Development and validation of a rapid reverse-phase HPLC method for the determination of methotrexate from nanostructured liquid crystalline systems.

A reversed-phase liquid chromatography (RP-LC) method was successfully developed and validated for the determination of methotrexate in nanostructured liquid crystalline systems composed by polyether functional siloxane and silicone polyether copolymer. The LC method was performed on RP C18-ODS column, Agilent Zorbax® (4.6 x 250 mm, 5 µm), maintained at room temperature, with a mobile phase constituted by a mixture of 50 mM ammonium acetate buffer (pH 6.0) and methanol (77:23,v/v) with a flow rate of 1.0 mL/min, using ultraviolet detection at 313 nm. The parameters used in the validation process were linearity, specificity, intra and inter-day precision, accuracy, robustness. The quantitation and detection limits yielded good results. The calibration plot assumed linear behavior from 5.0-150.0 µg. mL-1 (r2 = 0.9999). The methotrexate was subjected to oxidation, acid, base and neutral degradation, photolysis and heat as stress conditions. There were no interfering peaks at or near the retention time of methotrexate. The nanostructured liquid crystalline systems did not interfere with the analysis and the recovery was quantitative. The intra and inter-day assay relative standard deviation were less than 0.20 %. The method developed proved to be simple, sensitive, accurate, precise, reproducible and therefore adequate for routine analysis of methotrexate in nanostructured liquid crystalline systems.

Structural characterization and in vivo evaluation of retinyl palmitate in non-ionic lamellar liquid crystalline system.

Carrier systems for lipophilic drugs, such as the liquid crystalline systems (LCS) have been extensively studied to improve effect and selectivity. Retinyl palmitate (RP) is widely used in pharmaceutical and cosmetics products to improve the skin elasticity. The aim of this study was the development, characterization and the in vivo effectiveness of RP in non-ionic LCS structures. LCS containing polyether functional siloxane as oil phase, silicon glycol copolymer as surfactant and water in the ratio 30:10:60, with and without RP were studied. The results of the polarized light microscopy, small-angle X-ray scattering and rheology analysis indicated the presence of typical LCS structures with lamellar arrangement. Regardless of the presence of RP, the rheological studies showed the pseudo plastic behavior of the systems. However, highest hysteresis area was verified when comparing the system in the presence and in the absence of RP. Stability study SAXS monitored, carried out up to 30 days in various storage temperature conditions (25±2 °C, 37±2 °C and 5±2 °C) demonstrated the great structural stability of the LCS systems. The in vivo effectiveness analysis suggests that the RP-loaded LCS provided a significant reduction of the orbicular wrinkles in human volunteers (P=0.048).

Effect of cationic micelles on the decomposition of alpha-aminophenyl cephalosporins.

The intramolecular rates of degradation of alpha-aminophenyl cephalosporins were determined with and without hexadecyltrimethylammonium bromide (CTAB). Micellar-derived spectral shifts were used to measure the bind of the ionic forms as well as to determine the effect of CTAB on the apparent dissociation constant of the antibiotics. The rate of the degradation of cephalexin (Cp), cefadroxil (Cf), and cephradine (Cph), increased with surfactant concentration reaching a plateau at high surfactant concentrations. In the plateau region, the rate constant was salt sensitive decreasing with NaBr concentrations. These effects were quantitatively analyzed within the framework of the pseudo-phase model with explicit considerations of ion exchange. All the experimental results were fitted to this model. The intramolecular degradation of Cf, Cp and Cph was catalyzed by 96-, 59-, and 29-fold, respectively. A working hypothesis to rationalize these effects was suggested. The obtained results demonstrate that the quantitative analysis can be used to assess, predict and control the effects of surfactants on the drug stability.

Structure and viscoelastic behavior of pharmaceutical biocompatible anionic microemulsions containing the antitumoral drug compound doxorubicin.

Structure and viscoelastic properties of negatively charged oil-in-water (o/w) microemulsions have been investigated. Microemulsions (ME) containing soya phosphatidylcholine (SPC), eumulgin HRE 40 (EU) and sodium oleate (SO) as surfactant, cholesterol (CHO) as oil phase, and aqueous buffer with and without the antitumoral doxorubicin (DOX) have been studied. The effect of the oil phase/surfactant ratio (O/S) and the DOX incorporation on the structural and rheological properties have been studied in several compositions of ME systems. The rheological analyses were performed through the oscillation stress sweep, creep recovery test, and viscosity test. The combination of the DOX incorporation with the high O/S ratio provided a further viscoelastic structure with linear behavior. Independently of the O/S ratio the oil phase diameter increases according to a sigmoid profile, stabilizing up to 340 min. The apparent viscosity decreases a minimum value with the shear rate, but increases with both the O/S ratio and the DOX incorporation in the system. The structural and rheological properties of the studied MEs were directly dependent on the O/S ratio and can be used to improve the application of the system in the pharmaceutical field.

