Preparazione e caratterizzazione di forme farmaceutiche solide orali a partire da materiale composito

2006/2007La somministrazione per via orale è la preferita per il trattamento farmacologico cronico. Circa il 40% dei nuovi principi attivi, tra i quali numerosi potenti farmaci lipofilici, è caratterizzato da bassa solubilità in acqua e la somministrazione per via orale di tali farmaci è frequentemente associata a bassa biodisponibilità. Infatti l’assorbimento di un principio attivo rilasciato da una forma farmaceutica orale dipende essenzialmente da due fattori: la dissoluzione del p.a. nel tratto gastrointestinale e la sua permeabilità attraverso la mucosa. Sulla base di questi due parametri i principi attivi sono stati distinti in quattro classi (Biopharmaceutical Classification System). In particolare per la seconda classe di composti, la dissoluzione nel lume gastro-intestinale è lo step limitante il processo di assorbimento. Per questa classe, numerosi approcci tecnologici sono riportati in letteratura allo scopo di aumentare la biodisponibilità orale di farmaci lipofilici come per esempio l’incorporazione in veicoli lipidici inerti come olii, la formulazione di sistemi auto-emulsificanti, di emulsioni e microemulsioni, liposomi, complessi di inclusione e sistemi dispersi farmaco-carrier. Rispetto a tali metodi convenzionali, il lavoro svolto ha riguardato la preparazione di sistemi attivati a base di ubidecarenone e ciclosporina ricorrendo alla tecnologia NEC (Nanoemulsified Composites) in collaborazione con la ditta Remedia s.r.l. titolare della tecnologia brevettata. La tecnologia NEC si basa sull’incorporazione di una doppia microemulsione (o/a/o) in un carrier microporoso. Successivamente l’attenzione è stata focalizzata sull’innovativa applicabilità delle microonde alla preparazione di sistemi binari farmaco:carrier. Il riscaldamento per mezzo delle microonde sfrutta le proprietà che le sostanze chimiche hanno di assorbire l’energia direttamente dalle onde elettromagnetiche le quali sono in grado di aumentare l’agitazione termica, e quindi la temperatura. L’energia fornita dalle radiazioni viene ceduta direttamente alla sostanza ed in tempi molto brevi. Tutte le sostanze caratterizzate da un dipolo, anche minimo, possono assorbire microonde. In tale contesto, oggetto della ricerca è stata l’attivazione dell’ibuprofene e piroxicam. Per tutti i farmaci considerati è stato inizialmente effettuato uno studio di messa a punto delle sostanze e delle condizioni operative ottimali, atte a fornire un prodotto finale lavorabile (prodotto composito). Alla preparazione dei sistemi ha fatto seguito la caratterizzazione chimico-fisica, necessaria per appurare lo stato solido del principio attivo. In particolare le tecniche adottate sono state: calorimetria a scansione differenziale (DSC), raggi X su polvere (PXRD), microscopia elettronica (SEM), hotstage microscopi (HSM), laser-light scattering. Successivamente le formulazioni approntate sono state caratterizzate anche dal punto di vista tecnologico e dissolutivo in termini di studi di cinetica di solubilizzazione e di rilascio. Inoltre in alcuni casi il prodotto è stato testato in vivo su ratti. Ed è stata anche valutata, in alcuni casi, la possibilità di realizzare forme farmaceutiche solide ad uso orale quali capsule e compresse. I risultati ottenuti sono qui di seguito riassunti. UBIDECARENONE La tecnologia preparativa adottata unitamente alla selezione dei componenti, hanno dimostrato il raggiungimento dell’obiettivo del presente lavoro e cioè l’aumento della biodisponibilità in vivo dell’Ubidecarenone. Le caratteristiche tecnologiche del materiale composito hanno permesso un’agevole realizzazione di capsule rigide ma, con un’ulteriore selezione di eccipienti per compressione diretta, si potrà realizzare anche la forma di dosaggio in compresse. CICLOSPORINA Il prodotto composito preparato è dunque risultato essere in grado di aumentare la biodisponibilità in vivo della ciclosporina rispetto alla materia prima commerciale. IBUPROFENE Dai risultati ottenuti si può concludere che la tecnica utilizzata e i polimeri scelti hanno portato ad un grado di amorfizzazione del farmaco tale da essere responsabile dell’incremento del profilo di dissoluzione in vitro delle dispersioni solide (IBU:PVP/VA e IBU: HP-β-CD) rispetto ai campioni di confronto. PIROXICAM Anche in questo studio è stato possibile verificare l’applicabilità delle MW alla creazione di sistemi dispersi solvent-free. Gli indiscussi vantaggi al ricorso ad un reattore a MW focalizzate (CEM) in termini di tempi e potenze applicate sono stati verificati. Il raggiungimento dell’obiettivo del lavoro e cioè l’aumento della biodisponibilità in vitro del piroxicam è stato ottenuto associando alla tecnologia adottata il polimero PVP/VA 64.XX Ciclo197

