.vfa.de/de/arzneimittel-forschung/datenbanken-zu-arzneimitteln/nisdb/nis-details/_892 .
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INTRODUCTION: Interferon beta (IFN beta) preparations are an established group of drugs used for immunomodulation in patients with multiple sclerosis (MS). Subcutaneously (sc) applied interferon beta-1a (IFN beta-1a sc) has been in continuous clinical use for 25 years as a disease-modifying treatment. AREAS COVERED: Based on data published since 2018, we discuss recent insights from analyses of the pivotal trial PRISMS and its long-term extension as well as from newer randomized studies with IFN beta-1a sc as the reference treatment, the use of IFN beta-1a sc across the patient life span and as a bridging therapy, recent data regarding the mechanisms of action, and potential benefits of IFN beta-1a sc regarding vaccine responses. EXPERT OPINION: IFN beta-1a sc paved the way to effective immunomodulatory treatment of MS, enabled meaningful insights into the disease process, and remains a valid therapeutic option in selected vulnerable MS patient groups.
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Electrochemical cell-based biosensors have attracted increasing interest within the last 15 years, with a large number of reports generally dealing with the sensors' sensitivity, selectivity, stability, signal-to-noise ratio, spatiotemporal resolution, etc. However, only a few of them are now available as commercial products. In this review, technological advances, current challenges, and opportunities of electrochemical cell-based biosensors are presented. The article encompasses emerging studies on cell-based biological field-effect devices, cell-based impedimetric sensors, and cell-based microelectrode arrays, mainly focusing on the last five years (from 2016 to mid-2021). In addition, special attention lies in recent progress at the single-cellular level, including intracellular monitoring with high spatiotemporal resolution as well as integration into microfluidics for lab-ona- chip applications. Moreover, a comprehensive discussion on challenges and future perspectives will address the future potential of electrochemical cell-based biosensors.
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Técnicas Biosensibles , Técnicas ElectroquímicasRESUMEN
INTRODUCTION: Consistent treatment adherence is an important determinant of durable response in multiple sclerosis (MS). Published data indicate that adherence to > 80% of prescribed doses may be considered optimal. Feedback of electronic application monitoring data to patients has been considered a promising means to support high adherence. METHODS: The 2-year prospective non-interventional study REBIFLECT conducted at outpatient neurological centers (731 patients at 134 study sites in Germany) investigated whether treatment adherence to subcutaneous (sc) interferon beta-1 injection during a 1-year period is enhanced by regular physician-patient talks reflecting dosing data recorded by the application device in the context of clinical data or disease parameters. Qualitative adherence was defined as number of weeks with properly distributed injections per total number of weeks with prescribed injections. Quantitative adherence was defined as number of administered injections per prescribed injections. RESULTS: Overall median qualitative adherence was 90.5%. Approximately 70% of patients with adherence data available in the respective periods had a qualitative treatment adherence of 80-100%. With a mean of 97.9% quantitative adherence was very high and remained stable in the 2-year observation period. The stability of this effect is demonstrated by the subgroup with just one reflection talk (≥ 100%) and only a slight decrease in the subgroup with more than five reflection talks (97.9%). CONCLUSION: Treatment adherence with the Rebismart® device was generally very high, consistent with other non-interventional studies. The first reflection talk supported by RebiSmart® induces excellent adherence, stabilized by repetition. Reflection to patients of subcutaneous interferon beta-1a treatment monitored by RebiSmart® is recommended to ensure prolonged strong treatment adherence.
