Manuel Cordoba-Diaz

Validation of a high-performance liquid chromatographic method for the determination of norfloxacin and its application to stability studies (photo-stability study of norfloxacin)

The development and validation study of a sensitive, rapid, reproducible, easy and precise reversed-phase high-performance liquid chromatographic assay for norfloxacin (NFLX) samples from photo-stability of solid dosage forms, without using gradient elution, extraction methods and without using counter-ion has been carried out. The method showed excellent linearity (r2> or =0.999) in the range 1-20 microg ml(-1) using a Lichrosorb-RP-8 column (10 microm, 20 cm x 4.6 mm) and UV-detection (278 nm) at ambient temperature. This method showed good efficiency for the analysis of photodegraded NFLX samples, and was applied to study the photo-stability of NFLX tablets under different conditions (direct sun light, ultraviolet light and fluorescent light). It was proven that the use of a disintegrant can increase the photo-stability of the NFLX in the tablets. This effect was studied in directly compressible tablets with microcrystalline cellulose (MCC) and mannitol for direct compression.

Validation protocol of an automated in-line flow-through diffusion equipment for in vitro permeation studies

Transdermal drug delivery experiments are often tedious and time consuming in terms of sampling, labor, etc. In this way, the automation of such experiments has increased in the last few years. A protocol suitable to validate an automated diffusion equipment with seven in-line flow-through cells is described. The proposed protocol was divided into two parts. First, validation of each component which makes up the whole equipment, including the study of the statistical variability of the internal volumes between the cells, the temperature into the different chambers, the time and sample volumes, etc. In the second part, a series of permeation studies were carried out comparing the performance of the system against a classical Franz-type diffusion cell. Ketoprofen was used as a model drug. It was proved the low variability of the replicates obtained with the automated flow-through diffusion cells. The best work conditions as flow rate into the receptor chamber, temperature, etc., as well as the best mathematical approach for the diffusion data, were determined. The advantages in terms of time saved and easiness of validation of the flow-through cell design in comparison to the Franz-type cell were evidenced.

Nano and microparticulate chitosan-based systems for antiviral topical delivery

Acyclovir (ACV) is one of the drugs of choice for the treatment of epidermal, ocular or systemic herpetic infections. Nevertheless, its trans-mucosal limited absorption and the scarce contact time of the formulation with the mucosal surface - especially in the ocular mucosa - constitute a big limitation of the antiviral efficiency. The most effective way to solve these problems is to increase the quantity and the residence time of the drug over the ocular surface. In order to cope with all these requirements, micro-particles (MPs) and nano-particles (NPs) containing ACV have been developed using cross-linked chitosan with tripolyphosphate (TPP) due to the biocompatibility, bio-adhesion ability and the potential power as penetration enhancer of this polymer. Particles were characterized by Fourier-transformed infrared (FTIR) spectroscopy, X-ray diffraction, SEM, Zeta potential and particle size. Encapsulation efficiency and release profiles in flow through diffusion cells were also determined. Besides the Slug Mucosal Irritation (SMI) assay has been applied as an alternative to the Draize test to predict the mucosal irritation of the selected formulation. FTIR and X-ray results suggested an electrostatic interaction ACV-Chitosan that made ACV be molecularly dispersed within the polymer matrix. Encapsulation efficiency was 75% for MP and 16% for NP. Release profiles in flow through diffusion cells were also determined. From the diffusion profiles, it was found that the amounts of ACV effectively diffused in 24h were 30, 430 and 80 μg for the ACV solution, MP and NP respectively. SMI results showed that chitosan-based particles induced moderate irritation and mild tissue damage, what supposes that ACV-MP constitute a promising alternative for further development of an antiviral formulation.

Determination of norfloxacin by fluorescence in the presence of different antacids: quantification of analytical interferences.

