Synthesis and in vitro studies on a potential dopamine prodrug.

Dopamine delivery to the central nervous system (CNS) undergoes the permeability limitations of blood-brain barrier (BBB) which is a selective interface that excludes most water-soluble molecules from entering the brain. Neutral amino acids permeate the BBB by specific transport systems. Condensation of dopamine with neutral amino acids could afford potential prodrugs able to interact with the BBB endogenous transporters and easily enter the brain. The synthesis and characterization of the dopamine derivative 2-amino-N-[2-(3,4-dihydroxy-phenyl)-ethyl]-3-phenyl-propionamide (7) is described. The chemical and enzymatic stability of 7 was evaluated. The molecular weight (300 Da) and Log Papp (0.76) indicated that the physico-chemical characteristics of compound 7 are adequate to cross biological membranes. Compound 7 was enzymatically cleaved to free dopamine in rat brain homogenate (t1/2 = 460 min). In human plasma, the t1/2 of 7 was estimated comparable to that reported for L-DOPA. In view of a possible oral administration of 7, studies of its chemical behavior under conditions simulating those of the gastrointestinal tract showed that no dopamine production occurred; furthermore, 7 is able to permeate through a simulated intestinal mucosal membrane. The collected data suggest that compound 7 could beconsidered a very valuable candidate for subsequent in vivo evaluation.

Transbuccal tablets of carbamazepine: formulation, release and absorption pattern.

Tranbsuccal drug administration is an attractive method, as it has several advantages especially with respect to peroral delivery. Here we report: i) the aptitude of carbamazepine (CBZ) to penetrate porcine buccal mucosa and reconstituted human oral (RHO) epithelium; ii) three different tablet formulations for transbuccal administration; iii) the drug release rate from tablets. CBZ permeation through the buccal mucosa was investigated by using two different bi-compartmental open models: Franz cells for porcine buccal mucosa and Transwell diffusion cells system for RHO epithelium. Results, expressed as drug flux (Js) and permeability coefficients (Kp), indicated that CBZ well penetrates the membrane and arrives in the acceptor phase. Js and Kp resulted 7x10(-2) mg/cm2h and 0.23 cm/h for in vitro experiments and 1.81 x 10(-2) mg/cm2h and 4.57 x 10(-2) cm/h for ex vivo experiments. The flux is extensively affected by the membrane thickness. The CBZ release from three different formulations of tablets, prepared with loaded microspheres, loaded matrices, and conventional compressed physical mixture of components was studied. Using the new formulated "non-conventional" tablets prolonged drug release was obtained. Loaded matrix tablets discharged CBZ faster than microsphere tablets (17% and 12% in about 2.5 h respectively). Results indicate the possibility of administering CBZ on buccal mucosa.

Effects of gamma-irradiation on trehalose-hydroxyethylcellulose microspheres loaded with vancomycin.

Ionizing radiation can be used as a drug sterilization technique, provided that the drug itself is not modified and that no toxic products are produced; moreover, if the irradiated product is a drug delivery system, the drug release characteristics must not be significantly altered by radiation. The aim of this work was to study the effects of sterilization by ionizing radiation on hydroxyethylcellulose/trehalose spherical micromatrices, containing the antibiotic vancomycin. Our experimental results showed that gamma-rays did not alter the chromophore groups of vancomycin (UV measurements), and did not modify the kinetic behavior of drug release from microspheres. Moreover, no significant changes in the shape and in the size distribution of microspheres were found after irradiation. The electron spin resonance (ESR) spectroscopy was proven to be a valid identification method of the executed radiation treatment, even after 5 years. The experimental results showed that the therapeutic application of the pharmacological system investigated was not compromised by irradiation, and that ESR spectroscopy can be used to distinguish irradiated from non-irradiated products.

Neurons and ECM regulate occludin localization in brain endothelial cells.

We report that extracellular matrix and neurons modulate the expression of occludin, one of the main components of tight junctions, by rat brain endothelial cells (RBE4.B). Of the three extracellular matrix proteins which we tested (collagen I, collagen IV, and laminin), collagen IV stimulated at the best the expression of occludin mRNA. The corresponding protein, however, was not synthesized. Significant amounts of occludin accumulated only when RBE4.B cells were cultured on collagen IV-coated inserts, in the presence of cortical neurons, plated on laminin-coated companion wells. Finally, occludin segregated at the cell periphery, only when endothelial cells were co-cultured with neurons for > or = 1 week.

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.

Response charactterization of ammonium tartrate solid state pellets for ESR dosimetry with radiotherapeutic photon and electron beams.