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Esclerosis Múltiple Recurrente-Remitente , Esclerosis Múltiple , Trastornos Relacionados con Sustancias , Adyuvantes Inmunológicos , Retroalimentación , Humanos , Inyecciones Subcutáneas , Interferón beta-1a/uso terapéutico , Esclerosis Múltiple/tratamiento farmacológico , Esclerosis Múltiple Recurrente-Remitente/tratamiento farmacológico , Estudios ProspectivosRESUMEN
In this study, an online multi-sensing platform was engineered to simultaneously evaluate various process parameters of food package sterilization using gaseous hydrogen peroxide (H2O2). The platform enabled the validation of critical aseptic parameters. In parallel, one series of microbiological count reduction tests was performed using highly resistant spores of B. atrophaeus DSM 675 to act as the reference method for sterility validation. By means of the multi-sensing platform together with microbiological tests, we examined sterilization process parameters to define the most effective conditions with regards to the highest spore kill rate necessary for aseptic packaging. As these parameters are mutually associated, a correlation between different factors was elaborated. The resulting correlation indicated the need for specific conditions regarding the applied H2O2 gas temperature, the gas flow and concentration, the relative humidity and the exposure time. Finally, the novel multi-sensing platform together with the mobile electronic readout setup allowed for the online and on-site monitoring of the sterilization process, selecting the best conditions for sterility and, at the same time, reducing the use of the time-consuming and costly microbiological tests that are currently used in the food package industry.
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An efficient preservation of a cell-based biosensor chip to achieve a ready-to-use on-site system is still very challenging as the chip contains a living component such as adherent mammalian cells. Herein, we propose a strategy called on-sensor cryopreservation (OSC), which enables the adherent cells to be preserved by freezing (-80 °C) on a biosensor surface, such as the light-addressable potentiometric sensor (LAPS). Adherent cells on rigid surfaces are prone to cryo-injury; thus, the surface was modified to enhance the cell recovery for OSC. It relies on i) the integration of elastic electrospun fibers composed of polyethylene vinyl acetate (PEVA), which has a high thermal expansion coefficient and low glass-transition temperature, and ii) the treatment with O2 plasma. The modified sensor is integrated into a microfluidic chip system not only to decrease the thermal mass, which is critical for fast thawing, but also to provide a precisely controlled micro-environment. This novel cryo-chip system is effective for keeping cells viable during OSC. As a proof-of-concept for the applicability of a ready-to-use format, the extracellular acidification of cancer cells (CHO-K1) was evaluated by differential LAPS measurements after thawing. Results show, for the first time, that the OSC strategy using the cryo-chip allows label-free and quantitative measurements directly after thawing, which eliminates additional post-thaw culturing steps. The freezing of the chips containing cells at the manufacturing stage and sending them via a cold-chain transport could open up a new possibility for a ready-to-use on-site system.
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Técnicas Biosensibles , Animales , Criopreservación , Congelación , Polímeros , PotenciometríaRESUMEN
The feasibility of light-addressed detection and manipulation of pH gradients inside an electrochemical microfluidic cell was studied. Local pH changes, induced by a light-addressable electrode (LAE), were detected using a light-addressable potentiometric sensor (LAPS) with different measurement modes representing an actuator-sensor system. Biosensor functionality was examined depending on locally induced pH gradients with the help of the model enzyme penicillinase, which had been immobilized in the microfluidic channel. The surface morphology of the LAE and enzyme-functionalized LAPS was studied by scanning electron microscopy. Furthermore, the penicillin sensitivity of the LAPS inside the microfluidic channel was determined with regard to the analyte's pH influence on the enzymatic reaction rate. In a final experiment, the LAE-controlled pH inhibition of the enzyme activity was monitored by the LAPS.
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Técnicas Biosensibles , Microfluídica , Penicilinasa/análisis , Potenciometría , Fuerza Protón-MotrizRESUMEN
α-hydroxy ketones (HK) and 1,2-diols are important building blocks for fine chemical synthesis. Here, we describe the R-selective 2,3-butanediol dehydrogenase from B. clausii DSM 8716T (BcBDH) that belongs to the metal-dependent medium chain dehydrogenases/reductases family (MDR) and catalyzes the selective asymmetric reduction of prochiral 1,2-diketones to the corresponding HK and, in some cases, the reduction of the same to the corresponding 1,2-diols. Aliphatic diketones, like 2,3-pentanedione, 2,3-hexanedione, 5-methyl-2,3-hexanedione, 3,4-hexanedione and 2,3-heptanedione are well transformed. In addition, surprisingly alkyl phenyl dicarbonyls, like 2-hydroxy-1-phenylpropan-1-one and phenylglyoxal are accepted, whereas their derivatives with two phenyl groups are not substrates. Supplementation of Mn2+ (1 mM) increases BcBDH's activity in biotransformations. Furthermore, the biocatalytic reduction of 5-methyl-2,3-hexanedione to mainly 5-methyl-3-hydroxy-2-hexanone with only small amounts of 5-methyl-2-hydroxy-3-hexanone within an enzyme membrane reactor is demonstrated.
