Development and validation of a HPLC method for the determination of cyclosporine a in new bioadhesive nanoparticles for oral administration.

A simple and reliable high performance liquid chromatography method was developed and validated for the rapid determination of cyclosporine A in new pharmaceutical dosage forms based on the use of poly (methylvinylether-co-maleic anhydride) nanoparticles. The chromatographic separation was achieved using Ultrabase C18 column (250×4.6 mm, 5 µm), which was kept at 75°. The gradient mobile phase consisted of acetonitrile and water with a flow rate of 1 ml/min. The effluent was monitored at 205 nm using diode array detector. The method exhibited linearity over the assayed concentration range (22-250 µg/ml) and demonstrated good intraday and interday precision and accuracy (relative standard deviations were less than 6.5% and the deviation from theoretical values is below 5.5%). The detection limit was 1.36 µg/ml. This method was also applied for quantitative analysis of cyclosporine A released from poly (methylvinylether-co-maleic anhydride) nanoparticles.

Cuidados paliativos en pacientes trasplantados de órgano sólido / No disponible

No disponible

[Evaluation of the contralateral breast with magnetic resonance in patients with newly diagnosed unilateral breast cancer]. / Evaluación de la mama contralateral mediante resonancia magnética en pacientes con diagnóstico reciente de cáncer mamario unilateral.

Breast Magnetic Resonance (BMR) imaging is a useful tool in the evaluation of breast cancer before surgical treatment. BMR imaging plays an important role in the evaluation of the extension of the malignant lesions, and the study of multifocality and multicentricity. BMR may have a role in the detection of synchronous contralateral breast cancer that is occult to conventional imaging methods (mammography and ultrasonography). In this study we review 13 series of different authors in which they have used BMR in the evaluation of the contralateral breast in patients with newly diagnosed breast cancer. Two thousand five hundred and eleven patients were evaluated with BMR and 123 contralateral cancers, that were occult to conventional methods, were detected with this technique (4,9 %). Therefore, BMR imaging of the breast is useful as a complementary tool because of its high sensitivity in local staging of a breast cancer and its ability in the detection of synchronous contralateral breast cancer in patients with newly diagnosed breast cancer.

Evaluation of CD7 and terminal deoxynucleotidyl transferase (TdT) expression in CD34+ myeloblasts from patients with myelodysplastic syndrome.

There is an emerging use of flow cytometry to evaluate patients with myelodysplastic syndrome (MDS). We have studied CD7 and TdT expression in the CD34+ myeloid blast cell population in 55 bone marrow samples of patients with MDS. CD7 and/or TdT were detected in 38 out of 55 patients (69%). CD7 expression was not related to other bad prognosis data but conversely, we found an association between TdT+ CD34 myeloblasts and high-risk MDS patients according to the International Prognostic Scoring System. Therefore, CD7 and TdT may help to establish the diagnosis of MDS and, TdT expression also seems to be a useful marker in distinguishing risk groups.

Humoral immune response in hens naturally infected with Salmonella Enteritidis against outer membrane proteins and other surface structural antigens.

A simple procedure for obtaining surface exposed antigens of Salmonella Enteritidis is described. A heat treatment of whole bacteria in saline solution induced the release of small membrane vesicles containing outer membrane components as well as surface appendage components, such as fimbriae and flagellin. The characterization of the structural components of this extract, called HE, was established by SDS-PAGE and immunoblotting using polyclonal and monoclonal specific antibodies. Five major groups of proteins were identified: flagellin, porins, OmpA, SEF21 and SEF14 fimbriae. The immunogenicity of these proteins was studied by immunoblotting with serum samples from naturally infected hens. Flagellin, porins, OmpA, SEF14 and SEF21 fimbriae were immunogenic in the S. Enteritidis infected hens (frequency of reactants: 47.3, 97.3, 64.7, 50.0 and 60.8%, respectively); porins also reacted with sera from non infected hens (66.7%). The immunogenicity of these antigens in infected birds provide promise that they may serve as components of an effective subcellular vaccine for poultry salmonellosis.

