Stability of furosemide and aminophylline in parenteral solutions / Estabilidade de furosemida e aminofilina em soluções parenterais

Parenteral solutions (PS) are one of the most commonly used drug delivery vehicles. Interactions among the drug, components in the drug's formulation, and the PS can result in the formation of inactive complexes that limit efficacy or increase side effects. The aim of this work was to evaluate possible interactions between the drugs and PS, assess drug stability and to identify degradation products after 20 h at room temperature. Furosemide (FSM) and Aminophylline (APN) were added to PS containing either 20 percent mannitol or 0.9 percent NaCl at pH 6.5-7.5 and 10-11. Their behavior was studied individually and as an admixture, after 1 h oxidation with H2O2, using a spectrophotometer and HPLC. Individually, FSM and APN added to 20 percent mannitol and 0.9 percent NaCl solutions had the highest stability at pH 10-11. When FSM and APN were combined, the behavior of FSM was similar to the behavior observed for the drug individually in the same solutions. With the drugs combined in 20 percent mannitol pH 10-11, HPLC showed that both drugs were stable after a 20 h period yielding two distinct peaks; in oxidized samples, the elution profile showed four peaks with retention times unrelated to the untreated samples.

Soluções parenterais de grande volume são frequentemente utilizadas no ambiente hospitalar para a veiculação de fármacos. No entanto, possíveis incompatibilidades entre as estruturas dos fármacos, em diferentes veículos de administração, podem gerar possíveis associações antagônicas ou sinérgicas, resultando em alterações das propriedades físico-químicas, consequentemente, dos efeitos farmacológicos e das respostas clínicas esperadas. Este artigo avaliou a estabilidade e a possível formação de produtos de degradação entre os fármacos furosemida e aminofilina quando estes foram veiculados em soluções parenterais, após o preparo e após o período de 20 h. Furosemida e aminofilina foram adicionadas às soluções de 20 por cento manitol e 0,9 por cento NaCl nos valores de pH 6,5-7,5 e 10-11. A estabilidade dos fármacos foi avaliada individualmente, combinada e após degradação com peróxido de hidrogênio através de espectrofotometria de UV e HPLC. Furosemida e aminofilina individualmente avaliadas mostraram alta estabilidade em ambas as soluções estudadas nos valores de pH 10-11. Quando os fármacos foram combinados o comportamento da furosemida foi similar ao observado na ausência de aminofilina. Os fármacos combinados em 20 por cento manitol pH10-11 por HPLC foram estáveis após o período de 20 h. Após degradação o perfil de cromatograma encontrado foi diferente do observado na ausência de degradação mostrando que o método é indicativo de estabilidade.

Limitations in the Use of Fluorescein Diacetate/Propidium Iodide (FDA/PI) and Cell Permeable Nucleic Acid Stains for Viability Measurements of Isolated Islets of Langerhans.

BACKGROUND: A review of current literature shows that the combined use of the cell permeable esterase-substrate fluorescein diacetate (FDA) and the cell impermeant nucleic acid stain propidium iodide (PI) to be one of the most common fluorescence-based methods to assess the viability of isolated islets of Langerhans, and it is currently used for islet product release prior to transplantation in humans. However, results from this assay do not correlate with islet viability and function or islet transplantation success in animals or humans (Eckhard et al. 2004; Ricordi et al. 2001). This may be in part attributed to considerable differences as well as discrepancies in the use of these reagents on islets. We critically surveyed the literature and evaluated the impact of a number of variables associated with the use of FDA/PI to determine their reliability in assessing islet cell viability. In addition, we evaluated other fluorescent stains, such as SYTO(R)13, SYTO(R)24 and SYBR(R)14 as possible alternatives to FDA. RESULTS: We found that the stability of stains in storage and stock solutions, the number of islets stained, concentration of stains, staining incubation time, the buffer/media used, and the method of examining islets were significant in the final scoring of viability. For archival file photos, the exposure time and camera/software settings can also impact interpretation of viability. Although our results show that FDA does detect intracellular esterase activity and staining with PI does assess cell membrane integrity, the results obtained from using these stains did not correlate directly with expected islet function and viability per transplantation into diabetic athymic nude mice (Papas et al. 2007). In addition, the use of two nucleic acid stains, such as SYTO(R)13 and PI, for live/dead scoring exhibited staining anomalies which limit their accuracy in assessing islet viability. CONCLUSIONS: From a review of the literature and from our observations on the impact of reagent handling and various staining and imaging parameters used to visually evaluate islets, consistent interpretation of islet cell membrane integrity and viability is dependent upon a number of factors. We discuss the utility and limitations of these reagents in evaluating islet cell membrane integrity and viability.