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A combined calorimetric gas- and spore-based biosensor array is presented in this work to monitor and evaluate the sterilization efficacy of gaseous hydrogen peroxide in aseptic filling machines. H2O2 has been successfully measured under industrial conditions. Furthermore, the effect of H2O2 on three different spore strains , namely Bacillus atrophaeus, Bacillus subtilis and Geobacillus stearothermophilus, has been investigated by means of SEM, AFM and impedimetric measurements. In addition, the sterilization efficacy of a spore-based biosensor and the functioning principle are addressed and discussed: the sensor array is convenient to be used in aseptic food industry to guarantee sterile packages.
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Técnicas Biosensibles , Calorimetría , Peróxido de Hidrógeno/aislamiento & purificación , Esporas Bacterianas/efectos de los fármacos , Bacillus/efectos de los fármacos , Bacillus/crecimiento & desarrollo , Bacillus subtilis/efectos de los fármacos , Bacillus subtilis/crecimiento & desarrollo , Desinfectantes/química , Desinfectantes/aislamiento & purificación , Gases/química , Gases/aislamiento & purificación , Geobacillus stearothermophilus/efectos de los fármacos , Geobacillus stearothermophilus/crecimiento & desarrollo , Humanos , Peróxido de Hidrógeno/farmacología , Infertilidad , Esporas Bacterianas/crecimiento & desarrollo , EsterilizaciónRESUMEN
Applying biosensors for evaluation of the extracellular acidification of microorganisms in various biotechnological fermentation processes is on demand. An early stage detection of disturbances in the production line would avoid costly interventions related to metabolically inactive microorganisms. Furthermore, the determination of the number of living cells through cell plating procedure after cultivations is known as time- and material-consuming. In this work, a differential light-addressable potentiometric sensor (LAPS) system was developed to monitor the metabolic activity of Corynebacterium glutamicum (C. glutamicum ATCC13032) as typical microorganism in fermentation processes. In this context, the number of living cells in suspensions was directly determined utilizing the read-out principle of the LAPS system. The planar sensor surface of the LAPS design allows to fixate 3D-printed multi-chamber structures, which enables differential measurements. In this way, undesirable external influences such as pH variations of the medium and sensor signal drift can be compensated.
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Técnicas Biosensibles/métodos , Corynebacterium glutamicum/aislamiento & purificación , Potenciometría/métodos , Corynebacterium glutamicum/metabolismo , Medios de Cultivo/química , Fermentación , Concentración de Iones de Hidrógeno , LuzRESUMEN
INTRODUCTION: Patient adherence is a key determinant of treatment success in multiple sclerosis (MS). The RebiSmart® autoinjector facilitates patient self-injection of subcutaneous interferon ß-1a (sc IFN ß-1a) and allows quantitative measurement of adherence via its automated dosing log. We evaluated patient adherence and patient-reported cognitive and health-economic outcomes over 2 years in patients using RebiSmart®. METHODS: In this non-interventional, single-arm study, enrolled patients were 12-65 years of age, had relapsing-remitting MS or a single demyelinating event, and had been prescribed 44 or 22 µg sc IFN ß-1a. Quantitative adherence (proportion of scheduled injections administered) and qualitative adherence (proportion of weeks with treatment schedule correctly followed) were monitored over 2 years. Other end points included self-assessed adherence, patient-reported outcomes (fatigue, depression and quality of life), cognitive outcomes and health-economic outcomes. RESULTS: A total of 368 of 392 (93.9%) enrolled patients were analyzed. Mean quantitative adherence was 85.3% overall (months 1-24), 89.6% for months 1-12 and 83.3% for months 13-24. No major impact on quantitative adherence was observed for sex, age (< 37 years vs. ≥ 37 years), prior medication or participation in the patient support program RebiSTAR®. Mean qualitative adherence was 67.0% overall (months 1-24). Self-assessed adherence was reported as being higher than RebiSmart®-monitored adherence. There was a trend toward more MS-related visits to physicians among patients with high adherence. CONCLUSIONS: Patients using RebiSmart® demonstrated high adherence to treatment that was associated with a slight improvement in information processing speed and working memory and an overall tendency for more intensive self-management. FUNDING: Merck Serono GmbH, Germany, an affiliate of Merck KGaA, Darmstadt, Germany.
