Viviana De Caro

Functional feature of a novel model of blood brain barrier : studies on permeation of test compounds

Drug delivery to the central nervous system (CNS) is subject to the permeability limitations imposed by the blood-brain barrier (BBB). Several systems in vitro have been described to reproduce the physical and biochemical behavior of intact BBB, most of which lack the feature of the in vivo barrier. We developed a fully formed monolayer of RBE4.B immortalized rat brain microvessel endothelial cells (ECs), grown on top of polycarbonate filter inserts with cortical neuronal cells grown on the outside. Neurons induce ECs to synthesize and sort occludin to the cell periphery. Occludin localization is regulated by both compositions of the substratum and soluble signals released by cortical co-cultured neurons. The observed effects do not require strict physical contact among cells and neurons. To assess the physiological function of the barrier we examined the transendothelial transfer of three test compounds: dopamine, L-tryptophan and L-DOPA. Polycarbonate filter inserts, where ECs were co-cultured with neurons, were assumed as open two compartments vertical dynamic models. Permeation studies demonstrated that the ECs/neurons co-cultures possess permeability characteristics approaching those of a functional BBB: the system behaved as a selective interface that excludes dopamine permeation, yet permits L-tryptophan and L-DOPA to cross. The movement of test compounds from the donor to the acceptor compartment was observed at a distinct time from the start of co-culture. Transfer was determined using standard kinetic equations. Different performance was observed after 5 and 7 days of co-culture. After 5 days dopamine, L-tryptophan and L-DOPA passively permeate through the membrane as indicated by fittings with a first-order kinetic process equation. After 7 days of co-culture, occludin localizes at ECs periphery, dopamine does not cross the barrier to any further extent, while the transfer of L-tryptophan and L-DOPA fits well with a saturable Michaelis-Menten kinetic process, thus indicating the involvement of a specific carrier-mediated transport mechanism. Permeation studies confirmed that culture of ECs in the presence of neurons induces the characteristic permeability limitations of a functional BBB.

Bioavailability in vivo of naltrexone following transbuccal administration by an electronically-controlled intraoral device: A trial on pigs

Naltrexone (NLX), an opioid antagonist, is widely used in the treatment of opiate addiction, alcoholism and smoking cessation. Its current peroral administration induces various adverse side effects and has limited efficacy since bioavailability and patient compliance are poor. The development of a long-acting drug delivery system of NLX may overcome the current drawbacks and help in the improvement of treatment of addiction. The primary endpoints of this study were: a) to compare the NLX bioavailability and pharmacokinetics after delivering a single transbuccal dose, released by a prototype of intraoral device, versus an intravenous (I.V.) bolus of the same drug dose; b) to verify the functioning of a prototype of a new intraoral device in vivo; c) to evaluate the permeation enhancement effect of iontophoresis; d) to assess any histomorphological changes in the buccal mucosa after transbuccal delivery. The system was tested on 6 pigs in a cross-over trial. Venous blood samples were drawn at a fixed timetable from the beginning of drug administration and analyzed for the presence of NLX, using an LC/MS/MS method. A punch biopsy was performed for histological analysis after the final experiment. The administration of I.V. NLX induced a sharp increase in blood levels after 5 min and then a steep decrease. In contrast, transmucosal delivery resulted in a gradual increase in blood NLX levels, reaching its peak after 90 min, followed by a slow decrease. After 6h the blood levels of NLX delivered through the buccal mucosa were higher as compared to I.V. administration. No signs of flogosis or tissue damage were histologically highlighted. These results suggest that buccal delivery by an intraoral electronic device could potentially induce long-lasting, continuous and controlled blood levels of NLX, avoiding at the same time spikes of drug plasma levels typical of the I.V. administration route.

Trehalose-hydroxyethylcellulose microspheres containing vancomycin for topical drug delivery.

A new formulation, in which vancomycin is entrapped into trehalose and hydroxyethylcellulose (Natrosol) spherical matrices, is described. Microspheres were produced by the solvent evaporation method. The entrapped drug was fully recovered following microspheres dissolution. Differential scanning calorimetry analyses proved that Natrosol maintains trehalose in its amorphous form. The stabilizing effects of trehalose on vancomycin were evaluated even after long storage and heating of microspheres. Calorimetric data indicated no decomposition of the entrapped drug. In vitro drug release, already performed by using a general two-compartment linear time-invariant open model, suggests that the new delivery system is suitable for topical application on extensive and purulent or burn wounds, when the skin is heavily damaged and the barrier disrupted. The system activation is determined by osmotic phenomena. The prepared new delivery system seems to have characteristics suitable for topical applications on extensive and purulent wounds. The system is able to take away serous exudates from wounds, thus letting the matrix to swell and form a viscous gel-like dispersion that, in turn, enables drug diffusion.

Evaluation of galantamine transbuccal absorption by reconstituted human oral epithelium and porcine tissue as buccal mucosa models: part I.