Verificação da atividade antibacteriana de sabonete líquido contendo extrato glicólico de Dimorphandra mollis Benth. / Verification of the antibacterial activity of liquid soap containing glycolic extract of Dimorphandra mollis Benth.

Dimorphandra mollis Benth., Composita e, falso barbatimão, é utilizada topicamente como cicatrizante, adstringente e antimicrobiano. No presente estudo, verificou-se a atividade antibacteriana de sabonete líquido contendo extrato glicólico de D. mollis (EGD) em diferentes concentrações (8, 15 e 20%) e em diferentes pHs (6 e 8). Foram preparadas cinco formulações (F) de sabonete: F1 - triclosan (0,1%), F2 - EGD (8%), F3- EGD (15%), F4 - EGD (20%) e F5 - sem conservante. Cascas de D. mollis foram secas em estufa de ar circulante e pulverizadas. Os extratos brutos foram preparados por turbo-extração utilizando-se etanol. Após filtração, os extratos foram concentrados em evaporador rotatório, liofilizados e ressuspendidos em propilenoglicol para a obtenção do extrato glicólico. A atividade antibacteriana foi verificada pelo método de difusão em ágar, empregando cilindros em placa. Placas contendo Staphylococcus aureus, Pseudomonas aeruginosa e Escherichia coli foram incubadas a 37ºC durante 24 horas. Após incubação, as leituras foram realizadas com paquímetro, observando-se o diâmetro do halo de inibição de crescimento bacteriano. Verificou-se que o sabonete líquido contendo triclosan provocou inibição do crescimento bacteriano em ambos os pHs; já os sabonetes sem conservante e contendo EGD, independente da concentração e do pH empregados, não apresentaram atividade antibacteriana.

Dimorphandra mollis Benth., Compositae, false barbatimão, has been used topically as a healing, astringent and antibacterial. In this study, antibacterial activity was verified on liquid soap containing glycolic extract of D. mollis (DGE) at different concentrations (8, 15 and 20%) and at different pH levels (6 and8). Five soap formulations (F) were prepared: F1 -tryclosan (0.1%), F2 - DGE (8%), F3 - DGE (15%),F4 - DGE (20%) and F5 - without preservatives. Bark of D. mollis were dried in a circulating air oven and ground. The rude extracts were prepared by turboextraction with ethanol. After screening, the extract were concentrated in rotating evaporator, lyophilized and resuspended in propileneglycol to obtain the glycolic extract. The antimicrobial activity was verified by diffusion in agar method, using cylinder in plate. Plates containing Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli were incubated at 37ºC for 24 hours. After incubation, the results were analysed with a pachy meter, observing the bacterial grouth inhibition halo diameter. It was verified that the liquid soap containing tryclosan caused on inhibition of bacterial growth at both pH levels; the soaps without preservatives and containing DGE, independently of the concentration and pH levels used, did not present antibacterial activity.

Oil-in-water lecithin-based microemulsions as a potential delivery system for amphotericin B.

In this work the structural features of microemulsions (MEs) containing the pharmaceutical biocompatible Soya phosphatidylcholine/Tween 20 (1:1) as surfactant (S), Captex 200 as oil phase (O), and phosphate buffer 10mM, pH 7.2 as aqueous phase (W) were studied. Systems obtained with different proportions of the components were described by pseudo-ternary phase diagrams in order to characterize the microemulsions studied here. MEs were prepared with and without the polyene antifungal drug amphotericin B (AmB). The maximum AmB incorporation into the ME system was dependent on both the oil phase and surfactant proportions with 6.80 and 5.7 mg/mL in high contents, respectively. The incorporation of AmB into the ME systems significantly increased the profile of the droplet size of the ME for all ranges of surfactant proportions used in the formulations. The microstructures of the system were characterized by dynamic light scattering (DLS) and rheological behavior. The DLS results showed that the size of the oil droplets increases 4.6-fold when AmB is incorporated into the ME system. In all cases the increase in the proportion of the oil phase of the ME leads to a slight increase in the diameter of the oil droplets of the system. Furthermore, for both the AmB-loaded and AmB-unloaded MEs, the size of the oil droplets decrease significantly with the increase of the S proportion in the formulations, demonstrating the efficiency of the surfactant in stabilizing the ME. Depending on the ME composition, an anti-thixotropic behavior was found. The maximum increases of the consistency index caused by the increase of the oil phase of the ME were of 17- and 25-times for the drug-loaded and drug-unloaded MEs, respectively. However, the observed effect for the drug-loaded ME was about 4.6 times higher than that for the drug-unloaded one, demonstrating the strong effect of the drug on the rheological characteristics of the ME system. Therefore, it is possible to conclude that the investigated ME can be used as a very promising vehicle for AmB.