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Adyuvantes Inmunológicos/uso terapéutico , Interferón beta-1a/uso terapéutico , Esclerosis Múltiple/tratamiento farmacológico , Cooperación del Paciente/estadística & datos numéricos , Calidad de Vida , Adolescente , Adulto , Anciano , Femenino , Alemania , Humanos , Masculino , Persona de Mediana Edad , Esclerosis Múltiple Recurrente-Remitente/tratamiento farmacológico , Medición de Resultados Informados por el Paciente , Resultado del Tratamiento , Adulto JovenRESUMEN
INTRODUCTION: Although multiple sclerosis (MS) remains incurable, interferon beta (IFNß) has been at the forefront of treatment for many years. Different formulations of IFNß allow for different levels of exposure: low-dose/frequency with some agents, and high-dose/frequency with others. Areas covered: This review article discusses existing and emerging efficacy and safety data for IFNß in MS. Clinical evidence of IFNß efficacy has been generated and accumulated over many decades. During this time, key clinical trials have demonstrated the benefits of high-dose and/or high-frequency dosing of IFNß-1a or ß-1b, compared with lower levels of exposure, on outcome measures such as relapse rates, disability progression, disease progression and magnetic resonance imaging lesion outcomes. IFNß therapy is well tolerated and has one of the best characterized safety profiles of all first line therapies. The overall severity of adverse events (AEs) does not appear to be affected by different IFNß exposures. Typical AEs that patients may experience with IFNß are mild, reversible and manageable. Expert commentary: IFNß is one of the best characterized treatments for MS, with a large body of clinical and real-world evidence supporting the risk-benefit profile. High-dose/frequency regimens may provide better long-term outcomes.
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Inmunoterapia/métodos , Interferón beta/uso terapéutico , Esclerosis Múltiple/terapia , Ensayos Clínicos como Asunto , Cálculo de Dosificación de Drogas , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Testimonio de Experto , Humanos , Esclerosis Múltiple/inmunología , Medición de Riesgo , Resultado del TratamientoRESUMEN
A capacitive electrolyte-insulator-semiconductor (EIS) field-effect biosensor for acetoin detection has been presented for the first time. The EIS sensor consists of a layer structure of Al/p-Si/SiO2/Ta2O5/enzyme acetoin reductase. The enzyme, also referred to as butane-2,3-diol dehydrogenase from B. clausii DSM 8716T, has been recently characterized. The enzyme catalyzes the (R)-specific reduction of racemic acetoin to (R,R)- and meso-butane-2,3-diol, respectively. Two different enzyme immobilization strategies (cross-linking by using glutaraldehyde and adsorption) have been studied. Typical biosensor parameters such as optimal pH working range, sensitivity, hysteresis, linear concentration range and long-term stability have been examined by means of constant-capacitance (ConCap) mode measurements. Furthermore, preliminary experiments have been successfully carried out for the detection of acetoin in diluted white wine samples.