In vivo sustained release of adenoviral vectors from poly(D,L-lactic-co-glycolic) acid microparticles prepared by TROMS.

We have prepared and characterised injectable adenovirus-loaded polymeric microparticles to be used for in vitro and in vivo gene transfer studies. Microparticles were prepared by the water-in-oil-in-water solvent evaporation method using a novel system where the emulsification process is carried out by the turbulent injection of the phases in the total recirculation one-machine system (TROMS) apparatus. In vitro studies were performed to assess the amount of infectious adenovirus released from the microparticles, showing that these microparticles release higher amounts of infectious adenovirus than microparticles prepared by standard emulsification techniques. We also tested whether sustained release in vivo could overcome the short-lived gene expression profile which is typical of adenovirus delivery into muscle. Intramuscular injection of adenovirus-loaded microparticles in immunocompetent mice showed transgene (beta-galactosidase) expression for at least 7 weeks in two out of four muscles injected with adenovirus-loaded microparticles prepared by TROMS, but not in control muscles injected with purified adenovirus stocks.

Hemoglobin A1c levels in non-diabetic patients with end-stage renal disease (ESRD) receiving hemodialysis.

Hemoglobin A1c (HbA1c) level represents an established tool to monitor glycemic control in diabetic patients, but the previous commonly used tests of HbA1c in patients with end-stage renal disease (ESRD) may not be reliable because of the presence of anemia, assay interference from uremia, and decreased red blood cell (RBC) life span. HbA1c level measured by turbidimetric immunoassay method is not affected by the above factors. We enrolled 40 non-diabetic ESRD patients receiving hemodialysis and 55 non-diabetic patients without ESRD for this study. HbA1c was analyzed by turbidimetric immunoassays with Synchron CX system. We found that the average HbA1c level in non-diabetic ESRD patients receiving hemodialysis was 5.99% and in the control group was 5.45% (p<0.05). There was no significant difference in fasting glucose levels and Hct % between the two groups (p>0.05). Our limited data indicate that HbA1c levels are elevated in nondiabetic ESRD patients receiving hemodialysis. We propose that the elevated HbA1c level may be due to the repetitive exposure of patients' RBCs to the high glucose level in dialysate (200 mg/dl) or may reflect true glucose intolerance in non-diabetic patients with ESRD.

Influence of the surface characteristics of PVM/MA nanoparticles on their bioadhesive properties.

The aim of this work was to investigate the influence of the cross-linkage of poly(methylvinylether-co-maleic anhydride) (PVM/MA) nanoparticles with increasing amounts of 1,3-diaminopropane (DP) and, eventually, bovine serum albumin (BSA) on their gastrointestinal transit and bioadhesive properties. The fluorescently-labelled formulations were orally administered to rats and, at different times, the amount of nanoparticles in both the lumen content and adhered to the gut mucosa were quantified. The gut transit was evaluated by calculating the gastric (k(ge)) and intestinal (k(ie)) emptying rates. The adhered fraction of nanoparticles in the whole gut was plotted versus time and, from these curves, the intensity, capacity and extent of the adhesive interactions were estimated. The bioadhesive potential of PVM/MA was much higher when formulated as nanoparticles (NP) than in the solubilised form in water. However, k(ge) and k(ie) increased by increasing the extent of cross-linkage of nanoparticles with DP, while the capacity to develop adhesive interactions and the intensity of the adhesive phenomenon were significantly higher for non-hardened than for DP-cross-linked carriers. In contrast, the BSA-coating of cross-linked nanoparticles significantly decreased k(ge) and k(gi), whereas the intensity of the bioadhesive phenomenon was significantly higher than for NP. In summary, the adhesivity of the nanoparticles appears to modulate their gastrointestinal transit profile.

Optimization of topical cidofovir penetration using microparticles.