Solid state pellets (1 mm thick) for electron spin resonance (ESR) dosimetry were made using ammonium tartrate as the radiation-sensitive substance. Their behaviour was experimentally investigated as a function of dose with 60Co gamma rays. The calibration function obtained permits measurements of absorbed dose in the 2-50 Gy range, with a combined uncertainty of +/-4%. The lowest detectable dose was about 0.5 Gy. These properties are comparable with or even better than those of ESR dosimeters made from other materials. The time stability of the ESR signal of ammonium tartrate dosimeters at different storage conditions after irradiation was studied. A rather complex behaviour was observed, which suggests that more species of free radicals are produced by radiation and that migration processes may be effective. No dependence of the response on beam quality was found for high-energy photon and electron beams produced by a linear accelerator used in radiotherapy, whereas dose was underestimated with low-energy x-rays.

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.

Synthesis and characterization of aminoacidic pro-drugs of valproic acid.

Some new derivatives of valproic acid (1) have been synthesized by condensation reaction of L-phenylalanine ethyl ester (2), L-tryptophan methyl ester (3), L-leucine methyl ester (4), L-methionine methyl ester (5) or L-histidine methyl ester (6) with 1 in the presence of dicyclohexylcarbodiimide (DCC). The reaction afforded in good yield prodrugs containing an amino acidic moiety. The structure of the new compounds was determined with the aid of spectroscopic and analytical data. To evaluate the stability following p.o., administration, the synthesized molecules were tested for gastro-intestinal hydrolysis. No significant general acid-base hydrolysis for the peptide bond was observed. The molecules offer a potentially useful mean to obtain highly selective drug release to the brain.

L-pyroglutamyl-L-tryptophan derivatives as potential drug carriers. II.: Permeation behaviour and stability in the gastro-intestinal tract.

The in vitro permeation behaviour of L-tryptophan (1). L-pyroglutamic acid (2), L-pyroglutamyl-L-tryptophan (3), and the corresponding ethyl ester derivatives (4, 5 and 6 respectively) was studied to collect the essential informations for oral administration of these molecules. Dipeptides 3 and 6 offer a potentially useful mean to facilitate diffusion across the blood-brain barrier (BBB) and enhance the rate of entry of drug molecules into the central nervous system (CNS). The transfer rate constants (Kd) from simulated gastro-intestinal juices to simulated plasma, throughout artificial wall lipid membranes, were defined. The Kd values suggested that the molecules are absorbed both in gastric and intestinal environments in about comparable amounts. Since peptide bonds are often rapidly broken down by enzymes of most biological fluids the enzymatic hydrolysis characteristics in natural gastro-intestinal environment were studied. While hydrolysis of the ester bond of 6 was about 12%, after 5 h no important hydrolysis was observed for peptide bond of 3 and 6.

White beeswax microspheres: a comparative in vitro evaluation of cumulative release of the anticancer agents fluorouracil and ftorafur.

White beeswax microspheres were investigated as a vehicle for the controlled release of anticancer agents. The in vitro diffusion of fluorouracil (1) and its prodrug ftorafur (2) was studied. The transfer rate constants from simulated gastro-intestinal juices to simulated plasma, throughout artificial wall lipid membranes, were defined. The constant's values suggested that the two drugs are poorly absorbed from stomach and main absorption occurred in the intestinal tract. Microspheres of 1 and 2 were prepared by a meltable dispersion of white beeswax and a wetting agent. The method is simple, inexpensive, rapid and reproducible. The drug release from the prepared microspheres was evaluated in vitro. More than 95% of the isolated microspheres were of particle size range 100-425 microns. The average drug content was 4%. Drug dissolution was greatly retarded as a result of microsphere formation.

In vitro evaluation of cumulative release of valproic acid and vitamin E from hexadecanol microspheres. Part 2: Antiepileptic agents.

Hexadecanol microspheres were investigated as a vehicle for the controlled release of valproic acid. Microsphere of the antiepileptic agent were prepared by the meltable dispersion method in water using wetting agents. To prevent hepatotoxicity, microspheres containing both the drug and vitamin E were also prepared. The method in simple, inexpensive, rapid and reproducible. The drug release was evaluated in vitro. Kinetic results were analyzed to distinguish between the first-order release model and the square root of time relationship. More than 99% of the isolated microspheres were particle size range 200-710 microns. The average drug content was 24%. Drug bioavailability was greatly affected as a result of microspheres formation.

New prospective in treatment of Parkinson's disease: studies on permeation of ropinirole through buccal mucosa.