Structural changes of biocompatible neutral microemulsions stabilized by mixed surfactant containing soya phosphatidylcholine and their relationship with doxorubicin release.

Depending on the composition, the mixture of surfactant, oil and water, may form supramolecular aggregates with different structures which can significantly influence the drug release. In this work several microemulsion (ME) systems containing soya phosphatidylcholine (SPC) and eumulgin HRE40 (EU) as surfactant, cholesterol (O) as oil phase, and ultra-pure water as an aqueous phase were studied. MEs with and without the antitumoral drug doxorubicin (DOX) were prepared. The microstructures of the systems were characterized by photon correlation spectroscopy, rheological behavior, polarized light microscopy, small-angle X-ray scattering (SAXS) and X-ray diffraction (XRD). The results reveal that the diameter of the oil droplets was dependent on the surfactant (S) amount added to formulations. The apparent viscosity was dependent on the O/S ratio. High O/S ratio leads to the crystallization of cholesterol polymorphs phases which restricts the mobility of the DOX molecules into the ME structure. Droplets with short-range spatial correlation were formed from the ME with the low O/S ratio. The increase of the cholesterol fraction in the O/S mixture leads to the formation of ordered structures with lamellar arrangements. These different structural organizations directly influenced the drug release profiles. The in vitro release assay showed that the increase of the O/S ratio in the formulations inhibited the constant rate of DOX release. Since the DOX release ratio was directly dependent on the ratio of O/S following an exponential decay profile, this feature can be used to control the DOX release from the ME formulations.

Doxorubicin biocompatible O/W microemulsion stabilized by mixed surfactant containing soya phosphatidylcholine.

Microemulsions (ME) containing soya phosphatidylcholine (SPC)/polyoxyethylenglycerol trihydroxystearate 40 (EU)/sodium oleate (SO) as surfactant cholesterol (CHO) as oil phase and aqueous buffer were studied. Pseudo-ternary phase diagrams of the investigated systems were obtained at constant SPC/EU/SO weight ratio 3.5:3.5:3.0 by titration, in order to characterize the proportions between the components to form clear systems. The dynamic light scattering results showed that the size of the oil droplets decreases significantly with the ratio of surfactant/oil phase added to system. Depending on the composition ME system could exhibit a thixotropic behavior. The apparent viscosity increased 25- and 13-folds with cholesterol concentration for drug-free and drug-load ME, respectively. It was also verified that the octanol/aqueous buffer partition coefficient (KO/B) of doxorubicin (DOX) was pH dependent increasing abruptly above pH 6.0. It was possible to incorporate 2.24 mg/ml of DOX into ME. The incorporation of DOX in the ME systems increased the droplets size for all surfactant concentrations used in the system. The results suggest that DOX interacts with the microstructure of the ME at the studied pH increasing significantly the drug solubility. It was possible to conclude that the investigated ME can be a very promising vehicle as drug-carrier for administration of doxorubicin.

Desenvolvimento e validação de método de análise quantitativa para triancinolona em micropartículasbiodegradáveis por CLAE / Development and validation of HPLC method for quantitative analysis of triamcinolone in biodegradable microparticles

Neste trabalho, foi desenvolvido um método de cromatografia líquida de alta eficiência (CLAE) para a determinação quantitativa da triancinolona contida em micropartículas de ácido polilático-co-glicólico (PLGA). As condições cromatográficas utilizadas foram Colunade Fase Reversa C18, 250mm x 4,6mm com diâmetro partícula 5 m. O forno de coluna foi termostatizado a 35 mais ou menos 2ºC, a fase móvel usada foi metanol:água 45:55 (v:v), com fluxo isocrático de 1 mL.min-1 e volume de injeção de 10 ì L. Foi utilizado detector de UV-Vis selecionado em 239nm. A curva padrão obtida apresentou linearidade (r2 maior que 0,999) na faixa de concentração 100-2.500 ng.mL-1. O método proposto apresentou precisão adequada com desvio padrão relativo menor que 3 por cento. Os resultados mostraram que a exatidão do método apresentou valores de recuperação dentro dos limites recomendáveis em toda a faixa de concentração estudada. O método de CLAE mostrou especificidade e seletividade com linearidade dentro da faixa de concentração de trabalho utilizada e precisão e exatidão que permitem a quantificação da triancinolona em micropartículas de PLGA.