Over the last decade, interest in delivering drugs through buccal mucosa has increased. As a major limitation in buccal drug delivery could be the low permeability of the epithelium, the aim of this study was to evaluate the aptitude of galantamine, useful in Alzheimers disease, to penetrate the buccal mucosa. The evaluation of the ability of galantamine to permeate through the buccal epithelium was investigated using two permeation models. Firstly, in vitro permeation experiments were carried out using reconstituted human oral non-keratinised epithelium and Transwell diffusion cells system. Results were validated by ex vivo experiments using porcine buccal mucosa as membrane and Franz type diffusion cells as permeation model. The entity of buccal permeation was expressed in terms of drug flux (J(s)) and permeability coefficients (K(p)). Data collected by in vitro and ex vivo experiments were in agreement and suggested that buccal mucosa does not block diffusion of galantamine. The effects of drug application on histology of tissue specimens used in every experiment were also studied: no sign of flogosis and no significant cytological or architectural changes were highlighted.

Physical methods to promote drug delivery on mucosal tissues of the oral cavity

Introduction: The success of drug delivery through the mucosal tissue of the oral cavity represents a current challenge as well as a great future perspective. The need for more rapid onset of action and improved absorption of medications has resulted in great development of drug delivery technologies that use physical methods to overcome the barrier properties of oral mucosae. Areas covered: This review discusses the various physical techniques which have been, and are being, explored to sustain drug delivery in the oral cavity. In particular, supersaturation, eutectic formation, iontophoresis, electroporation, sonophoresis, laser radiation, photomechanical waves and needleless injection are considered. Following a careful selection of the most appropriate site and technique, in agreement with local variations of the oral mucosal permeability features, physical methods to promote drug delivery can improve treatment of diseases. Expert opinion: Although physical methods are very promising to promote drug delivery through keratinized epithelial tissues, they are not extensively used on the oral cavity mucosae. The authors feel that, in the near future, these methods could be further developed to provide noninvasive and convenient means for locoregional/systemic delivery of drugs with poor bioavailability profile, short half-life and multiple doses scheduling. This review will help the readers in the selection of a suitable physical method for improving drug delivery in the oral cavity for future chances. The authors imagine that new formulations or devices will be marketed in the coming years.

5-Fluorouracil Buccal Tablets for Locoregional Chemotherapy of Oral Squamous Cell Carcinoma: Formulation, Drug Release and Histological Effects on Reconstituted Human Oral Epithelium and Porcine Buccal Mucosa

5-Fluorouracil (5-FU) is currently used for treatment of oral squamous cell carcinoma (OSCC). 5-FU is given by i.v. although the systemic administration is associated with severe toxic effects and no topical formulations of 5-FU for buccal drug delivery have been reported. In this study we would report the development of buccal tablets suitable for direct application of low-doses of 5-FU on cancer lesions. The topical administration could be effective on tumor area while systemic undesired side effects are avoided. Preliminarily, the limited tendency of 5-FU to cross the buccal tissue was established using reconstituted human oral epithelium (RHOE, in vitro) and porcine buccal mucosa (ex vivo) as mucosal models. The values of steady-state flux and permeability coefficient suggested the scarce aptitude of 5-FU to reach the systemic circulation. Matrix buccal tablets, were designed for 5-FU local delivery, developed and prepared. Release tests showed a highly reproducible Higuchian drug discharge. After tablet administration on buccal tissue specimens, the occurrence of histo-morphological effects of 5-FU was highlighted. Apoptotic events were registered in all samples treated while only negligible amounts of 5-FU permeated the buccal membrane and reached the simulated plasma. The results suggest that loaded matrix tablets containing 5% of 5-FU could be a useful means in topical treatment of OSCC.

Potential dopamine prodrug-loaded liposomes: preparation, characterization, and in vitro stability studies

Dopamine delivery to the central nervous system (CNS) undergoes the permeability limitations of the blood-brain barrier (BBB). Condensation of dopamine with neutral amino acids could afford potential prodrugs able to interact with the BBB endogenous transporters and easily enter the brain. To improve the bioavailability of the dopamine prodrug, 2-amino-N-[2-(3,4-dihydroxy-phenyl)-ethyl]-3-phenyl-propionamide (DOPH), it was encapsulated in unilamellar liposomes of dimiristoylphosphatidylcholine (DMPC) and cholesterol. Vesicles were characterized by dynamic light scattering in order to evaluate their dimensions and vesicle stability, by zeta-potential measurements, by means of electronic microscopy after freeze-fracture and differential scanning calorimetry. The influence of vesicle composition on DOPH chemical and enzymatic stability was also evaluated. The formulated liposome suspensions were found to be stable, monodisperse systems with a negative zeta potential. From the obtained results, it is possible to conclude that, in studied samples, DOPH inclusion in liposomes offers the possibility of preventing photodegradation and of enhancing in vitro plasma stability. These studies suggest the potential of these formulations as a method to prevent DOPH chemical degradation and enzymatic metabolism.