Cidofovir is a new class of antiviral agent with potent in vitro and in vivo activity against a broad spectrum of herpes viruses. The aim of this work was to obtain a prolonged therapeutic effect of cidofovir in the basal epidermis after its topical application. For this purpose, poly(lactide-co-glycolide) (PLGA) microparticles were prepared by solvent evaporation and spray-drying methods. Microparticles prepared by spray-drying showed a encapsulation efficiency of 80%. Conversely, for all the microspheres prepared by the W/O/W solvent evaporation method the encapsulation efficiency was low. Also, microparticles prepared by spray-drying showed a higher burst release. Skin penetration and distribution experiments were carried out with cidofovir-loaded microparticles prepared by spray-drying, since these carriers presented the best characteristics in terms of size and encapsulation efficiency. A cidofovir solution in 0.2% PVA served for comparison. Penetration experiments were carried out in Franz type diffusion cells with an available diffusion area of 1.76 cm(2), using porcine skin. The results obtained showed that the amount of cidofovir penetrated, over a 24 h time period, was higher with the drug solution than with microparticles. Cidofovir distribution in porcine skin, after topical application of microparticles and drug solution for 24 h, was determined by horizontal slicing of the skin. The profiles obtained for the two formulations showed that the quantity of cidofovir retained in the skin decreased with the depth. Besides the amount of cidofovir found in the basal epidermis (120-150 microm) was much higher with microparticles than with the control solution. These data showed that cidofovir-loaded microparticles could improve cidofovir topical therapy since these vehicles increased drug retention in the basal epidermis and decreased its penetration through the skin.

In vitro evaluation of gentamicin released from microparticles.

In this study, the preparation, characterization and drug release behaviour of gentamicin (GM)-loaded poly(D,L-lactide-co-glycolide) microspheres are described. The microspheres were produced using a double emulsion solvent evaporation technique. All the microspheres preparation resulted in spherical shape and the mean diameter was 3 microm (for empty microspheres) and between 5 and 9 microm for microparticles loaded with GM. The encapsulation efficiency (EE) ranged from 3.4 to 90% depending on the formulation. Increasing the volume of the external aqueous phase, increased the EE. Encapsulation also depended on the pH value of the internal aqueous phase, the highest value was achieved when maintained the internal aqueous phase at pH 6, where GM was more soluble. Moreover, increasing nominal GM loading yielded lower encapsulation efficiencies. The release profiles of GM from microparticles resulted in biphasic patterns. After an initial burst, a continuous drug release was observed for up to 4 weeks. Finally, the formulations with higher loading released the drug faster.

Probability tree algorithm for general diffusion processes.

Motivated by path-integral numerical solutions of diffusion processes, PATHINT, we present a tree algorithm, PATHTREE, which permits extremely fast accurate computation of probability distributions of a large class of general nonlinear diffusion processes.

Chromosomal changes pattern and gene amplification in T cell non-Hodgkin's lymphomas.

T cell non-Hodgkin's lymphomas are a heterogeneous group of lymphomas with poor prognosis, and whose genetic alterations are not well understood. Comparative genomic hybridization (CGH) is a technique that allows the identification of DNA imbalances without cytogenetic studies. We have studied 37 samples from 29 T cell non-Hodgkin's lymphomas (25 peripheral and four lymphoblastic lymphomas) by CGH in order to detect DNA sequence copy number changes of putative importance in the biology and prognosis of these neoplasms. We detected abnormal CGH profiles in 16/27 (59%) of samples at diagnosis, a ratio that increased to 66% (23/37) when we included the relapsed samples. The most common recurrent changes were gains related to the X chromosome, either the whole chromosome or partially the Xq26-27 bands (19%). Other recurrent changes included gains of bands 9q34, gains of chromosomes 17, 19, and 20, and complete or partial deletions of chromosome 13 (10%). Cancer-related genes located at Xq26-28 region were analyzed by Southern blot and fluorescence in situ hybridization (FISH). Low level amplification of some of these genes was detected by this technique confirming the results obtained by CGH in this region. The detection of abnormal CGH profiles in these T cell lymphomas could have clinical implications. Patients with abnormal CGH profiles showed significant associations with advanced stage of disease, overexpression of P53, and higher proliferative index.