Marine Polysaccharides in Microencapsulation and Application to Aquaculture: “From Sea to Sea”

This review’s main objective is to discuss some physico-chemical features of polysaccharides as intrinsic determinants for the supramolecular structures that can efficiently provide encapsulation of drugs and other biological entities. Thus, the general characteristics of some basic polysaccharides are outlined in terms of their conformational, dynamic and thermodynamic properties. The analysis of some polysaccharide gelling properties is also provided, including the peculiarity of the charged polysaccharides. Then, the way the basic physical chemistry of polymer self-assembly is made in practice through the laboratory methods is highlighted. A description of the several literature procedures used to influence molecular interactions into the macroscopic goal of the encapsulation is given with an attempt at classification. Finally, a practical case study of specific interest, the use of marine polysaccharide matrices for encapsulation of vaccines in aquaculture, is reported

"The good, the bad and the ugly" of chitosans

The objective of this paper is to emphasize the fact that while consistent interest has been paid to the industrial use of chitosan, minor attention has been devoted to spread the knowledge of a good characterization of its physico-chemical properties. Therefore, the paper attempts to critically comment on the conflicting experimental results, highlighting the facts, the myths and the controversies. The goal is to indicate how to take advantage of chitosan versatility, to learn how to manage its variability and show how to properly tackle some unexpected undesirable features. In the sections of the paper various issues that relate chitosan properties to some basic features and to advanced solutions and applications are presented. The introduction outlines some historical pioneering works, where the chemistry of chitosan was originally explored. Thereafter, particular reference is made to analytical purity, characterization and chain modifications. The macromolecular characterization is mostly related to molecular weight and to degree of acetylation, but also refers to the conformational and rheological properties and solution stability. Then, the antimicrobial activity of chitosan in relation with its solubility is reviewed. A section is dedicated to the formulation of chitosan biomaterials, from gel to nanobeads, exploring their innovative application as active carrier nanoparticles. Finally, the toxicity issue of chitosan as a polymer and as a constructed nanomaterial is briefly commented in the conclusions

The effect of mushroom culture filtrates on the inhibition of mycotoxins produced by Aspergillus flavus and Aspergillus carbonarius

Two of the mycotoxins of greatest agroeconomic significance are aflatoxin B-1 (AFB(1)), and ochratoxin A (OTA). It has been reported that extracts from some wood-decaying mushrooms, such as Lentinula edodes and Trametes versicolor showed the ability to inhibit AFB(1) or OTA biosynthesis. Therefore, in our study, a wide screening of 42 isolates of different ligninolytic mushrooms was assayed for their ability to inhibit the synthesis of OTA in Aspergillus carbonarius and AFB(1) in Aspergillus flavus, in order to find a metabolite that can simultaneously inhibit both mycotoxins. The results showed that four isolates produce metabolites able to inhibit the synthesis of OTA, and 11 isolates produced metabolites that inhibited AFB(1) by >50%. Two strains, the Trametes versicolor strain TV117 and the Schizophyllum commune strain S.C. Ailanto, produced metabolites able to significantly inhibit (>90%) the synthesis of both mycotoxins. Preliminary results suggest that the mechanism of efficacy of the S. commune rough and semipurified polysaccharides could be analogous to that found previously for Tramesan(R), by enhancing the antioxidant response in the target fungal cells. The overall results indicate that S. commune's polysaccharide(s) could be a potential agent(s) in biological control and/or a useful component of the integrated strategies able to control mycotoxin synthesis