Preliminary study on the potential utility of GFP as a biosensor for drug stability in parenteral solutions.

In the health care setting, drugs added to large volume parenteral solutions (LVPS) are routinely administered to improve therapeutic effects and provide a faster clinical response. The development of analytical techniques that permit the detection of incompatibilities between drugs and parenteral solutions is necessary to guarantee their correct association with minimum adverse effects. Green fluorescent protein (GFP) has been used as a biological indicator of sterilization and disinfection processes because it exhibits a high thermal stability and is easily detected using UV light and spectrofluorometry. The response of GFP structure and/or protonation state to physicochemical changes in the solution favors its potential use as a biosensor for drug stability in parenteral solutions. The stability of the diuretic drugs furosemide and aminophylline, individually or combined, added to parenteral solutions of 20% mannitol and 0.9% NaCl was monitored by absorbance and RP-HPLC immediately and after 20 h of storage at room temperature, with and without 1 h exposure to a strong oxidant, H2O2. Changes in GFP fluorescence intensity were evaluated under the same conditions for purified GFP added to aliquots of the drug/LVPS solutions. Results show that GFP fluorescence intensity was proportional to the loss in drug stability over time and thus may potentially be added to a lot sample of a drug/parenteral solution as an immediate on-site test for defective product.

Study on the thermal stability of green fluorescent protein (GFP) in glucose parenteral formulations.

Large volume parenteral solutions (LVPS) that are widely used in the healthcare system must be processed by moist-heat treatment to an assured sterility level in which the efficacy is measured by a bioindicator (BI) that provides fast, accurate and reliable results. This study evaluated the thermal stability of green fluorescent protein (GFP) into glucose-based LVPS (1.5-50%) solutions to determine its utility as a BI for thermal processes. GFP, expressed by Escherichia coli, isolated/purified by TPP/HIC, was diluted in buffered (each 10mM: Tris-EDTA, pH 8; phosphate, pH 6 and 7; acetate, pH 5) and in water for injection (WFI; pH 6.70+/-0.40) glucose solutions (1.5-50%) and exposed to constant temperatures from 80 degrees C to 95 degrees C. The thermal stability was expressed in decimal reduction time (D-value, time required to reduce 90% of the GFP fluorescence intensity). At 95 degrees C, the D-values for GFP in 1.5-50% glucose were: (i) 1.63+/-0.23 min (pH 5); (ii) 2.64+/-0.26 min (WFI); (iii) 2.50+/-0.18 min (pH 6); (iv) 3.24+/-0.28 min (pH 7); (v) 2.89+/-0.44 min (pH 8). By the convenient measure of fluorescence intensity and its thermal stability, GFP has the potential as a BI to assay the efficacy of moist-heat processing of LVPS at temperatures < or =100 degrees C.

Evaluation of the pH- and thermal stability of the recombinant green fluorescent protein (GFP) in the presence of sodium chloride.

The thermal stability of recombinant green fluorescent protein (GFP) in sodium chloride (NaCl) solutions at different concentrations, pH, and temperatures was evaluated by assaying the loss of fluorescence intensity as a measure of denaturation. GFP, extracted from Escherichia coli cells by the three-phase partitioning method and purified through a butyl hydrophobic interaction chromatography (HIC) column, was diluted in water for injection (WFI) (pH 6.0-7.0) and in 10 mM buffer solutions (acetate, pH 5.0; phosphate, pH 7.0; and Tris-EDTA, pH 8.0) with 0.9-30% NaCl or without and incubated at 80-95 degrees C. The extent of protein denaturation was expressed as a percentage of the calculated decimal reduction time (D-value). In acetate buffer (pH 4.84+/-0.12), the mean D-values for 90% reduction in GFP fluorescence ranged from 2.3 to 3.6 min, independent of NaCl concentration and temperature. GFP thermal stability diluted in WFI (pH 5.94+/-0.60) was half that observed in phosphate buffer (pH 6.08+/-0.60); but in both systems, D-values decreased linearly with increasing NaCl concentration, with D-values (at 80 degrees C) ranging from 3.44, min (WFI) to 6.1 min (phosphate buffer), both with 30% NaCl. However, D-values in Tris-EDTA (pH 7.65+/-0.17) were directly dependent on the NaCl concentration and 5-10 times higher than D-values for GFP in WFI at 80 degrees C. GFP pH- and thermal stability can be easily monitored by the convenient measure of fluorescence intensity and potentially be used as an indicator to monitor that processing times and temperatures were attained.