FcgammaRIIA exogenously expressed in HeLa cells activates the mitogen-activated protein kinase cascade by a mechanism dependent on the endogenous expression of the protein tyrosine kinase Syk.

HeLa cells transfected to express the human Fc receptor FcgammaRIIA were stimulated with aggregates of IgG, IgG-ovalbumin equivalence immune complexes and monoclonal antibody reacting with FcgammaRIIA. All of these stimuli activated the cells as judged from the band-shift characteristic of the activation of the p42-MAP/ERK kinase. Since this response is currently associated with the activation of the protein tyrosine kinase Syk, the expression of which is currently thought to be restricted to hemopoietic cells, the results were considered as an indirect evidence of the expression in HeLa cells of either Syk or another protein tyrosine kinase accounting for the same function. Transfection with a dominant negative Syk mutant abrogated the response to FcgammaRIIA cross-linking, whereas overexpression of Syk did not increase the extent of the response. Further evidence of the expression of syk was obtained by the reverse transcription PCR approach and sequencing of the DNA bands. Moreover, immunoprecipitation with anti-Syk antibody of the cell lysates obtained after cross-linking of FcgammaRIIA followed by immunoblotting with anti-phosphotyrosine antibody showed the phosphorylation of a protein band migrating as Syk. These data indicate that expression of FcgammaRIIA on epithelial HeLa cells conveys signals to the p42-MAP/ERK kinase by a mechanism involving the recruitment of Syk. In contrast, cross-linking of this receptor does not yield productive signals coupled to other responses associated to the FcgammaR system such as triggering of the arachidonic acid cascade, activation of the NF-kappaB system and production of chemotactic cytokines.

Pharmacokinetic/pharmacodynamic modeling of antipyretic and anti-inflammatory effects of naproxen in the rat.

Pharmacokinetic/pharmacodynamic modeling was used to characterize the antipyretic and anti-inflammatory effects of naproxen in rats. An indirect response model was used to describe the antipyretic effects of naproxen after short intravenous infusions. The model assumes that basal temperature (T(a)) is maintained by the balance of fever mediators given by a constant (zero order) rate of synthesis (K(syn)), and a first order rate of degradation (K(out)). After an intraperitoneal injection of lipopolysaccharide, the change in T(a) was modeled assuming an increase in fever mediators described as an input rate function [IR(t)] estimated nonparametrically. An inhibitory E(max) model adequately described the inhibition of IR(t) by naproxen. A more complex model was used to describe the anti-inflammatory response of oral naproxen in the carrageenin-induced edema model. Before carrageenin injection, physiological conditions are maintained by a balance of inflammation mediators given by K(syn) and K(out) (see above). After carrageenin injection, the additional synthesis of mediators is described by IR(t) (see above). Such mediators induced an inflammatory process, which is governed by a first order rate constant (K(IN)) that can be inhibited by the presence of naproxen in plasma. The sigmoidal E(max) model also well described the inhibition of K(IN) by naproxen. Estimates for IC(50) [concentration of naproxen in plasma eliciting half of maximum inhibition of IR(t) or K(IN)] were 4.24 and 4.13 microg/ml, for the antipyretic and anti-inflammatory effects, respectively.

Gliadin nanoparticles as carriers for the oral administration of lipophilic drugs. Relationships between bioadhesion and pharmacokinetics.