Structure of the capsular polysaccharide of the KPC-2-producing Klebsiella pneumoniae strain KK207-2 and assignment of the glycosyltransferases functions

Klebsiella pneumoniae strain KK207-2 was isolated in 2010 froma bloodstreaminfection of an inpatient at an Italian hospital. It was previously found to produce the KPC-2 carbapenemase and to belong to clade 1 of sequence type 258. Genotyping of the conserved wzi and wzc genes from strain KK207-2 yielded contrasting results: the wzc-based method assigned the cps207\u20132 to a new K-type, while the wzi-based method assigned it to the known K41 K-type. In order to resolve this contradiction, the capsular polysaccharide of K. pneumoniae KK207-2 was purified and its structure determined by using GLC-MS of appropriate carbohydrate derivatives, ESI-MS of both partial hydrolysis and Smith degradation derived oligosaccharides, andNMR spectroscopy of oligosaccharides, and the lithium degraded, native and de-O-acetylated polysaccharide. All the collected data demonstrated the following repeating unit for the K. pneumoniae KK207-2 capsular polysaccharide: OAc 6 [3)-\u3b2-D-Gal-(1-4)-\u3b2-D-Glc-(1-]n 4 I 1 \u3b2-D-Glcp-(1-6)-\u3b1-D-Glcp-(1-4)-\u3b2-D-GlcpA-(1-6)-\u3b1-D-Glcp The polysaccharide contains about 0.60 acetyl groups per repeating unit on C6 of the Gal residue. The reactions catalysed by each glycosyltransferase in the cpsKK207-2 gene cluster were assigned on the basis of structural homology with other Klebsiella K antigens

Rapid generation of Shigella flexneri GMMA displaying natural or new and cross-reactive O-Antigens

Generalized modules for membrane antigens (GMMA) are exosomes released from engineered Gram-negative bacteria and represent an attractive vaccine platform for the delivery of the O-Antigen (OAg), recognized as the key target for protective immunity against several pathogens such as Shigella. Shigella is a major cause of disease in Low- and Middle-Income countries and the development of a vaccine needs to deal with its large serotypic diversity. All S. flexneri serotypes, except serotype 6, share a conserved OAg backbone, corresponding to serotype Y. Here, a GMMA-producing S. flexneri scaffold strain displaying the OAg backbone was engineered with different OAg-modifying enzymes, either individually or in combinations. This strategy rapidly yielded GMMA displaying 12 natural serotypes and 16 novel serotypes expressing multiple epitopes combinations that do not occur in nature. Importantly, a candidate GMMA displaying a hybrid OAg elicited broadly cross-bactericidal antibodies against a large panel of S. flexneri serotypes

Structural characterisation of novel exopolysaccharide biosynthesized by potential probiotic strain Lactobacillus fermentum D12

Lactobacillus fermentum D12, biosynthesize exopolysaccharides, is released in large amounts in MRS broth supplemented with glucose. High Performance Size Exclusion Chromatography (HPSEC), 1H-NMR, GC and GC-MS analysis revealed that this strain produces three different types of EPOLs; the first homopolysaccharide (HoPOL) of a molecular weight of 400 kDa and two different low molecular weight heteropolysaccharides (HePOLs) of less than 2 kDa. 2D-NMR spectroscopy analysis revealed that HoEPOL, with the highest molecular mass, is composed of repeating units of D-glucose linked by an α-1,4-glycosidic bond, where 20% of the glucose subunits is acetylated at C-3. Further chromatographic analyses and NMR experiments showed that each HePOL contained mannose, glucose and galactose in an averaged relative molar ratio of 1.78:0.87:1 and 6.38:1.6:1, respectively. Since a probiotic strain survival in rigorous gastrointestinal (GI) conditions is the first probiotic selection criterion to be met, and with respect to efficient survival of D12 strain in GIT in vitro (bacteria counts ≥106 CFU ml-1) the potential probiotic role of Lb. fermentum D12 was evaluated. Also, sensitivity to different antibiotics, minimum inhibitory concentrations (MICs), antagonistic activity and analysis of fermentation of different carbohydrates using API 50 CHL media of this potential probiotic strain was assessed