On the incorporation of the non-steroidal anti-inflammatory naproxen into cationic O/W microemulsions.

Microemulsions (ME) containing hexadecyltrimethylammonium bromide (HTAB)/ethanol as surfactant, isopropylmyristate (IM) or butylstearate (BS) as oil phase and aqueous buffer were studied. Pseudo-ternary phase diagrams of the investigated systems were obtained at constant surfactant/cosurfactant molar ratio (1:5) by titration in order to characterize the proportions between the components to obtain clear systems. Oil in water microemulsions were prepared in a wide range of phase volume (phi). UV-vis absorption spectra of naproxen at pH 5.5 showed that the solubility of Np increases significantly in the presence of O/W ME in high phase volumes. For both, IM and BS microemulsions, the dynamic light scattering experiments showed that the size of the oil droplets remains constant in low values of phi, increasing abruptly in high phi values. Phase solubility study revealed that for both IM and BS microemulsions, the drug incorporation followed a straight-line profile in all range of phi. The data could be analyzed through the phase-separation model and the association constants (K) calculated varied from 27 to 90 M(-1), depending on the pH and on the microemulsion oil phase.

Studies on the encapsulation of diclofenac in small unilamellar liposomes of soya phosphatidylcholine.

The encapsulation of acid (AD) and sodium diclofenac (SD) in small unilamellar liposomes (SUV) as well as the interactions of the drug with the bilayer was studied. SUV was prepared by sonication from multilamellar liposomes containing soya phosphatidylcholine and diclofenac at various proportions. The size distribution obtained from dynamic light scattering showed that the incorporation of SD decreases significantly the size of the liposomes suggesting that the drug interacts with the bilayer of the liposomes. This size decrease is related with the phase transition of liposomes to mixed micelar solution. The encapsulation of the hydrophilic dye indocyanine green in the aqueous compartment of liposomes showed that the rate of captured dye decreases with SD concentration suggesting the transition of liposomes to mixed micelles. The (31)P NMR analysis indicates that SD interacts with the phosphate of phosphatidylcholine head groups. A schematic model for interaction of SD with phosphatidylcholine of the liposomes in which the diclofenac anion interacts with the ammonium group of the phospholipid and the dichlorophenyl ring occupies a more internal site of bilayer near phosphate group was proposed.

Study of the diclofenac/phospholipid interactions with liposomes and monolayers.

The interaction of diclofenac sodium (SD) with soya phosphatidylcholine (SPC) has been studied with floating Langmuir monolayers and liposomes. SD was either introduced into the subphase of SPC monolayers or co-spread with SPC on an aqueous subphase. In both cases, SD caused the surface pressure isotherm to become more expanded, thus demonstrating the affinity between SD and SPC. The incorporation of SD caused SPC liposomes to have a decreased diameter according to light scattering experiments. When SPC liposomes were injected into an aqueous subphase, their destruction yielding surface-active monomers could be monitored by changes in surface pressure. SD-loaded liposomes displayed a much faster kinetics when the surface density of surface-active monomers was plotted against time, with rate constants increasing significantly with the SD concentration. The kinetic profile can be quantitatively analyzed by plotting ln[1 - (gamma/gamma infinity)] versus t1/2.

Effect of hexadecyltrimethylammonium bromide-based microemulsions on the rate of decomposition of the beta-lactam antibiotic cephaclor.

Microemulsions of hexadecyltrimethylammonium bromide (HTAB)/n-butanol/hexadecane/water catalyze the intramolecular degradation of cephaclor. The rate increase is a sensitive function of the microemulsion volume fraction and salt concentration. The effects of microemulsions, analyzed quantitatively using a pseudophase ion-exchange model, assumed that the extent of ion dissociation from the microemulsions varies with volume fraction. Comparison of micellar and microemulsion effects on the same reaction shows that microemulsions are less effective catalysts. Acceleration decreased significantly by increasing the relative proportion of n-butanol ratio in microemulsions and by addition of n-butanol in HTAB micelles. Comparison of the activation parameters of the reaction in aqueous solution, microemulsions, and micelles suggests that catalysis by both aggregates is driven mainly by entropic contributions.