Norfloxacin is a fluorquinolone that can interfere with certain antacids (derivatives of Al and Mg) because its dissolution profiles are dependent on pH. Furthermore, it can form insoluble complexes that modify its absorption and bioavailability. Two sensitive and selective analytical methods using fluorescence (FL) and UV spectrophotometry (UV) have been developed to study the dissolution behaviour in gastric juice of different formulations of norfloxacin in tablets. There are no significant differences when the samples are measured by both methods and their ruggedness in the presence of some excipients is proven. From this, it is concluded that they are effective for this study. When different antacids are added to the dissolution medium, using UV and FL methods with the same samples, totally different dissolution profiles appear. Using FL, it would appear that up to 400% of the amount of norfloxacin in the tablet is released. These profiles are misleading because the uniformity of dosage units was tested before the dissolution studies. It is proven that the antacids dissolved in gastric juice do not produce fluorescence, but cause important analytical interferences with norfloxacin. This may be because their association with Al3+ or Mg2+ forms a new compound. Nevertheless, it is observed that this effect is more important in some antacids (Almagate, Magaldrate). This may be because their ability to deliver Al to the medium is greater.

Cytotoxicity of an ebulin l-anti-human CD105 immunotoxin on mouse fibroblasts (L929) and rat myoblasts (L6E9) cells expressing human CD105.

Tumour growth is characterised by the formation of a fine vessel network or neovasculature which nourishes tumour cells. Two kinds of novel anti-angiogenic therapies are based on the prevention of vessels growth and on the destruction of those vessels already formed. We report here on the design and construction of a novel immunotoxin formed with the non-toxic type II ribosome-inactivating protein ebulin l and the mouse anti-human CD105 monoclonal antibody 44G4. The 44G4-ebulin immunotoxin was formed by covalent linking of both proteins with N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) and was purified by chromatography on Superdex 200 HiLoad. The analysis of the anti-ribosomal effects in a cell-free translation system indicated that conjugation does not affect the activity of ebulin l. The immunotoxin displays cytotoxicity with nanomolar IC50 values on human CD105+ cells like the mouse fibroblasts L929 cells transfected with the short form of human CD105 and the rat myoblasts L6E9 transfected with the long form of human CD105. In contrast, cells lacking human CD105 were 2-2.5 logs less sensitive to the immunotoxin. Free ebulin displays IC50 values in the range 10(-6) M. Since CD105 is being considered as a potential target for the anti-vascular therapy of tumours, the present immunotoxin could be a promising tool for the anticancer therapy, especially due to the very low in vivo toxicity of ebulin l as compared ricin and other toxins used for immunotoxins.

A stability study of tetracycline and tetracycline cyclodextrins in tablets using a new HPLC method

A sensitive, rapid, reproducible, easy and precise reverse-phase high-performance liquid chromatographic assay for stability studies of tetracycline hydrochloride (TC.HCl) formulated with different excipients and techniques without using gradient elution, extraction methods, and at ambient temperature has been developed and validated. The method was especially developed for the analysis of TC.HCl and its main degradation product, 4-epi-anhydrotetracycline, due to its toxicity, in samples obtained from stability studies of solid dosage forms (tablets). The influence of the excipients used for the pharmaceutical design of the different tablet formulations and the use of hydroxypropyl-beta-cyclodextrin on the stability were evaluated. A significant improvement of the stability of TC.HCl was found in some tablet formulations. The precision and accuracy of the method was also studied for the encapsulated TC.HCl, and no significant interferences were found. The results obtained suggested that the developed HPLC method is selective and specific for the analysis of TC.HCl samples, and that it can be applied for long-term and accelerated protocols for stability studies.

Modification of fluorescent properties of norfloxacin in the presence of certain antacids

Modification of the fluorescent properties of norfloxacin samples in the presence of different antacids in terms of dissolution rates has been reported in a previous paper. The formation of chelates with Al3+ and Mg2+ ions has been previously suggested as a mechanism of interaction. In the present paper, the chelation was studied with different types and amounts of antacids and the stability of the non-absorbable chelates with each antacid was studied. Six dose fractions of each antacid were used in samples with the same norfloxacin concentration (9 microg ml(-1)). All samples were measured using both UV/Vis-spectrophotometry and spectrofluorimetry, and compared to a standard solution of norfloxacin (10 microg ml(-1)) without antacids, used as a reference in the calibration of the spectrofluorimeter. The results showed that the fluorescence signal features remarkable differences depending on the kind and the concentration of antacid, as well as on the time of contact. It was found that increasing amounts of antacids increased the fluorescence signal of norfloxacin samples. The evolution of the fluorescence signal in function of the antacid concentration showed a maximum and a posterior decrease. It was observed that, for a higher concentration of antacid in the medium, a higher signal was obtained and lower stability of the compound norfloxacin-antacid was observed. The data obtained strongly indicated that the binding of Al3+ and Mg2+ ions to the carboxylic groups of norfloxacin produced non-absorbable chelates. This effect might reduce the drug bioavailability.