Structural characterisation of novel exopolysaccharide biosynthesized by potential probiotic strain Lactobacillus fermentum D12

Lactobacillus fermentum D12, biosynthesize exopolysaccharides, is released in large amounts in MRS broth supplemented with glucose. High Performance Size Exclusion Chromatography (HPSEC), 1H-NMR, GC and GC-MS analysis revealed that this strain produces three different types of EPOLs; the first homopolysaccharide (HoPOL) of a molecular weight of 400 kDa and two different low molecular weight heteropolysaccharides (HePOLs) of less than 2 kDa. 2D-NMR spectroscopy analysis revealed that HoEPOL, with the highest molecular mass, is composed of repeating units of D-glucose linked by an α-1,4-glycosidic bond, where 20% of the glucose subunits is acetylated at C-3. Further chromatographic analyses and NMR experiments showed that each HePOL contained mannose, glucose and galactose in an averaged relative molar ratio of 1.78:0.87:1 and 6.38:1.6:1, respectively. Since a probiotic strain survival in rigorous gastrointestinal (GI) conditions is the first probiotic selection criterion to be met, and with respect to efficient survival of D12 strain in GIT in vitro (bacteria counts ≥106 CFU ml-1) the potential probiotic role of Lb. fermentum D12 was evaluated. Also, sensitivity to different antibiotics, minimum inhibitory concentrations (MICs), antagonistic activity and analysis of fermentation of different carbohydrates using API 50 CHL media of this potential probiotic strain was assessed

Influence of Bacterial Biofilm Polysaccharide Structure on Interactions with Antimicrobial Peptides: A Study on Klebsiella pneumoniae

Biofilms are complex systems produced by bacteria and constituted by macromolecular matrix embedding cells. They provide advantages to bacteria including protection against antimicrobials. The protection given by biofilms produced by Klebsiella pneumoniae strains towards antimicrobial peptides of the innate immune system was investigated. In particular, the role of matrix bacterial exopolysaccharides was explored. Three clinical strains producing exopolysaccharides with different chemistry were selected and the interaction of purified biofilm polysaccharides with two bovine cathelicidins was studied by circular dichroism spectroscopy and microbiological assays to establish their influence on the peptide’s antimicrobial activity. The spectroscopic data indicated a different extent of interaction with the two peptides, in a manner dependent on their sugar composition, and in particular the presence of rhamnose residues correlated with a lower interaction. The extent of interaction was then related to the protection towards antimicrobial peptides, conferred by the addition of the different exopolysaccharides, in minimum inhibitory concentration (MIC) assays against a reference Escherichia coli strain. Microbiological results were in very good agreement with spectroscopic data, confirming the active role of matrix polysaccharides in determining a biofilm’s protective capacity and indicating lower protection levels afforded by rhamnose containing exopolysaccharides

“The Good, the Bad and the Ugly” of Chitosans

The objective of this paper is to emphasize the fact that while consistent interest has been paid to the industrial use of chitosan, minor attention has been devoted to spread the knowledge of a good characterization of its physico-chemical properties. Therefore, the paper attempts to critically comment on the conflicting experimental results, highlighting the facts, the myths and the controversies. The goal is to indicate how to take advantage of chitosan versatility, to learn how to manage its variability and show how to properly tackle some unexpected undesirable features. In the sections of the paper various issues that relate chitosan properties to some basic features and to advanced solutions and applications are presented. The introduction outlines some historical pioneering works, where the chemistry of chitosan was originally explored. Thereafter, particular reference is made to analytical purity, characterization and chain modifications. The macromolecular characterization is mostly related to molecular weight and to degree of acetylation, but also refers to the conformational and rheological properties and solution stability. Then, the antimicrobial activity of chitosan in relation with its solubility is reviewed. A section is dedicated to the formulation of chitosan biomaterials, from gel to nanobeads, exploring their innovative application as active carrier nanoparticles. Finally, the toxicity issue of chitosan as a polymer and as a constructed nanomaterial is briefly commented in the conclusions