Stability of green fluorescent protein (GFP) in chlorine solutions of varying pH.

Green fluorescent protein (GFP) is an excellent biosensor as a result of its ability to be easily monitored in a wide variety of applications. Enzymes and proteins have been used as biological indicators to evaluate the immediate efficacy of industrial procedures, such as blanching, pasteurization, and disinfection treatments, as well as to monitor the satisfactory preservation of a product subjected to disinfection or sterilization. The purpose of this work was to study GFP stability in chlorinated water for injection (WFI) and chlorinated buffered solutions at various pH ranges, with and without agitation, to evaluate the exposure time required for chlorine to decrease 90% of its fluorescence intensity (decimal reduction time, D-value, min, 25 degrees C). Fluorescence intensity (Ex/Emmax = 394/509 nm) was measured immediately after the addition of GFP (8.0-9.0 microg/mL) into buffered or unbuffered chlorine solutions with or without constant stirring. With solutions constantly stirred, GFP fluorescence decreased abruptly on contact with chlorine in concentrations greater than 150 ppm, with D-values between 1.3 min (147 ppm chlorine) and 1.7 min (183 ppm chlorine). In phosphate buffered chlorine solutions (pH = 7.15 +/- 0.08), GFP retained its structure between 52 and 94 ppm, but protein stability decreased 10-fold when exposed to 110 ppm chlorine. The recovery of GFP fluorescence intensity due to renaturation was observed between 30 and 100 ppm chlorine in WFI (final pH = 11.01 +/- 0.23) without stirring. Stirring enhanced the contact between GFP and chlorine throughout the assay and provided a more accurate D-value evaluation. GFP performed as a suitable fluorescent marker for monitoring disinfection effectiveness.

Detection of nisin expression by Lactococcus lactis using two susceptible bacteria to associate the effects of nisin with EDTA.

Nisin, a bacteriocin produced during the exponential growth phase of Lactococcus lactis ATCC 11454, inhibits the growth of a broad range of Gram-positive bacteria. Gram-negative bacteria can also be inhibited by nisin with EDTA. In this study, nisin production was assayed by the agar diffusion method using Lactobacillus sake ATCC 15521 and a recombinant Escherichia coli DH5-alpha expressing the recombinant green fluorescent protein as the nisin-susceptible test organisms. The titers of nisin expressed and released in culture media were quantified and expressed in arbitrary units (AU/mL of medium) and converted to standard nisin concentration (Nisaplin, 25 mg of pure nisin with an activity of 1 x 10(6) AU/mL). The expression and release of nisin by L. lactis in skimmed milk (9.09% total solids) with Man Rugosa Shepeer-Bacto Lactobacilli broth (1:1) was monitored in a 5 L New Brunswick fermentor. Combining EDTA with nisin increased the bactericidal effect of nisin on the bacteria examined. The presence of EDTA was necessary to inhibit E. coli growth with nisin. L. sake was shown to be a good indicator for the evaluation of nisin release in the culture media, including with the addition of EDTA.

Stability of recombinant green fluorescent protein (GFPuv) in glucose solutions at different concentrations and pH values.

The stability at room temperature (25 degrees C) of recombinant green fluorescent protein (GFPuv), expressed by Escherichia coli cells and isolated by three-phase partitioning extraction with hydrophobic interaction column, was studied. The GFPuv was diluted in buffered (each 10 mM: Tris-HCl, pH 8.0; phosphate, pH 6.0 and 7.0 and acetate, pH 5.0) and in unbuffered (water for injection [WFI]; pH 6.70 +/- 0.40) glucose solutions (from 1.5 to 50%). By assaying the loss of fluorescence intensity as a measure of denaturation, the stability of GFPuv in these solutions was evaluated relative to glucose concentration, pH, osmolarity, density, conductivity, and viscosity. The extent of protein denaturation (loss of fluorescence intensity) was expressed in decimal reduction time (D-value), the time required to reduce 90% of the initial fluorescence intensity of GFPuv. The D-value between 56 and 83 h of GFPuv at 1.5-15% glucose in WFI was equivalent to 20-30% glucose in a phosphate. The stability of GFPuv in 50% glucose was similar for all buffers studied and four times higher than in WFI. By the convenient measure of fluorescence intensity, GFPuv can be used as an indicator to report the extent of denaturation rates of other proteins in glucose solutions.

Production of nisin by Lactococcus lactis in media with skimmed milk.

