Veicolazione di farmaci antitumorali in sistemi a rilascio controllato per terapie innovative dei tumori

Abstract

Studi precedenti svolti nel nostro laboratorio hanno mostrato che l’interazione tra lecitina e chitosano nella proporzione 20:1 portava alla formazione di nanoparticelle (NCL). Questi nanosistemi lecitina/chitosano possiedono caratteristiche di dimensione e carica superficiale che potrebbero essere particolarmente adatte alla veicolazione transmucosale dei farmaci. Lo scopo di questo progetto è stato quello di studiare nanoparticelle (NCL) caricate con tamoxifene (TAM), con l’obiettivo di produrre una formulazione adatta alla somministrazione orale. L’idea è stata quella di veicolare le nanoparticelle in microparticelle costituenti una polvere che al contatto con fluidi biologici restituissero il sistema colloidale di partenza. La presenza di nanoparticelle potrebbe consentire al farmaco di migliorare la propria biodisponibilità e ridurre la variabilità interpaziente, aumentandone cosi l’efficacia terapeutica e diminuendone gli effetti tossici. Innanzitutto è stato studiato il comportamento di rilascio del farmaco dal sistema NCL-TAM in differenti condizioni: fluido gastrico simulato, fluido intestinale simulato con e senza enzimi. Come enzimi sono stati scelti lipasi e lisozima, capaci di degradare in modo selettivo i componenti delle nanoparticelle. Il sistema colloidale prodotto caricato con l’antitumorale è stato anche studiato dal punto di vista della citotossicità su cellule di carcinoma mammario sensibili al tamoxifene (MCF7). L’uptake cellulare da parte di cellule di carcinoma del colon usate come modello dell’epitelio intestinale (CaCo-2) è stato altresì detrminato. Inoltre è stato condotto uno studio ex vivo su intestino di ratto per studiare la capacità del farmaco di passare attraverso la membrana intestinale quando somministrato sotto forma di nanoparticelle. In questi esperimenti è stata anche valutata l’incidenza della bioadesione del sistema nanoparticellare alla mucosa intestinale, sul passaggio del farmaco. Infine, è stata realizzata una polvere atta alla somministrazione orale costituita da microparticelle cariche di nanoparticelle. Dapprima è stato studiato il comportamento del sistema NCL-TAM con alcuni eccipienti idrofili, come α–ciclodestrina, β–ciclodestrina, maltodestrina e trealosio; in seguito si è passati alla produzione di polveri, mediante una tecnica di essicamento spray. Queste polveri sono costituite da microparticelle idrofile caricate con nanoparticelle veicolanti tamoxifene. Infine le microparticelle ottenute sono state caratterizzate per quanto riguarda le dimensioni, morfologia e comportamento in fluidi biologici simulati.

In Italy 7 women on 100 develop mammary cancer in the course of life. The grow of cancer is influenced by the concentration of hormones, and it has been demonstrated that the modulation of hormones levels is able to hold back the progress of the disease. Tamoxifen, an antiestrogen drug, is a first line drug used for mammary cancer, and it is also approved for cancer prevention. One of the major limits of the actual oral tablet therapy is the difference of inter- patient drug effects, probably due to the variability in metabolization capacity of patients. The formulation of tamoxifen with nanoparticles is expected to decrease this inconvenience by increasing the intestinal absorption and decreasing the metabolism of TAM. Previous studies made in our lab showed that the interaction between chitosan and lecithin in aqueous solution leads to the autoassembling of nanostructures composed by layers of both excipents. The aim of this project was to produce and study nanoparticles (NCL) loaded with tamoxifen (TAM) and to include nanoparticles in a formulation for oral delivery. The idea was to deliver nanoparticles by encapsulation in dry microparticles, i.e. in form of powder, that in contact with biological fluids give back the original colloidal system. In this work various properties of tamoxifen-loaded nanoparticles have been investigated like drug release, cytotoxicity and permeation through excised rat intestine. In this last experiment were evaluated the influence of nanoparticles on the passage of tamoxifen through the rat intestine tissue and the bioadhesive behaviour of nanoparticles. At the end some spray drying experiments were devoted to the production of a powder suitable for the oral administration made of microparticles containing tamoxifen-loaded nanoparticles. The results obtained in this work allowed to acquire new knowledge on the behaviour of chitosan-lecithin nanoparticles formulation. The release experiment showed that only the presence of specific enzymes trigger the release of tamoxifen from nanoparticle by demolition of structure on nanosystem. Indeed without enzyme like lipase or lysozyme the drug rests inside the nanoparticle until 24 hours. In viov it expected that tamoxifen will be available only when some enzyme like pancreatin are presents in intestinal lumen, while tamoxifen will be protected from gastric ambient. The possibility of improvement of drug intestinal permeation was evidenced by ex vivo intestinal experiment (Ussing chamber). With this experiment was possible demonstrate that the bioadhesion of the colloidal system on the mucous, close intestinal tissue, is crucial for the enhancement of drug permeation. Ex vivo experiments showed also that when we used nanoparticles, not only was obtained an increase of drug permeability, but the ratio between tamoxifen and its metabolites was completely changed. Indeed the experiment showed that in the receptor chamber, there was much more tamoxifen than 4-OH tamoxifen (metabolite) if compared to experiments in which only a tamoxifen suspension was used in the donor. This could help to decrease the variability of therapeutic effects observed in patients.It was possible to include tamoxifen-loaded nanoparticles in microparticles obtained by spray drying of water soluble excipients. The powder obtained could be used for the preparation of oral dosage forms, such as capsules, granules, tablets. The next step in the development of an oral formulation containing nanoparticles should take in account in vivo experiment to assess the effect on drug pharmacokinetics and to study the effect on model cancer in animals.