Effects of DA-Phen, a dopamine-aminoacidic conjugate, on alcohol intake and forced abstinence

The mesolimbic dopamine (DA) system plays a key role in drug reinforcement and is involved in the development of alcohol addiction. Manipulation of the DAergic system represents a promising strategy to control drug-seeking behavior. Previous studies on 2-amino-N-[2-(3,4-dihydroxy-phenyl)-ethyl]-3-phenyl-propionamide (DA-Phen) showed in vivo effects as a DA-ergic modulator. This study was aimed at investigate DA-Phen effects on operant behavior for alcohol seeking behavior, during reinstatement following subsequent periods of alcohol deprivation. For this purpose, male Wistar rats were tested in an operant paradigm of self-administration; behavioral reactivity and anxiety like-behavior during acute abstinence were evaluated. A characterization of DA-Phen CNS targeting by its quantification in the brain was also carried out. Our findings showed that DA-Phen administration was able to reduce relapse in alcohol drinking by 50% and reversed the alterations in behavioral reactivity and emotionality observed during acute abstinence. In conclusion, DA-Phen can reduce reinstatement of alcohol drinking in an operant-drinking paradigm following deprivation periods and reverse abstinence-induced behavioral phenotype. DA-Phen activity seems to be mediated by the modulation of the DAergic transmission. However further studies are needed to characterize DA-Phen pharmacodynamic and pharmacokinetic properties, and its potential therapeutic profile in alcohol addiction.

Aloin delivery on buccal mucosa: ex vivo studies and design of a new locoregional dosing system

Abstract Context: Chemoprevention of potential malignant disorders or cancerous lesions that affect oral mucosae requires extended duration of treatment. Locoregional delivery of natural products could represent a promising strategy for this purpose. Objective: To investigate the aptitude of aloin to permeate through, or accumulate in, the buccal mucosa and to develop a new prolonged oro-mucosal drug delivery system. Materials and methods: Permeation/accumulation of aloin from Curacao Aloe (containing 50% barbaloin) was evaluated ex vivo, using porcine buccal mucosa as the most useful model to simulate human epithelium. Oro-mucosal matrix tablets were prepared by dispersing aloin (10% w/w) in Eudragit® RS 100 as, biocompatible, low permeable, pH-independent, and non-swelling polymer. The prepared tablets were evaluated for drug–polymer compatibility, weight variation, drug uniformity content, diameter, thickness, hardness, friability, swelling, mucoadhesive strength, and drug release. Results: Aloin has low tendency to cross buccal mucosa, permeation is marginal, and high drug amounts remain entrapped into the epithelium. Matrix tablets characteristics were in agreement with pharmacopoeial requirements. Drug release showed highly reproducible Higuchian profile. Delivery through matrix tablets promoted drug accumulation in the mucosal tissue. Discussion and conclusion: Following application of matrix tablets on porcine buccal mucosa, the amount of discharged drug recovered in the tissue should be sufficient to produce the desired effects, providing therapeutic drug levels directly at the site of action. Aloin-loaded tablets are valid candidates for prevention/treatment of potentially malignant disorders and oral cancer and could potentially lead to clinically relevant drug delivery system as coadjuvant of conventional chemotherapy/radiation therapy.

N-valproyl-L-phenylalanine as new potential antiepileptic drug: synthesis, characterization and in vitro studies on stability, toxicity and anticonvulsant efficacy.

Valproic acid (VPA) is considered first-line drug in treatment of generalized idiopathic seizures such as absence, generalized tonic-clonic and myoclonic seizures. Among major antiepileptic drugs, VPA is also considered effective in childhood epilepsies and infantile spasms. Due to its broad activity, VPA acts as a mood stabilizer in bipolar disorder and it is useful in migraine prophylaxis. Despite its long-standing usage, severe reactions to VPA, such as liver toxicity and teratogenicity, are reported. To circumvent side effects due to structural characteristics of VPA, we synthesized in good yield a new VPA-aminoacid conjugate, the N-valproyl-L-Phenylalanine, and characterized by FT-IR, MS, (13)C and (1)H- NMR analyses. The Log D(pH7.4) value (0.19) indicated that new molecule was potentially able to cross biological membranes. The resistance to chemical and enzymatic hydrolysis of N-valproyl-L-phenylalanine was also assessed. All trials suggested that the compound, at the pH conditions of the entire gastro-intestinal tract, remained unmodified. Furthermore, the new compound did not undergo enzymatic cleavage both in plasma and in cerebral medium up to 24 h. The toxicity assay on primary cultures of astrocytes indicated that the synthetized conjugate was less toxic than both free VPA and L-Phenylalanine. In this paper, the anticonvulsant activity of the new compound against epileptic burst discharges evoked in vitro in rat hippocampal slices was also evaluated. These preliminary results underline that N-valproyl-L-phenylalanine as new potential antiepileptic agent could represent a good candidate to further investigations.