Nisin is a bacteriocin that inhibits the germination and growth of Gram-positive bacteria. With nisin expression related to growth conditions of Lactococcus lactis subsp. lactis, the effects of growth parameters, media components, and incubation time were studied to optimize expression. L. lactis ATCC 11454 was grown (100 rpm at 30 degrees C for 36 h) in both M17 and MRS standard broth media (pH 6.0-7.0) supplemented with sucrose (1.0-12.5 g/L), potassium phosphate (0.13 g/L), asparagine (0.5 g/L), and sucrose (0.24 g/L), and diluted 1:1 with liquid nonfat milk. Liquid nonfat milk, undiluted, was also used as another medium (9% total solids, pH 6.5). Nisin production was assayed by agar diffusion using Lactobacillus sake ATCC 15521 (30 degrees C for 24 h) as the sensitive test organism. The titers of nisin expressed and released in culture media were quantified and expressed in arbitrary units (AU/L of medium) and converted into known concentrations of "standard nisin" (Nisaplin, g/L). The detection of nisin activity was <0.01 AU/L in M17 and MRS broths, and 7.5 AU/L in M17 with 0.14% sucrose or 0.13% other supplements, and the activity increased to 142.5 AU/L in M17 diluted with liquid nonfat milk (1:1). The 25% milk added to either 25% M17 or 25% MRS provided the highest levels of nisin assayed.

NMR Wool Tube: a novel method for NMR solution analysis of derivatized glass surfaces.

Glass wool was placed within an NMR tube as a solid support for the covalent attachment of a molecule to allow for a simple one-dimensional 1H FT NMR solution analysis. This novel procedure avoids the use of expensive sample tubes or platforms, as required for magic angle or fast spinning, exotic pulse sequences, isotopic labeling or the use of a large number of scans to provide the ability to analyze the structure, mobility, ligand binding, and solvent interactions of the surface bound molecule.

Evaluation of recombinant green fluorescent protein, under various culture conditions and purification with HiTrap hydrophobic interaction chromatography resins.

To determine the influence of various culture conditions, transformed cells of Escherichia coli expressing recombinant green fluorescent protein (GFPuv) were grown in nine cultures with four variable conditions (storage of inoculated broth at 4 degrees C prior to incubation, agitation speed, isopropyl-beta-D-thiogalactopyranoside [IPTG] concentration, and induction time). The pelleted cells were resuspended in extraction buffer and subjected to the three-phase partitioning (TPP) extraction method. To determine the most appropriate purification resin, protein extracts were eluted through one of four types of HiTrap hydrophobic interaction chromatography (HIC) columns prepacked with methyl, butyl, octyl, or phenyl resins and analyzed further on a 12% sodium dodecylsulfate polyacrylamide gel. With Coomassie staining, a single band between 27 (standard GFPuv) and 29 kDa (molecular weight standard) was visualized for every HIC column sample. TPP extraction with HIC elution provided about 90% of the GFPuv recovered and eight-fold GFPuv enrichment related to the specific mass. Rotary speed and IPTG concentration showed, respectively, greater negative and positive influences on GFPuv expression at the beginning of the logarithmic phase for the set culture conditions (37 degrees C, 24-h incubation).

Thermal stability of recombinant green fluorescent protein (GFPuv) at various pH values.

The thermal stability of the recombinant green fluorescent protein (GFPuv) expressed by Escherichia coli cells and isolated by three-phase partitioning extraction with hydrophobic interaction chromatography was studied. The GFPuv (3.5-9.0 microg of GFPuv/mL) was exposed to various pH conditions (4.91-9.03) and temperatures (75-95 degrees C) in the 10 mM buffers: acetate (pH 5.0-7.0), phosphate (pH 5.5-8.0), and Tris-HCl (pH 7.0-9.0). The extent of protein denaturation (loss of fluorescence intensity) was expressed in decimal reduction time (D-value), the time exposure required to reduce 90% of the initial fluorescence intensity of GFPuv. For pH 7.0 to 8.0, the thermostability of GFPuv was slightly greater in phosphate buffer than in Tris-HCl. At 85 degrees C, the D-values (pH 7.1-7.5) ranged from 7.24 (Tris-HCl) to 13.88 min (phosphate). The stability of GFPuv in Tris-HCl (pH >8.0) was constant at 90 and 95 degrees C, and the D-values were 7.93 (pH 8.38-8.92) and 6.0 min (pH 8.05-8.97), respectively. The thermostability of GFPuv provides the basis for its potential utility as a fluorescent biologic indicator to assay the efficacy of moist-heat treatments at temperatures lower than 100 degrees C.