PURPOSE: The aim of this work was to evaluate the bioadhesive properties of non-hardened gliadin nanoparticles (NPs) and cross-linked gliadin nanoparticles (CL-NP) in the carbazole pharmacokinetic parameters obtained after the oral administration of these carriers. METHODS: A deconvolution model was used to estimate the carbazole absorption when loaded in the different gliadin nanoparticles. In addition, the elimination rates of both adhered and non-adhered nanoparticulate fractions within the stomach were estimated. RESULTS: Nanoparticles dramatically increased the carbazole oral bioavailability up to 49% and provided sustained release properties related to a decrease of the carbazole plasma elimination rate. The carbazole release rates from nanoparticles (NP and CL-NP), calculated by deconvolution, were found to be of the same order as the elimination rates of the adhered fractions of nanoparticles in the stomach mucosa. In addition, good correlation was found between the carbazole plasmatic levels, during the period of time in which the absorption process prevails, and the amount of adhered carriers to the stomach mucosa. CONCLUSION: Gliadin nanoparticles significantly increased the carbazole bioavailability, providing sustained plasma concentrations of this lipophilic molecule. These pharmacokinetic modifications were directly related to the bioadhesive capacity of these carriers with the stomach mucosa.

[Controlled liberation of active principles by means of new galenic formulations]. / Liberación controlada de principios activos mediante el empleo de formulaciones galénicas.

Drugs inside a conventional galenic form are distributed between specific biological targets and other anatomical tissues. With the aim to obtain a more rational and a better therapeutic, one of the most promising possibilities by using the concept of vectorization: association of an active principle to an appropriate vector with the object to increase its action efficiency and efficacy. By this means, they do not just increase the affinity of the drug to the target but also active principle gets protected from a potentially hostile environment (hydrolytic enzymes, acid pH, etc.). The success in the extension of the applications of the vectorización depends more and more of an appropriate design, for what the fundamental objective of this revision will be the one of presenting the general characteristics and some of the current applications in these new galenic forms.

Ganciclovir-loaded albumin nanoparticles: characterization and in vitro release properties.

Ganciclovir is one of the most widely used antiviral drug for the treatment of cytomegalovirus retinitis. Due to its short half-life in the vitreous, frequent administrations are necessary to maintain the therapeutic levels. In this context, the aim of this study was to characterise and in vitro evaluate the drug release properties of three different formulations of ganciclovir-loaded albumin nanoparticles. These carriers were prepared by a coacervation method and chemical cross-linking with glutaraldehyde. Depending on the step where the drug and/or cross-linking agent were added three different formulations were obtained, named models A, B and C. For model A nanoparticles, ganciclovir was incubated with the just-formed albumin nanoparticles. For the other two types of nanoparticulate formulations, the drug was added to a solution of albumin (model B) and glutaraldehyde (model C) prior to the formation of the carriers by coacervation. In all cases, the size of the different nanoparticulate formulations was comprised between 200 and 400 nm and the yield ranged from 50%, in model A, to 65% in model B. Concerning the ganciclovir loading, model B nanoparticles offered the higher capacity to carry this antiviral drug (around 30 microg ganciclovir/mg nanoparticle). On the contrary, the drug loading calculated for model A nanoparticles was only 14.6 microg/mg. The in vitro release profiles of the nanoparticles showed a biphasic pattern, with an initial and rapid release, followed by a slower step for up 5 days. This burst effect was especially relevant in model A (around 60% in 1 h), followed by model B (40%) and less important in model C (20%). The addition of trypsin to the release medium did not have a significant influence on the release characteristics. However, the release of the drug was increased in acidic or basic mediums, due to the disruption of the covalent binding between ganciclovir and the protein matrix via glutaraldehyde. This strong linkage was also confirmed by TLC experiences. In summary, a first step of incubation between the drug and the protein, prior to the preparation of nanoparticles, enabled us to obtain albumin carriers able to release ganciclovir in a sustained way.

Bioadhesive potential of gliadin nanoparticulate systems.