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Acetoína/aislamiento & purificación , Oxidorreductasas de Alcohol/química , Técnicas Biosensibles , Acetoína/química , Capacidad Eléctrica , Enzimas Inmovilizadas/química , Semiconductores , Silicio/química , Dióxido de Silicio/químicaRESUMEN
Extended release mini matrix tablets for 8-Prenylnaringenin (8-PN), an extremely poorly soluble weakly acidic drug, were developed by using polyvinylacetate/polyvinylpyrrolidone as matrix former. Mini matrix tablets were manufactured by direct compression or wet granulation technique. With conventional modified release formulations, the drug demonstrated pH-dependent release due to pH-dependent solubility of the drug substance (i.e., increasing solubility at higher pH-values). In order to achieve pH-independent drug release two classes of pH-modifying agents (water-soluble vs. water-insoluble) were studied with respect to their effect on the dissolution of 8-PN. Addition of water-soluble salts of weak acids (sodium carbonate and sodium citrate) failed in order to achieve pH-independent 8-PN release. In contrast, addition of water insoluble salts of a strong base (magnesium hydroxide and magnesium oxide) was found to maintain high pH-values within the mini matrix tablets during release of 8-PN at pH 1 over a period of 10 h. The micro-environmental conditions for the dissolution of the weakly acidic drug were kept almost constant, thus resulting in pH-independent drug release. Compound release from mini matrix tablets prepared by wet granulation was faster compared to the drug release from tablets prepared by direct compression.
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Preparaciones de Acción Retardada , Excipientes/química , Tecnología Farmacéutica , Fosfatos de Calcio/química , Carbonatos/química , Química Farmacéutica , Citratos/química , Flavanonas/química , Concentración de Iones de Hidrógeno , Cinética , Lactosa/química , Hidróxido de Magnesio/química , Óxido de Magnesio/química , Silicatos de Magnesio/química , Povidona/química , Citrato de Sodio , Solubilidad , Almidón/química , Comprimidos , Tecnología Farmacéutica/métodos , Agua/químicaRESUMEN
On the basis of Pluronic P104 as primary emulsifier and Lauroglycol 90 as amphiphilic oil phase, two nanoemulsion systems were developed with Pluronic((R)) L62 or L81 as secondary emulsifiers. The possible nanoemulsion region of combinations of these excipients was described in ternary phase diagrams. Three formulations were selected from the nanoemulsion region and their potential impact on oral absorption was examined in the Caco-2 monolayer model of the small intestine. The apparent permeability of the BCS class III compound Atenolol was enhanced 2.5-fold, of BCS class II compound Danazol 3.2-fold and of BCS class I compound Metoprolol 1.4-fold. The three formulations were very well tolerated by the Caco-2 cells, which was confirmed by TEER measurements, a MTT test and a LDH release test.
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Danazol/farmacocinética , Antagonistas de Estrógenos/farmacocinética , Mucosa Intestinal/metabolismo , Nanomedicina , Poloxámero , Células CACO-2 , Danazol/administración & dosificación , Emulsiones , Antagonistas de Estrógenos/administración & dosificación , Humanos , Tamaño de la PartículaRESUMEN
The gene encoding a putative (R,R)-butane-2,3-diol dehydrogenase (bdhA) from Bacillus clausii DSM 8716T was isolated, sequenced and expressed in Escherichia coli. The amino acid sequence of the encoded protein is only distantly related to previously studied enzymes (identity 33-43%) and exhibited some uncharted peculiarities. An N-terminally StrepII-tagged enzyme variant was purified and initially characterized. The isolated enzyme catalyzed the (R)-specific oxidation of (R,R)- and meso-butane-2,3-diol to (R)- and (S)-acetoin with specific activities of 12U/mg and 23U/mg, respectively. Likewise, racemic acetoin was reduced with a specific activity of up to 115U/mg yielding a mixture of (R,R)- and meso-butane-2,3-diol, while the enzyme reduced butane-2,3-dione (Vmax 74U/mg) solely to (R,R)-butane-2,3-diol via (R)-acetoin. For these reactions only activity with the co-substrates NADH/NAD+ was observed. The enzyme accepted a selection of vicinal diketones, α-hydroxy ketones and vicinal diols as alternative substrates. Although the physiological function of the enzyme in B. clausii remains elusive, the data presented herein clearly demonstrates that the encoded enzyme is a genuine (R,R)-butane-2,3-diol dehydrogenase with potential for applications in biocatalysis and sensor development.