The aptitude of ropinirole to permeate the buccal tissue was tested using porcine mucosa mounted on Franz-type diffusion cells as ex vivo model. Drug permeation was also evaluated in presence of various penetration enhancers and in iontophoretic conditions. Ropinirole, widely used in treatment of motor fluctuations of Parkinson's disease, passes the buccal mucosa. Flux and permeability coefficient values suggested that the membrane does not appear a limiting step to the drug absorption. Nevertheless, an initial lag time is observed but the input rate can be modulated by permeation enhancement using limonene or by application of electric fields. Absorption improvement was accompanied by the important reduction of the lag time; at once the time required to reach the steady state plasma concentration was drastically decreased. On the basis of these results we could assume that clinical application of ropinirole by buccal delivery is feasible.

New prospectives in the delivery of galantamine for elderly patients using the IntelliDrug intraoral device: in vivo animal studies.

The transbuccal delivery of drugs could assist several categories of chronic, especially elderly, patients in adhering to a correct dosage regimen. In particular, patients suffering from dementia have several difficulties in following the prescribed dosage, in addition to problems associated with swallowing tablets. Galantamine is currently used for treating patients with mild to moderate Alzheimer's-type dementia. The transbuccal delivery of this drug could be an interesting non- invasive and safe administration route. Several studies have been performed in vitro and ex vivo within the framework of a European Commission funded Project (IntelliDrug-FP6), aimed at developing a device which would be fitted for controlled delivery of drugs by an electronic and software-driven system. The primary objective of this study was to evaluate the efficacy of a prototype of the IntelliDrug device in vivo on 6 pigs, following a single Galantamine dose to be delivered through the buccal mucosae, as compared to intravenous drug injection. The secondary objectives were: a) to verify Galantamine bioavailability through buccal delivery; b) to evaluate the permeation enhancement effect of iontophoresis; and, finally, c) to assess any histomorphological changes in the buccal mucosae after transbuccal delivery. The results suggested that transbuccal delivery has the potential to cause long-lasting and controlled blood levels of Galantamine. The latter crosses the entire buccal mucosae, reaching systemic circulation after about 30 minutes and its plasmatic peak approximately 120 minutes after administration. The histological analysis of the buccal mucosae did not reveal any evidence of flogosis or tissue injury. Our results have clearly confirmed that the buccal delivery of Galantamine is a reliable tool with which to overcome the drawbacks associated with the conventional administration route. In general, transbuccal drug delivery has been shown to be an interesting, non-invasive and safe administration route for delivering systemically-acting drugs.

A new delivery system of clobetasol-17-propionate (lipid-loaded microspheres 0.025%) compared with a conventional formulation (lipophilic ointment in a hydrophilic phase 0.025%) in topical treatment of atrophic/erosive oral lichen planus. A Phase IV, randomized, observer-blinded, parallel group clinical trial.

BACKGROUND: Topical application of clobetasol-17-propionate has been diffusely reported as an efficacious therapy in atrophic/erosive oral lichen planus (OLP), without exposing the patient to systemic side-effects. However, prolonged contact and respective topical effects on the oral mucosa should be avoided. OBJECTIVES: The aim of the present study was to evaluate efficacy and compliance of new lipid microspheres loaded with 0.025% of clobetasol propionate (formulation A) compared with a commonly used formulation (a sort of dispersion of a lipophilic ointment in a hydrophilic phase) with the same amount of drug (formulation B) in the topical treatment of OLP. PATIENTS AND METHODS: Fifty patients with symptomatic OLP were enrolled in a controlled single-blind phase IV clinical trial. After a dropout of five patients, a total of 45 patients [12 males and 33 females; mean age 61.1 years (+/- 12.3 SD; range 25-82)] were treated (17 with formulation A and 28 with formulation B, matched for gender and age; P > 0.2) with the same dosage regimen. At times T0, T1 and T2 we evaluated the following parameters: (i) pain score (by linear visual analogue scale; 0-100); (ii) clinical score; (iii) clinical resolution; and (iv) patient compliance. Statistical analysis was calculated using S-Plus 4.0 and SPSS 9.0 (Student's t-test, chi(2), Kolmogorov-Smirnow, Friedman, Student-Newman-Keuls, Mann-Whitney U-test and Spearman tests). RESULTS: Both formulations were found to be similar for parameters ii, iii and iv, although with a better general trend for formulation A; a significant difference was registered for formulation A in terms of a reduction in painful symptoms (parameter i) at time T2 (P = 0.02). CONCLUSIONS: Our results suggest that the new topical drug delivery system (formulation A) may enhance, at least in terms of symptom remission and compliance, the effectiveness of clobetasol propionate at a dose of 0.025% in OLP therapy.