The objective of this work was to prepare, characterise and evaluate the adhesive potential of gliadin nanoparticulate carriers. Firstly, lectin-nanoparticle conjugates were obtained by the carbodiimide (CDI) covalent binding of Dolichos biflorus lectin (DBA) to the surface of gliadin nanoparticles (NP) containing carbazole (as a model lipophilic drug). The DBA binding efficiency was favoured in mild acidic conditions. Similarly, a CDI concentration of about 0.63 mg/mg nanoparticles, acting during at least 1 h, provided binding efficiencies of about 50% bulk lectin. Under optimised experimental conditions, the DBA conjugates showed a size of around 500 nm and the amount of loaded carbazole and the DBA content were calculated to be around 15 and 23.5 microg/mg, respectively. The bioadhesive activity of NP and DBA conjugates was determined in samples of small and large rat intestinal mucosa. The amount of adsorbed NP was calculated to be around 8 and 4 g/m(2) in the small and large intestine, respectively. This high capacity to interact with the mucosa may be explained by gliadin composition. In fact, gliadin is rich in neutral and lipophilic residues. Neutral amino acids can promote hydrogen bonding interactions with the mucosa, while the lipophilic components can interact with the biological tissue by hydrophobic interactions. The bioadhesive activity of DBA conjugates was calculated to be about 2 g/m(2) in the small intestine and greater than 4 g/m(2) in the caecum and distal colon. These degrees of interaction were always significantly higher than those obtained with controls. Finally, DBA did not provide the specificity for interaction with Peyer's patches. In summary, gliadin nanoparticles show a high capacity of non-specific interaction with the intestine, whereas DBA binding to the surface of these carriers provided a greater specificity for colonic mucosa.

Subcellular localization of intracellular protein tyrosine phosphatases in T cells.

A high protein tyrosine phosphatase (PTPase) activity is required to maintain circulating T lymphocytes in a resting phenotype, and to limit the initiation of T cell activation. We report that 15 of the currently known 24 intracellular PTPases are expressed in T cells, namely HePTP, TCPTP, SHP1, SHP2, PEP, PTP-PEST, PTP-MEG2, PTEN, PTPH1, PTP-MEG1, PTP36, PTP-BAS, LMPTP, PRL-1 and OV-1. Most were found in the cytosol and many were enriched at the plasma membrane. Only TCPTP and PTP-MEG2 had subcellular localizations that essentially excludes them from a direct role in early T cell antigen receptor signaling events. Overexpression of 6 of the PTPases reduced IL-2 gene activation, 3 of them thereby identified as novel candidates for negative regulators of TCR signaling. Our findings expand the repertoire of PTPases that should be considered for a regulatory role in T cell activation.

Stimulation of Fc gamma R receptors induces monocyte chemoattractant protein-1 in the human monocytic cell line THP-1 by a mechanism involving I kappa B-alpha degradation and formation of p50/p65 NF-kappa B/Rel complexes.

THP-1 monocytic/macrophage cells were stimulated via their FcgammaR receptors with insoluble aggregates of human IgG and the production of the C-C chemokine monocyte chemoattractant protein (MCP)-1 assayed. A dose- and time-dependent production of MCP-1 comparable to that produced by the most potent agonists could be detected in the culture medium by a sensitive ELISA assay. This was accompanied by a parallel activation of the transcription factor NF-kappaB as judged from both the appearance of kappaB-binding activity containing p50/p65 NF-kappaB/Rel complexes in the nuclear extract and the disappearance of the NF-kappaB inhibitor IkappaB-alpha in the cell lysate. In contrast, IkappaB-beta and IkappaB-epsilon expression was not modified, thus pointing to the occurrence of a selective degradation of IkappaB-alpha under those conditions. Attempts to modulate MCP-1 production with compounds that display inhibitory effects on the activation of NF-kappaB such as the proteasome inhibitor N-acetyl-leucinyl-leucinyl-norleucinal, the antioxidant pyrrolidine dithiocarbamate and the salicylate derivative 2-hydroxy-4-trifluoromethylbenzoic acid showed a parallel effect on both MCP-1 production and NF-kappaB activation, thus pointing to the involvement of kappaB-binding sites on the transcriptional regulation of MCP-1 production. Our findings suggest the existence in monocytic cells of a signaling mechanism initiated by cross-linking of low-affinity FcgammaR, most likely of the FcgammaRII family since THP-1 cells do not express FcgammaRIII receptors, that involves activation of NF-kappaB associated to the proteolytic degradation of IkappaB-alpha and leads to the transcriptional up-regulation of MCP-1.