Influence of Chopper and Mixer Speeds and Microwave Power Level During the High-Shear Granulation Process on the Final Granule Characteristics

Although microwave drying technology has been used extensively, detailed studies in the pharmaceutical field are necessary to model the different operational parameters involved in microwave drying in combination with the high-shear granulation processes. The implications of the chopper and the mixer speeds during the granulation step and the microwave power level during the drying step on the final granule characteristics were investigated. alpha-Lactose monohydrate and microcrystalline cellulose were granulated at three different mixer and chopper speeds in a laboratory-scale high-shear mixer (Mi-Mi-Pro) and dried at three microwave power levels. The dried granules were characterized by friability tests, particle size analysis, bulk and tapped density studies, and porosimetry. Neither the mixer speed nor the chopper speed had a significant influence on the granule friability, which was low for all batches produced. The selected materials and experimental conditions induced a very robust granulation process, but the granule size distribution was influenced by the microwave power level. The reciprocal relationship between the dust formation and the microwave power level was analyzed using a central composite factorial design. The amount of dust remained low in all batches, but it influenced some of the inherent density properties and the volume reduction behavior of the granulation mass. In almost all cases, the Carr index decreased slightly with increasing microwave power. The major granule characteristics were not changed when different mixer or chopper speeds were changed, although the mixer speed did alter the intragranular pore size distribution.

Elderberries: A Source of Ribosome-Inactivating Proteins with Lectin Activity

Sambucus (Adoxaceae) species have been used for both food and medicine purposes. Among these, Sambucus nigra L. (black elder), Sambucus ebulus L. (dwarf elder), and Sambucus sieboldiana L. are the most relevant species studied. Their use has been somewhat restricted due to the presence of bioactive proteins or/and low molecular weight compounds whose ingestion could trigger deleterious effects. Over the last few years, the chemical and pharmacological characteristics of Sambucus species have been investigated. Among the proteins present in Sambucus species both type 1, and type 2 ribosome-inactivating proteins (RIPs), and hololectins have been reported. The biological role played by these proteins remains unknown, although they are conjectured to be involved in defending plants against insect predators and viruses. These proteins might have an important impact on the nutritional characteristics and food safety of elderberries. Type 2 RIPs are able to interact with gut cells of insects and mammals triggering a number of specific and mostly unknown cell signals in the gut mucosa that could significantly affect animal physiology. In this paper, we describe all known RIPs that have been isolated to date from Sambucus species, and comment on their antiviral and entomotoxic effects, as well as their potential uses.

The effect of photodegradation on the fluorescent properties of norfloxacin: (Photodegradation and fluorescence of norfloxacin)

Norfloxacin (NFLX), a broad spectrum antibacterial quinolone, is a very thermostable but photosensitive drug, especially in solution leading to the formation of an ethylenediamine degradate. The modification of the fluorescent properties of NFLX in acid solution after exposure to fluorescent light and the degradation mechanisms were studied. Two analytical methods were previously developed and validated for NFLX, ultraviolet spectrophotometry (UV) and spectrofluorimetry (FL). Data obtained using both methods in the analysis of remaining NFLX in terms of percent recoveries revealed that there was no statistically significant modifications of the UV signal and of the recoveries obtained by the method. However, an important increase of the fluorescent signal after light exposure of NFLX solutions appeared, which led to an increase of the average recovery up to 270% over 15 months. Using a previously validated HPLC method for the photostability studies of NFLX, a loss of 5% with respect to the initial drug amount was observed. The study of UV and fluorescence spectra evidenced the formation of the degradation product, which induced significant modification of the fluorescent properties of NFLX samples. These results clearly indicated that FL analysis definitively is the method of choice and can be used to study the photodegradation of NFLX.